Featured Abstract * (BUTTON) Article notes * (BUTTON) Copyright and License information Issue date 2023 Jul. © 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article under the terms of the [19]http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. [20]PMC Copyright notice PMCID: PMC10336391 Thursday 18 May 2023 Late Breaking Oral OT04: Therapeutics Chairs: Jonathan Anderson Location: Room 606/607 11:30 ‐ 13:00 OT04.5. Toward extracellular vesicles based delivery of CRISPR/Cas9 to target androgen receptor in prostate cancer cells Houjian Cai University of Georgia, Athens, USA Introduction: Prostate cancer (PCa) is the most diagnosed malignancy in the United States in men. About 20–30% of PCa patients will develop castration resistance. Androgen receptor (AR) signaling is still maintained in the majority of PCa cells. CRISPR/Cas9 machinery mediated genomic editing provides a genetic approach to remove AR and silence AR signaling, but an efficient and specific delivery of CRISPR machinery to PCa cells has not been established. Methods: Cas9 was genetically modified by a fusion with the octapeptide derived from the N‐terminus of Src kinase. 3T3 cells stably expressing luciferase was infected with lentivirus expressing Cas9, myristoylated Cas9 (mCas9), or mCas9 (G2A)/sgRNA‐Luc. 293T overexpressing AR cells were infected by lentivirus expressing mCas9/sgRNA‐AR, or transduced with extracellular vesicles (EVs) encapsulating mCas9/sgRNA‐AR ribonucleoprotein (RNP) complex. The targeted cells were analyzed for the expression of Cas9 and knockout efficiency of luciferase or AR. Functionality of Cas9/sgRNA were also examined by T7 endonuclease assay and Sanger sequencing. EVs were characterized by the size distribution, morphology, and EVs protein biomarkers. A membrane protein was also co‐expressed in the EVs‐producing cells. Its specificity in targeting recipient cells was analyzed. Results: We proved the principle that the genetically modified mCas9/sgRNA‐luciferase was encapsulated into EVs and functional to knock out luciferase. Next, the mCas9/sgRNA‐AR was capable of knocking out ectopically expressed and endogenous AR gene in the 293T‐AR cells and/or PCa cells. The indel of AR gene was illustrated by the T7 endonuclease assay and Sanger sequencing. The encapsulated Cas9 protein accounted for 0.5% of total EV protein and was resistant from the protease digestion. Finally, incorporation of a membrane protein confers specificity in targeting recipient cells. We will further determine that the EVs expressing the membrane protein and encapsulating CRISPR RNP silence AR in PCa cells. Summary/Conclusion: N‐myristoylated CRISPR‐Cas9 is functional and encapsulated into EVs. mCas9/sgRNA‐AR leads to the indel of AR gene in target cells. This study will potentially develop a technology to deliver CRISPR machinery for treatment of castration resistant prostate cancer by removing AR at the genomic DNA level. Funding: U01 [21]CA225784‐01; DOD W81XWH‐22‐PCRP‐EHDA; R21 AI157831‐01A1 and [22]AI171944‐01A1; The American Institute for Cancer Research; Georgia Research Alliance; and the Seed funding from the Georgia Regenerative Engineering & Medicine Center. keywords: Extracellular vesicles, CRISPR, Cas9/sgRNA, AR, prostate cancer OT05: Single EV Analysis Chairs: Tijana Jovanovic‐Talisman, Randy Carney Location: Ballroom 6BC 15:15 ‐ 16:45 OT05.5. Placenta‐derived extracellular vesicles indicate dysregulation of transplacental transfer Dennis Yüzen ^1; Isabel Graf^2; Anke Diemert^3; Petra Clara Arck^2 ^1Laboratory for Experimental Feto‐Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center of Hamburg‐Eppendorf, Germany; Institute of Immunology, University Medical Center of Hamburg‐Eppendorf,, Hamburg, Germany; ^2Laboratory for Experimental Feto‐Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center of Hamburg‐Eppendorf, Germany, Hamburg, Germany; ^3Department of Obstetrics and Prenatal Medicine, University Medical Center of Hamburg‐Eppendorf, Germany, Hamburg, Germany Introduction: A growing body of evidence supports the idea that placenta‐derived extracellular vesicles (EV) ensure successful pregnancy progression. Conversely, pregnancy adversities, e.g., preeclampsia or maternal infection, have been linked to placental dysfunction as indicated by altered placental EV production. Clinically, placental dysfunction manifests in an impaired transfer of nutrients and hormones from mother to the fetus. Recent studies further link placental dysfunction to an altered transplacental transfer of maternal cells to the fetus. These cells are referred to as maternal microchimeric cells (MMc) and have the potential to persist in the offspring for years. We investigated whether placenta‐derived EV serve as a proxy for transplacental cellular transfer in healthy and SARS‐CoV‐2 infected women. Methods: EV from third trimester serum samples of healthy pregnant women and pregnant women upon SARS‐CoV‐2 infection were isolated via cushion‐ultracentrifugation. Quality of EV‐isolation was ensured by Nanoparticle Tracking Analysis. Using placental alkaline phosphatase (PLAP)‐specific antibody staining, placenta‐derived EV were identified by Imaging Flow Cytometry analysis. Further, PLAP+ EV were correlated with MMc numbers from matching neonatal cord blood samples. Finally, differential quantitative proteomics will be used to identify possible markers on PLAP+ EV that are associated with an adequate placental transfer of MMc. Results: SARS‐CoV‐2 infection results in strongly reduced numbers of MMc but leads to significantly higher frequencies of PLAP+ EV in the maternal serum. In healthy mothers with female offspring, the number of total EV and PLAP+ EV are both significantly correlated with an a higher transplacental transfer of MMc. This effect was reversed in mothers suffering from SARS‐CoV‐2 infection. Summary/Conclusion: Reduced transfer of MMc following maternal infection is associated with an excessive production of placenta‐derived EV indicating a dysregulation in placental function. Decoupling of placenta‐derived EV and MMc transfer was specifically evident for women pregnant with a female fetus suggesting sex‐specific effects of EV formation following maternal infection. Funding: Funding of this study was made possible by grants provided by the German Research Foundation (KFO296: AR232/25‐2 and DI2103/2‐2) and the Authority for Science, Research and Equality, Hanseatic City of Hamburg, Germany. keywords: pregnancy, maternal microchimerism, SARS‐CoV‐2, placenta, trophoblast Sunday 21 May 2023 Late Breaking Oral LB1: Late Breaking Oral Symposia: Nervous System Chairs: Setty Margana, Ursula Sandau Location: Room 608/609 10:15 ‐ 11:45 LB1.1. Glial EVs travelling at the neuron surface Claudia Verderio ^1; Giulia D'Arrigo^2; Martina Gabrielli^3; Francesca Peri^4 ^1CNR Istituto di Neuroscienze, Vedano al Lambro, Italy; ^2Consiglio Nazionale delle Ricerche, Varese, Italy; ^3Consiglio Nazionale delle Ricerche, Milano, Italy; ^4University of Zurich, Switzerland Introduction: Extracellular vesicles (EVs) are important mediators of glia‐to‐neuron communication and are responsible for the spreading of misfolded proteins in the diseased brain. However, how glial EVs move across the extracellular space to reach target neurons and spread pathological signals remain largely elusive. Methods: Using optical tweezers combined to time‐lapse imaging we studied the interaction dynamics of single large glial EVs with cultured hippocampal neurons. EVs released by astrocytes/microglia were isolated by differential centrifugation at 10.000xg and characterized by TRPS, cryo‐EM and WB. Both neurons and EVs were treated with drugs interfering with cytoskeletal elements. Glial EVs carrying Aβ (Aβ‐EVs) and control EVs were stereotaxically injected in the mouse lateral entorhinal cortex (EC) and LTP measured in cortico‐hippocampal slices in both the EC and its target region, the dentate gyrus of the hippocampus (DG). Results: We found that glial EVs move along the surface of neurons, and that motion of most EVs is driven by the binding to a neuron receptor that is coupled to a moving cytoskeleton, whereas a minority of EVs independently moves in an actin‐dependent manner (D'Arrigo et al., 2021). To investigate whether EVs may use axonal projections to move throughout the brain, we exploited the ability of Aβ‐EVs to impair LTP and explored whether they may propagate LTP dysfunction along the EC‐DG circuit. Our data show that Aβ‐EVs injected in the EC propagate LTP impairment to the DG. However, when treated with annexin V that impairs their motion, Aβ‐EVs do not propagate the deficit along the EC‐DG circuit, implicating EV motion in the propagation of synaptic dysfunction (Gabrielli et al., 2022). Summary/Conclusion: We are currently imaging the dynamics of fluorescent microglial EVs (expressing farnesyl‐GFP or farnesyl‐mCherry) after injection into the brain of optically transparent Zebrafish larvae seeking direct evidence of glial EV motion along neuronal processes. Funding: This work was supported by Horizon2020 project #874721 PREMSTEM to CV keywords: glial EVs, optical tweezers, EV‐neuroin interaction LB1.2. Towards a human brain EV atlas: Characteristics of EVs from different brain regions Tanina arab ^1; Yiyao Huang^2; Ashley E. Russell^3; Emily R. Mallick^2; Rajini Nagaraj^4; Evan Gizzie^5; Javier Redding‐Ochoa^6; Juan C C. Troncoso^6; Olga Pletnikova^7; Andrey Turchinovich^8; David A A. Routenberg^5; Kenneth W. Witwer^2 ^1The Johns Hopkins University School of Medicine, Baltimore, USA; ^2Johns Hopkins University, Baltimore, USA; ^3Penn State Erie, The Behrend College, Laurel, USA; ^4MesoScale Diagnostics, LLC, Rockville, MD, USA, Bethesda, USA; ^5Meso Scale Diagnostics, Rockville, USA; ^6Johns Hopkins University School of Medicine, Baltimore, USA; ^7Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Baltimore, USA; ^8SciBerg, Mannheim, USA Introduction: The study of brain‐derived extracellular vesicles (bdEVs) is important since these small particles contain a diverse range of biomolecules and may contribute to intercellular communications in the brain in normal physiology and pathology. However, it is not yet known if bdEVs differ by brain region of origin. In this study, we obtained tissues from eight distinct regions from one donor and assessed the properties of their EVs. Methods: bdEVs were separated per our established protocol (Huang et al., JEV, 2020) from tissue of eight brain regions (n = 8, Johns Hopkins Brain Resource Center). Particle counts were assessed using nanoflow cytometry, while particle size distribution was measured by both nanoflow cytometry and transmission electron microscopy (TEM). bdEV morphology was observed by TEM. bdEV surface proteins were profiled using multiplexed ELISA and SP‐IRIS. EVs were profiled by transcriptomics and proteomics. Results: Particle yield was greatest for cerebellum EVs and smallest for orbitofrontal, postcentral gyrus, and thalamus regions. TEM suggested slightly different morphologies/electron density for some regions. Surface phenotyping showed that CD63 was less abundant across all regions than CD81 and CD9. bdEVs from different regions displayed different putative neuron, microglia, astrocyte, and endothelial cell markers, indicating potential cellular origin differences. For example, neuronal markers NCAM, CD271, and NRCAM were more abundant on EVs from the medulla, cerebellum, and occipital regions, respectively. Summary/Conclusion: The present study suggests that bdEVs may have distinct characteristics based on region of origin in the brain. These findings may offer insight into EV origins and cargo related to cellular composition. Our results suggest that studies of larger sample sizes and investigation of neuropathological cases are warranted. Funding: This work was supported in part by grants from the US National Institutes of Health: [23]AI144997, [24]MH118164, and [25]DA047807. keywords: Brain, Extracellular vesicles, tissue, corpus callosum, thalamus, orbitofrontal, postcentral gyrus, occipital gyrus, cerebellum, medulla, hippocampus. LB1.3. Tracking human neurologic disease status in mouse brain and plasma using reporter‐tagged EV‐associated biomarkers Katia Maalouf^1; Christine A. Vaine^2; Dawn M. Frederick^2; Akiko Yoshinaga^2; Wataru Obuchi^2; Shadi Mahjoum^3; Lisa Nieland^2; Jamal Al Ali^2; D. Cristopher Bragg^2; Xandra O. Breakefield^3; Koen Breyne ^1 ^1Massachusetts General Hospital and Harvard Medical School, Charlestown, USA; ^2Massachusetts General Hospital and Harvard Medical School, USA; ^3Massachusetts General Hospital and Harvard Medical School, Boston, USA Introduction: X‐linked dystonia‐parkinsonism (XDP) is a neurodegenerative disease caused by a retrotransposon insertion in intron 32 of the TAF1 gene. This insertion leads to the mis‐splicing of intron 32 (TAF1‐32i) and reduced TAF1 levels. TAF1‐32i transcript is unique to XDP patient cells and can be detected in their extracellular vesicles (EVs). Methods: We engrafted patient and control iPSC‐derived neural progenitor cells (hNPCs) into the striatum of immune‐compromised mice to investigate the status of EVs containing the XDP transcript signature. To track TAF1‐32i transcript spreading by EVs, we transduced brain‐implanted hNPCs with a lentiviral construct called ENoMi. ENoMi consists of a tetraspanin scaffold that is re‐engineered to distinguish hNPCs‐derived EVs from other cell‐derived EVs and protein contaminants in biofluids. Alongside improved detection by luciferase and fluorescent protein reporter tags of ENoMi‐hNPCs‐derived EVs, the ENoMi‐EV's surface is compatible with specific immunocapture purification facilitating TAF1‐32i analysis. Results: Through our ENoMi‐labeling method, TAF1‐32i was demonstrated to be present in EVs released from XDP hNPCs implanted in mouse brains. Post‐implantation of ENoMi‐XDP hNPCs, TAF1‐32i transcript was retrieved in EVs isolated from XDP NPC‐implanted mouse brain and blood, and a build‐up of TAF1‐32i was observed over time in the plasma. We compared and combined our EV isolation technique to analyze XDP‐derived TAF1‐32i with other techniques, including size exclusion chromatography, ExoDisc, and anti‐CD81 pulldown. Summary/Conclusion: Overall, our study demonstrates the successful engraftment of XDP patient‐derived hNPCs in mice as a tool for monitoring disease markers with EVs in biofluids. Funding: The Collaborative Center for X‐Linked Dystonia Parkinsonism. keywords: neurodegenerative disease ‐ brain‐derived EVs ‐ EV‐engineering ‐ biomarkers ‐ xenograft mouse model LB1.4. Mitovesicles secreted into the extracellular space of brains with mitochondrial dysfunction impair synaptic plasticity Efrat Levy ^1; Pasquale D'Acunzo^2; Elentina K. Argyrousi^3; Jonathan M. Ungania^4; Yohan Kim^5; Ottavio Arancio^3 ^1Center for Dementia Research, Nathan S. Kline Institute/Departments of Psychiatry, Biochemistry & Molecular Pharmacology, and the Neuroscience Institute, NYU Langone Medical Center, New York, USA; ^2Nathan S. Kline Institute, New York, USA; ^3Columbia University, USA; ^4Nathan S. Kline Institute, USA; ^5Center for Dementia Research, Nathan S. Kline Institute/Departments of Psychiatry, Biochemistry & Molecular Pharmacology, and the Neuroscience Institute, NYU Langone Medical Center, Park Ridge, USA Introduction: A key question in neurobiology is how pathology is initiated and propagated in the brain of patients with neurodegenerative disorders. Mitochondrial dysfunction and brain hypometabolism are hallmarks of aging, neurodegenerative, and neurodevelopmental disorders, causing the build‐up of damaged mitochondria and the production of high levels of reactive oxygen species over time. We previously demonstrated that the level and content of mitovesicles, extracellular vesicles of mitochondrial origin, isolated from brains with malfunctioning mitochondria are altered. Therefore, we investigated whether these mitovesicles have a pathogenic effect. Methods: Extracellular vesicles were isolated from the brain extracellular space of a mouse model of Down syndrome (Ts2 mice) and littermate controls (D'Acunzo P. et al. Nature protocols 2022; PMCID: PMC9633367). C57BL/6J coronal hippocampal slices were perfused with either mitovesicles, microvesicles, or exosomes, long‐term potentiation (LTP) was induced, and responses recorded. To test the involvement of monoamine oxidases, Ts2 mitovesicles were pretreated with either clorgiline (MAO‐A inhibitor) or pargyline (MAO‐B inhibitor) before LTP recordings. Results: Mitovesicles isolated from brains with dysfunctional mitochondria perturbed LTP of normal mouse hippocampi within a few minutes of treatment, compatible with an enzymatic, mitovesicle‐linked activity, and was abrogated when mitovesicles were deprived of their MAO‐B (but not MAO‐A) activity, thus defining the molecular mechanism. Microvesicles and exosomes isolated from the same brains and mitovesicles isolated from control brains did not have this electrophysiological effect. Summary/Conclusion: These data show that mitovesicles acquire pathogenic functions under conditions of mitochondrial dysfunction, potentially affecting memory formation and the propagation of neuropathology. Funding: NIH grants [[26]AG017617, [27]AG056732, [28]AG057517, [29]DA044489, NS110024]. keywords: Brain, neurodegeneration, aging, microvesicle, exosome, mitovesicle LB1.5. Cystatin C loaded in extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo Yuqi Gui^1; Yohan Kim^2; Santra Brenna^1; Maximilian Wilmes^3; Giorgio Zaghen^4; Lennart Kuchenbecker‐Pöls^3; Hannah Voß^5; Antonia Gocke^6; Hartmut Schlüter^5; Hermann C. Altmeppen^7; Tim Magnus^8; Efrat Levy^9; Berta Puig ^8 ^1Neurology Department, Experimental Research in Stroke and Inflammation (ERSI) group, University Medical Center Hamburg‐Eppendorf (UKE), HAMBURG, Germany; ^2Center for Dementia Research, Nathan S. Kline Institute/Departments of Psychiatry, Biochemistry & Molecular Pharmacology, and the Neuroscience Institute, NYU Langone Medical Center, Park Ridge, USA; ^3Neurology Department, Experimental Research in Stroke and Inflammation (ERSI) group, University Medical Center Hamburg‐Eppendorf (UKE), Germany; ^4Center for Dementia Research, Nathan S. Kline Institute/Departments of Psychiatry, Biochemistry & Molecular Pharmacology, and the Neuroscience Institute, NYU Langone Medical Center, USA; ^5Mass Spectrometry and Proteomics, University Medical Center Hamburg‐Eppendorf (UKE), Hamburg, Germany; ^6Mass Spectrometry and Proteomics, University Medical Center Hamburg‐Eppendorf (UKE), Germany; ^7Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf (UKE), Hamburg, Germany; ^8Neurology Department, Experimental Research in Stroke and Inflammation (ERSI) group, University Medical Center Hamburg‐Eppendorf (UKE), Hamburg, Germany; ^9Center for Dementia Research, Nathan S. Kline Institute/Departments of Psychiatry, Biochemistry & Molecular Pharmacology, and the Neuroscience Institute, NYU Langone Medical Center, New York, USA Introduction: Synaptic loss is an early event in the undersupplied but not yet irreversibly injured penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. In the present study, we aimed to detect proteins involved in endogenous protection mechanisms at the synapses in the penumbra after stroke and to analyze the potential beneficial effect of these candidates as a prospective stroke treatment. Methods: We performed mass spectrometry analyses of synaptosomes, isolated synaptic terminals, purified from the ipsilateral hemisphere of mice subjected to experimental stroke at different time points (24 hours, 4, and 7 days), and sham‐operated mice. Isolated primary neurons from mice were subjected to oxygen‐glucose deprivation (OGD) and treated with cystatin (CysC) either free or loaded in brain‐derived extracellular vesicles (BDEVs). Synapses were quantified after 3D reconstruction of confocal microscopy pictures. Mice subjected to stroke were treated with CysC‐loaded BDEVs by intracerebroventricular injection 6 hours after stroke and sacrificed 24h aftwerwards. Synaptic markers were analyzed by western blot and mass spectrometry analysis of the treated ipsilateral hemisphere was also performed. Results: Proteomic analyses indicated that, among the proteins differentially expressed between the two groups, the level of CysC was significantly increased at 24h after stroke, decreasing at 4 days before returning to steady‐state levels at 7 days, thus indicating a potential transient and intrinsic mechanism to rescue neurons. When recombinant CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, it significantly improved the preservation of synaptic structures. Moreover, the same effect was observed when brain‐derived extracellular vesicles (BDEVs)‐loaded with CysC were applied to the neuronal cultures. Finally, when recombinant CysC contained in BDEVs was administered to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25 and Homer‐1 in the penumbra area compared to the sham group. Summary/Conclusion: We show that CysC‐loaded EVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of its potential therapeutic use in stroke patients. keywords: extracellular vesicles, cystatin C, stroke, tMCAO, synaptosomes LB1.6. Characterization of mRNA, lncRNA, and circRNA in plasma and urine from participants with Alzheimer's disease, Parkinson's disease, and healthy controls Elizabeth Hutchins ^1; Rebecca Reiman^2; Cherae Bilagody^2; Jerry Antone^2; Bessie Meechoovet^2; Dorothy Marie Palomares^2; Megan Hall^3; Kendall Van Keuren‐Jensen^4 ^1Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, U, Phoenix, USA; ^2TGen, Phoenix, USA; ^3TGen, USA; ^4Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA Introduction: EVs originating from the cells in the CNS are released and carry cargo into the periphery, including metabolites, peptides, DNA and RNA. These messages originating from their tissue of origin can then be assessed and quantified in accessible biofluids, and are a promising area of research for monitoring neurodegenerative disease onset and progression. Biofluids such as plasma and urine are less invasive to collect and more readily available than cerebrospinal fluid and can potentially be utilized to track the onset and progression of neurodegenerative disease. We characterize the RNA cargo in two clinically relevant biofluids collected simultaneously, and across two timepoints. Methods: Plasma and urine samples were collected simultaneously, and across at two visits, from participants with Alzheimer's Disease (AD), Parkinson's Disease (PD), or healthy controls, from a contracted vendor. RNA was isolated from 1mL of plasma (Qiagen exoRNeasy Midi, 100% EtOH) or 10mL of urine (Qiagen exoRNeasy Maxi, 70% EtOH) and libraries were prepared for sequencing using Takara's SMARTer Stranded Pico v2 kit. Reads were aligned to the GRCh38 genome using STAR v2.6+ and genes quantified with featureCounts using a custom GENCODE v40 annotation with lncRNA from lncipedia v5.2, and circRNA were identified with CIRI2. Deeptools package was used to assess 5’ to 3’ gene coverage, with a bin resolution of 5bp. Results: We describe mRNA, lncRNA, and circRNA species identified in plasma and urine, their 5’ to 3’ gene coverage, likely tissue of origin and relevance to the CNS, and identify differentially expressed genes in AD and PD. Summary/Conclusion: Plasma and urine are clinically relevant biofluids that contain information from the CNS. More mRNA and lncRNA were detected in plasma than urine, though not significantly more. CircRNA are readily detected in plasma but relatively few are found in urine. Brain specific transcripts have a higher overall 5’ to 3’ coverage in plasma than urine. LB2: Late Breaking Oral Symposia: Clinical Application Chairs: Rosella Crescitelli, My Mahoney Location: Room 606/607 10:15 ‐ 11:45 LB2.1. First pilot case‐control interventional study using autologous extracellular vesicles as treatment option for chronic venous ulcers unresponsive to conventional treatments Lorenzo Gibello^1; Sergio D'Antico^2; Marika salafia^1; Rebecca Senetta^1; Margherita Alba Carlotta Pomatto^1; Giulia Orlando^1; Alessandro Sarcinella^1; Tatiana Lopatina^1; Pietro Quaglino^1; Marco Lorenzi^1; Fabio Verzini^1; Giovanni Camussi^3; Maria Felice MF Brizzi ^1 ^1University Of Turin, Torino, Italy; ^2AOU Torino, Torino, Italy; ^3Department of Medical Sciences, University of Turin, Torino, Italy Introduction: Current therapeutic approaches for chronic venous ulcers (CVUs) still require evidence of effectiveness. Diverse sources of extracellular vesicles (EVs) have been proposed for tissue regeneration, however the lack of potency tests, to predict in‐vivo effectiveness, and a reliable scalability have delayed their clinical application. This study aimed to investigate whether autologous serum‐derived EVs (s‐EVs), recovered from patients with CVUs, may be a proper therapeutic approach to improve the healing process. Methods: A pilot case‐control interventional study (CS2/1095/0090491) has been designed and s‐EVs recovered from patients. Patient eligibility included two or more distinct chronic lesions in the same limb with 11 months as median persistence of active ulcer before enrollment. Patients were treated three times a week, for 2 weeks. Results: Qualitative CVU analysis demonstrated that s‐EVs‐treated lesions displayed a higher percentage of granulation tissue compared to the control group (Sham) (s‐EVs 3 out of 5: 75–100% vs Sham: none), further confirmed at day 30. s‐EVs‐treated lesions also displayed higher sloughy tissue reduction at the end of treatment even increased at day 30. Additionally, s‐EV treatment led to a median surface reduction of 151 mm2 compared to 84 mm2 in the Sham group, difference even more evident at day 30 (s‐EVs 385 mm2 vs Sham 106 mm2 p = 0.004). Consistent with the enrichment of transforming growth factor‐β1 in s‐EVs, histological analyses showed a regenerative tissue with an increase in microvascular proliferation areas. Summary/Conclusion: This study first demonstrates the clinical effectiveness of autologous s‐EVs in promoting the healing process of CVUs unresponsive to conventional treatments. Funding: 2i3T and Unicyte keywords: chronic venous ulcers, autologous serum‐derived EVs, angiogenesis LB2.2. Natural killer cell‐derived extracellular vesicles in neuroblastoma immunotherapy Nasibeh Karimi ^1; Francesca Trampetti^1; Nicolo Toldo^1; Muller Fabbri^2 ^1Center for Cancer and Immunology Research, Children's National Research Institute, Children's National Hospital, Washington, DC, USA; ^2Center for Cancer and Immunology Research, Children's National Research Institute, Children's National Hospital, Washington, DC, Los Angeles, USA Introduction: In adoptive immunotherapy, natural killer cells (NK cells) have proven useful because they are capable of recognizing and killing tumor cells in a highly efficient manner. Neuroblastoma ‐derived extracellular vesicles (EVs) have been shown to dysregulate the function of NK cells. Since NK cell‐EVs can overcome the chemical and physical barriers in the tumor microenvironment, they may be more beneficial than NK cells for neuroblastoma immunotherapy. Methods: Since the NK cells are not abundant in the blood, an ex vivo expansion assay was used to produce a large quantity of functional NK cells. PBMC co‐culture with K562mb‐Il21 in the presence of IL‐2 for 21 days, and functional NK cells on days 14 and 21, were used for downstream experiments. The purity and phenotype of the NK cells were assessed by flow cytometry using CD56‐APC, CD16‐FITC, and CD3‐PE. Expanded NK cells were cultured in the presence of IL‐2 and EVs were isolated from the conditioned media by ultracentrifugation and an Iodixanol density cushion. EVs were characterized and quantified by Single‐particle interferometric reflectance imaging sensing (SP‐IRIS), electron microscopy, Nano particle tracking (NTA), Qubit protein quantification, and western blot (EV and non‐EV markers). Tumor growth and Real time quantification of cell death upon treatment with NK cells‐EVs were examined by continuous monitoring of the cells for 72 hours by using Incucyte technology. Results: PBMC co‐cultured with K562‐mb‐21 cells showed an expansion fold of 200 on day 14. Flow cytometry analysis of expanded NK cells on days 14 and 21 showed 99% purity and a high expression of CD56, an NK cell surface marker, with low contamination by CD3 (0.09%). In Western blotting analysis, classical EV markers such as CD9, CD63, CD81, and Flotillin‐1 were detected in NK cell‐EVs, while endoplasmic reticulum markers such as Calnexin were not detected. Additionally, SP‐IRIS confirmed that NK cell‐EVs were positive for tetraspanins CD9, CD63 and CD81. Vesicular structure of various sizes was observed by electron microscopy. Cell death was observed upon treatment with NK cell‐EVs in neuroblastoma cell lines. Summary/Conclusion: These results suggest that ex vivo expansion of NK cells leads to the production of large quantities of functional NK cells and NK‐EVs and that their cargo elicits cytotoxicity in neuroblastoma cell lines. LB2.3. PD‐L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD‐L1‐low and ‐negative patients with non‐small cell lung cancer Kerstin Menck ^1; Nadja Schöne^2; Marcel Kemper^2; Georg Evers^2; Carolin Krekeler^2; Arik Bernard Schulze^2; Georg Lenz^2; Eva Wardelmann^2; Claudia Binder^3; Annalen Bleckmann^4 ^1University Hospital Muenster, Muenster, Germany; ^2University Hospital Muenster, USA; ^3University Medicine Goettingen, Göttingen, Germany; ^4University Hospital Muenster, Münster, Germany Introduction: Immunotherapy has revolutionized the treatment of patients with non‐small cell lung cancer (NSCLC). The level of tissue PD‐L1 expression (tPD‐L1) positively correlates with the probability of treatment response. However, even tPD‐L1 low (1‐49%) and absent (< 1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study tested whether tumor‐associated extracellular vesicles (EVs) could fill this gap. Methods: We isolated small EVs (sEVs) and large EVs (lEVs) from cell culture supernatants as well as peripheral blood of NSCLC patients (n = 108), non‐cancer (n = 23) and healthy (n = 54) controls by differential ultracentrifugation and thoroughly characterized them by electron microscopy, NTA, and immunoblot. Antigen expression on patient‐derived lEVs was measured by standard flow cytometry and results were correlated with clinical data, including response to immunotherapy. Results: Using NSCLC cell lines, we identified a panel of tumor‐related antigens that were enriched on lEVs compared to sEVs. The levels of lEVs carrying these antigens were elevated in plasma of NSCLC patients and discriminated them from controls. Among the tested antigens, we focused on programmed cell death ligand 1 (PD‐L1), which is a well‐known direct target for immunotherapy. PD‐L1 was mainly found on a population of CD45‐/CD62P‐ lEVs and thus did not seem to originate from platelets or leukocytes. Patients with high levels of PD‐L1+ lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. The predictive value of lEV‐associated PD‐L1 was based on a single measurement at baseline and outperformed tissue PD‐L1, the current standard of care. This was particularly true in the subgroup of NSCLC patients with low or absent tPD‐L1 expression. Summary/Conclusion: Our data have identified PD‐L1+ lEVs as a novel predictive and prognostic marker for immunotherapy in NSCLC. Funding: This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation ‐ project 424252458), the Else Kröner‐Fresenius‐Stiftung (project 2019_A162), the innovative medical research (IMF) of the Medical Faculty, University of Münster (project ME 12 19 14), and Novartis (InCa Förderpreis 2021). keywords: NSCLC, microvesicles, biomarkers, flow cytometry, immunotherapy LB2.4. Improving melanoma prognosis and immunotherapy response prediction via a single Extracellular Vesicle digital enzyme‐linked immunosorbent assay Hanfei Shen ^1; Yasemin Atiyas^2; Andrew A. Lin^1; Zijian Yang^3; Jingbo Yang^1; Diao Liu^2; Wei Guo^1; David Issadore^1 ^1University of Pennsylvania, Philadelphia, USA; ^2University of Pennsylvania, USA; ^3Stanford University, Philadelphia, USA Introduction: Previous literature has shown plasma EV programmed death‐ligand 1 (PD‐L1) level to be positively correlated with tumor burden, and can serve as a potential predictor for melanoma patient response to anti‐PD‐1 immunotherapy. However, it remains challenging to accurately quantify plasma EV PD‐L1 level using conventional assays such as sandwich ELISA given the high concentration of background EVs in human plasma (10^10 EV/ml), which limits the clinical applicability. Methods: We designed a droplet‐based, high‐throughput optofluidic platform to detect and quantify EV subpopulations expressing specific surface proteins with single‐EV resolution. This device was applied to measure plasma EV PD‐L1 level with enhanced sensitivity and specificity. We determined the limit of detection (LOD) and limit of quantification (LOQ) of our digital assay using PD‐L1+ EV from culture media incubated with melanoma cell lines (WM9 and mel‐B7H1) known to express surface PD‐L1. Results: Proof‐of‐concept measurements have shown that our assay yields very low background (0.03% average EV per bead) and improved LOD and LOQ at least 100X improved compared to plate sandwich ELISA in the same in vitro sample across multiple cell culture models of melanoma. Summary/Conclusion: Our digital ELISA assay has superior sensitivity and specificity in quantifying EV PD‐L1 compared to plate sandwich ELISA. We aim to implement our assay to the measurement of plasma EV PD‐L1 for better prognosis and immunotherapy response prediction of metastatic melanoma. keywords: Blood, plasma, EV, protein, biomarker, PD‐L1, cancer, melanoma, immunotherapy, prognosis, ELISA, digital ELISA LB2.5. Extracellular vesicles as non‐viral alternative for therapeutic RNA delivery Olga Shatnyeva ^1; Farah Aqel^2; Kristin Schneider^1; Jana Kubackova^3; Kathrin Schwarz^4; Denise Hartung^2; Georg A Feichtinger^3; Carleen Kluger^5; Elena Kunold^6; Simone Strauch^1; Gregory Antonios^1; Katrin Ridders^1; Anthony Troegeler^7; Matthias Austen^1; Andreas Scheel^1; Cord Dohrmann^8 ^1Cell Therapy, Evotec International GmbH, Göttingen, Germany, Göttingen, Germany; ^2Evotec International GmbH, Göttingen, Germany; ^3Vectorology, In vitro Gene Therapy, Evotec GT GmbH, Orth an der Donau, Austria, Austria; ^4Cell Therapy, Evotec International GmbH, Göttingen, Germany, Germany; ^5Clinical proteomics, Evotec International GmbH, Munich, Germany, Munich, Germany; ^6Clinical proteomics, Evotec International GmbH, Munich, Germany, Germany; ^7In vitro Biology, Evotec (France) SAS, Toulouse, France, France; ^8Evotec International GmbH, Göttingen, Germany, Göttingen, Germany Introduction: Recently extracellular vesicles (EVs) have emerged as an attractive vehicle for drug delivery, including RNA. RNA‐based therapeutics have exciting potential to address targets currently undruggable by other modalities, but there are significant hurdles around deliverability, creating a need for appropriate delivery systems. Current approaches for loading of EVs with RNA suffer from poor efficiency. Our study aims to combine optimization of efficient EV production, purification, quality control and post‐isolation RNA loading to EVs by hybridization with lipid nanoparticles (LNP) for efficient functional delivery to target cells. Methods: Expi293 cells were used for EV production. Downstream purification was achieved by tangential flow filtration (TFF) in combination with differential ultracentrifugation. This was followed by extensive characterization by nanoflow cytometry (NanoFCM), transmission electron microscopy TEM, Western blot and mass spectrometry (MS). EGFP and Cre mRNA were loaded into purified Expi293 EV by co‐incubation of these EVs with LNPs carrying mRNA. A Ribogreen assay served as measure to determine efficiency of mRNA loading into EVs. Functional delivery of RNA cargo was analyzed by FACS and fluorescent microscopy in HEK293 reporter cells. Results: During the development of our EV characterization procedures, we found that a combination of NanoFCM, TEM, and MS in combination with cell‐based assays provides reproducible information about quality and functionality of EV batches. Efficient loading of synthetic mRNA cargo was achieved by EV incubation with LNP. Functional delivery of the EV‐encapsulated mRNA was associated with improved cell viability comparable to delivery via donor LNPs. Summary/Conclusion: Overall, in this study we evaluated a novel efficient post‐purification technique for mRNA cargo loading and in‐depth characterization of EV production batches. We envision that this approach will be useful for RNA drug delivery in a variety of therapeutic applications. keywords: functional RNA delivery, mRNA loading, mRNA encapsulation, EV therapeutics LB2.6. A clinically relevant biomanufacturing workflow to produce Natural Killer cells and Natural Killer cell‐derived extracellular vesicles for cancer immunotherapy Frederic St‐Denis‐Bissonnette ^1; Sarah Cummings^2; Shirley Qiu^2; Andrew Stalker^1; Gauri Muradia^2; Jelica Mehic^3; Shelby Kaczmarek^4; Dylan Burger^5; Seung‐Hwan Lee^4; Lisheng Wang^4; Jessie Lavoie^1 ^1Centre for Oncology, Radiopharmaceuticals and Research, Biologic and Radiopharmaceutical Drugs Directorate, Health Products and Food Branch, Health Canada, Ottawa, Canada; ^2Centre for Oncology, Radiopharmaceuticals and Research, Biologic and Radiopharmaceutical Drugs Directorate, Health Products and Food Branch, Health Canada, Canada; ^3Centre for Oncology, Radiopharmaceuticals and Research, Biologic and Radiopharmaceutical Drugs Directorate, Health Products and Food Branch, Health Canada, Ottawa, USA; ^4University of Ottawa, Canada; ^5University of Ottawa, Ottawa, Canada Introduction: Natural Killer (NK) cell‐derived extracellular vesicles (EVs) are emerging as biotherapeutics to treat cancer due to their cytotoxicity against cancer cells and immune‐modulatory activity towards immune cells. However, a scalable biomanufacturing workflow to produce clinical‐grade NK‐EVs for (pre‐)clinical applications is still needed. Methods: We established a novel, GMP‐compliant biomanufacturing workflow using a closed‐loop bioreactor to continuously produce NK‐EVs from the clinically relevant NK92‐MI cell line under serum‐free, xeno‐free, and feeder‐free conditions. NK‐EVs were isolated by size‐exclusion chromatography after differential centrifugation, coupled with ultrafiltration and filter‐based sterilization to concentrate and sterilize the NK‐EVs product. Characterization was performed to satisfy the MISEV2018 criteria, and data are accessible in the EV‐TRACK knowledgebase (ID: [30]EV230058). Results: To demonstrate the capacity and feasibility of this new biomanufacturing process, we generated three consecutive production lots using the same bioreactor cartridge, each lasting 5–7 days. As a result, we generated, each production lot, over 10^9 NK92 cells and large quantities of NK92‐EVs (>10^13). NK92 cells consistently displayed cytotoxic proteins (granzyme B and perforin) while demonstrating cytotoxicity against the human leukemic cell line K562. Our approach significantly improved the NK92 cell viability and cytotoxicity. Likewise, NK92‐EVs consistently demonstrated high‐purity (western blots and low endotoxin levels) and retention of the NK92 cell's characteristics such as expression of surface markers (CD2/CD45/CD56 and CD63/CD81), cytotoxic payload (granzyme B and perforin) and cytotoxicity to cancer cells. Summary/Conclusion: The scalable biomanufacturing workflow of NK cell and NK92‐EV contributes to the clinical translatability of adoptive cell and EV immunotherapies for cancer by providing a fast, affordable, and accessible strategy. Funding: This work was supported by operating grants from Genomics Research and Development Initiative (GRDI) Phase VII (2019–2024) obtained from the Government of Canada by JRL and LW; operating grants from Natural Sciences and Engineering Research Council RGPIN‐2019‐05220, Cancer Research Society/University of Ottawa 24064, and the Canadian Institute of Health Research—Operating Grant 175177 obtained by LW. keywords: Adoptive cell transfer (ACT); Clinical‐grade; Closed‐loop bioreactor; Extracellular Vesicles (EVs); Cancer Immunotherapy, Large‐scale production; Natural Killer (NK) cells LB3: Late Breaking Oral Symposia: Techniques and EV Heterogeneity Chairs: Andries Zijlstra, Daniel Chiu Location: Ballroom 6A 10:15 ‐ 11:45 LB3.1. Quantifying cancer‐associated Exosomes, Supermere and Lipoprotein markers with novel janus nanoparticle (JP) diffusometry assay and charge‐based electrokinetic biosensors Sonu Kumar; John Alex Sinclair; Nalin Maniya; Satyajyoti Senapati; Hsueh‐Chia Chang University of Notre Dame, USA Introduction: Several cancer markers, including EGFR, GPC1, Enolases, PON1, and TGFBI, have been found on the surface of exosomes, supermeres, and lipoproteins. These markers are often colocalized and reflect their parent cell's proteomic signature, providing necessary information for early non‐invasive cancer diagnosis. Classical assays such as ELISA, Western Blot, and proteomic mass spectrometry suffer from low sensitivity and cannot study the colocalization of proteins on these particles in any reasonable timeframe, which often requires vesicle isolation. Flow cytometry for these vesicles also gives inaccurate measurements due to the small size of these particles. Being able to quantify these particles without any isolation will greatly accelerate cancer research. Methods: Here, we propose two novel methods to measure the concentration of exosomes, supermeres, and HDL without any sample isolation. The first method uses a charge‐based sensor that utilizes voltage‐gating phenomena for ion exchange membranes to measure the concentration of these particles. Results: This method achieves a limit of detection of ∼1fM for exosomes and supermeres and ∼100 fM for lipoproteins. The second method uses the change in rotational blinking of Janus particles when an exosome binds to it, reaching a limit of detection of ∼1‐10 fM. Both methods have a 3–4 log10 dynamic range and can measure concentrations directly from human plasma in 30–60 minutes. The platform has high enough sensitivity to measure tumor‐specific surface markers from the entire EV/Supermere/Lipoprotein population in plasma, representing less than 0.01% of the ensemble. Preliminary results and ongoing preclinical studies with human patients include aEGFR and EGFR‐positive exosomes from Glioblastoma patients, PON1‐positive high‐density lipoprotein for coronary artery disease patients, GPC1‐positive exosomes for pancreatic cancer, CEA‐positive supermeres for colorectal cancer, and TGFBI‐positive supermeres for pan‐cancer detection. Summary/Conclusion: Current AUC for PON1‐HDL ∼ 0.99 for coronary artery disease and aEGFR‐exosomes ∼ 1.0 for glioblastoma. The diagnostic potential of supermeres and exosomes for cancer is compared for the same surface protein (GPC1 and CEA on exosomes vs supermeres) and more patients currently being tested. Funding: 1UH3CA241684‐01 LB3.2. Multi‐parametric flow cytometry identifies order and disorder of the lipid membrane (“lipid order”) as a key feature that distinguishes extracellular vesicle populations Ariana von Lersner^1; Patricia Midori M. M. Murobushi Ozawa^2; Sierra Lima^3; Tatyana Vagner^4; Bong Hwan Sung^1; Mohamed Wehbe^3; John T. Wilson^5; Jonathan Irish^3; Alissa M. Weaver^1; Dolores Di Vizio^6; Andries Zijlstra ^7 ^1Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA, Nashville, USA; ^2Vanderbilt University, Nashville, USA; ^3Vanderbilt University, USA; ^4Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, Los Angeles, USA; ^5Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA, USA; ^6Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles CA, USA, Los Angeles, USA; ^7Genentech, South San Francisco, USA Introduction: Extracellular vesicles (EVs) are generally thought to be heterogeneous due to the variation in their composition. Nevertheless, the classification of EV populations has been primarily defined by their size, their route of biogenesis or their method of their isolation. While EVs vary in many details, the enclosing lipid bilayer is their defining feature. We leveraged this feature to resolve EV heterogeneity. Methods: Using single‐EV flow cytometry, we demonstrate that lipid order, defined by lipid composition of the membrane, is a foundational feature of EV populations. Specifically: EVs prepared by DGUC and liposomes were stained with Di‐8‐ANNEPPS (di‐8, 0.25‐2μM) and subsequently evaluated by single‐EV flow cytometry using the Luminex Cellstream. Distinct EV populations were defined through dimensional reduction of 25 features across 5 emission channels collected for each EV. Liposomes comprised of DOPC, DPPC and cholesterol were used to create a reference standard for lipid order. Fluorescence anisotropy associated with lipid order was calculated as a change in the stokes shift and the lipid order/disorder (Lo/Ld) designation for each EV population was assigned using the liposomes reference standard. Results: Dimensional reduction of multi‐parametric data collected by single EV flow cytometry of vesicles stained with a lipophilic dye (di‐8) was found to readily resolve individual EV populations. An in‐depth evaluation of the lipid‐derived features revealed that EV populations of the same size exhibited distinct emission maximas indicative of fluorescence anisotropy associated with lipid order. Liposome standards modeling lipid order (DPPC) and lipid disorder (DOPC) demonstrated ratiometric fluorescence of di‐8 as a measurement of lipid order. This was further confirmed when increasing lipid order through the inclusion of cholesterol resulted in a corresponding shift in di‐8 fluorescent emission. Summary/Conclusion: In addition to defining a consistent profile of EV populations, matching the ratiometric fluorescence of EV populations to the cell membrane could be used to distinguish ectosomes from exosomes. Molecular perturbation of EV biogenesis through ablation of the GTPase Rab27a and overexpression of the tetraspanin CD63 was subsequently used to demonstrate that the EV populations defined by lipid order and the cargo they contain reflect the molecular state of a cell. Funding: This work was supported in part by grants from the National Institutes of Health (R01CA218526 to AZ and DDV, R01CA234557 to DDV, R01CA249424, R01CA249684, U01CA224276 to AMW) and the National Science Foundation (MCB‐2036809 to AMW and JTW). Drs. Zijlstra, Novitskaya and Jackson are employed by Genentech. keywords: Flow Cytometry, single‐EV analysis, lipid dye, heterogeneity, machine learning LB3.3. Deep learning complements NTA data to provide more complete nanoparticle characterization Takanori Ichiki ^1; Hiroaki Fukuda^2; Yasushi Shibuta^2; Hiromi Kuramochi^3 ^1The University of Tokyo, Bunkyo‐ku, Japan; ^2The University of Tokyo, Japan; ^3The University of Tokyo, Bunkyo, Japan Introduction: Nanoparticle tracking analysis (NTA) is crucial for characterizing extracellular vesicles (EVs), providing information about their size distribution and concentration. However, the Einstein‐Stokes equation assumes particles are spherical, which can lead to inaccuracies, particularly for non‐spherical particles or those with different densities. A new approach combining NTA and deep learning analysis has been developed to estimate non‐spherical particles mixed with spherical ones, and has been successfully applied to EV samples with high diversity. Methods: Spherical gold nanoparticles (80 nm in diameter), gold nanorods (45 nm in diameter × 180 nm in length), and polystyrene beads (100 nm in diameter) were measured with an NTA system, and their scattered light was imaged (0.01 fps, 100 frames) by dark field observation using a laser (405 nm). The center of gravity of the scattered light from each nanoparticle was then tracked to obtain time‐series 2D coordinates for each particle, and a training dataset was created containing each type of data. EVs were purified from the cell culture supernatant of breast cancer cells SkBr3 by ultracentrifugation to produce a suspension. Results: The NTA measurements of EVs derived from SkBr3 were performed, and the particle size and time‐series 2D coordinate data of each particle were obtained. The test data for deep learning was generated from the trajectory data by grouping the grains by 50 nm diameter based on the grain size measured by NTA. After training the above training data, shape prediction was performed for each EV using a model that combines 1D Convolution Neural Network and Bidirectional Long Short‐Term Memory. Particularly, a high mixing ratio of non‐spherical particles was observed in the small particle size region. This may be due to the inclusion of non‐spherical exomeres that are not surrounded by lipid bilayer. Summary/Conclusion: In conclusion, we applied a machine learning‐based shape prediction method to biological samples and investigated a methodology to achieve a multi‐parameter analysis of nanoparticles in liquids. This approach is promising to complement NTA data and provide a more complete characterization of nanoparticles. Funding: This research was supported by the Japan Science and Technology Agency (JST) through the open innovation platform for industry‐academia co‐creation (COI‐NEXT) Program (Grant Number JPMJPF2202)]. keywords: Nanoparticle tracking analysis, Deep learning LB3.4. Unveiling extracellular vesicles and lipid droplets Using synchrotron‐based techniques: a novel approach for characterisation studies William Phillips ^1; Nicholas P. Reynolds^2; Eduard Willms^3; Jereme Spiers^4; Lesley Cheng^5; Andrew F. Hill^6 ^1La Trobe Institute for Molecular Science, Bundoora, Australia; ^2La Trobe Institute for Molecular Science, Australia; ^3La Trobe Institute for Molecular Science, Melbourne, Australia; ^4Australian National University, Australia; ^5La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia; ^6Institute for Health and Sport, Victoria University, Melbourne, Australia Introduction: Extracellular vesicles (EVs) are diverse nanoparticles involved in intercellular communications and disease research. Their heterogeneity poses a challenge in isolation and characterization methods, ‘pure’ populations are difficult to isolate, further complicated by other nanoparticles such as lipoproteins and lipid droplets. We have conducted synchrotron‐based small angle X‐ray scattering (SAXS), and Fourier transform infrared spectroscopy (FTIR) to assess the heterogeneity of EVs isolated by various methods, and lipid droplets as a point of comparison. Methods: EVs were isolated from murine N2a neuroblast cells using SEC, Capto Core multimodal chromatography, ion‐exchange chromatography, or density gradient ultracentrifugation. Lipid droplets were isolated from cells utilising gradient centrifugation. FTIR and SAXS was performed at the Australian synchrotron. For SAXS, data was averaged and subtracted from background. The guinier region was used to generate P(r) for morphology analysis. Theoretical models using SAsview were also generated for fitting. SAXS data was further validated through cryo‐transmission electron microscopy (cryo‐TEM) FTIR samples were dried on BaF2 FTIR slides. 50 spectra were collected per sample. Peak integration, PCA and second derivative analysis were performed using Quasar. Results: Particle size calculated from SAXS P(r) suggested an overestimation of size by NTA. Theoretical model fitting correlated SAXS measurements. However, the Guinier region analysis suggested sample aggregation. Cryo‐TEM reinforced SAXS data, demonstrating varied morphologies and vesicles with multiple internal membranes. FTIR revealed similar spectral features between isolation methods and lipid droplets. PCA of integrated second derivative FTIR spectra revealed differences between isolation methods. Notably, column‐based isolations and lipid droplets were more similar than density gradient isolations. Summary/Conclusion: These results highlight EV heterogeneity and the associated challenges of isolating high‐purity EV subpopulations. SAXS determined the size of EVs but may not be able to provide further detail due to aggregation and heterogenicity. EVs isolated by the methods presented here are insufficiently homogenous to generate optimal SAXS data. FTIR revealed isolation techniques and lipid droplets generally share the same spectral features. However, second derivatives analysis suggest different methods produce isolates of varying compositions. These results highlight a significant challenge for EV research emphasising the need for basic‐EV research and further development of characterisation and isolation techniques. Funding: National Health and Medical Research Council Australian Nuclear Science and Technology Organisation LB3.5. Exploring the Unique Properties and Potential Applications of a New Class of vExosomes and Exomeres: Insights from Omics and Image Profiling Ahmed Fadiel ^1; Kenneth D. Eichenbaum^2 ^1University of Chicago, Chicago, USA; ^2Oakland University William Beaumont School of Medicine, Royal Oak, USA Introduction: Small extracellular vesicles are released by cells to mediate signaling for intercellular communication. A subset of these vesicles, “very small exosomes” or vExosomes, is of keen interest due to their particularly small size and unique protein and RNA cargoes. Another subset of these vesicles, exomeres, are defined as nanovesicles that lack a lipid bilayer. Exomeres are composed of a dense protein core surrounded by a sparse protein corona, which confers advantages that include enhanced stability, degradation resistance, and increased cargo loading capacity. Methods: We isolated exomeres from different sources using multiple isolation methods. The isolated and purified exomeres were subject to a comprehensive omics and advanced imaging analysis using Electron Microscopy, Atomic Force Microscopy, and Immuno‐EM to gain insights into their structural and functional properties. We used advanced imaging techniques such as electron microscopy, proteomics, and other profiling methods to characterize these unique vesicles. Results: Our analysis revealed two classes of exosomes, vExosomes ranging in size from 30–60 nm with unique protein and RNA cargo that distinguishes them from larger exosomes, and exomeres which were smaller than 30 nm. These findings suggest that vExosomes and exomeres may have essential roles in metabolic functions, including up‐ and down‐regulatory signaling processes. The profiling of their proteomes offers a promising avenue for biomarker discovery. Summary/Conclusion: Exosomes and exomeres are two classes of small extracellular vesicles that play critical roles in intercellular communication. vExosomes and exomeres display unique properties and envelope unique cargoes that distinguish them from larger exosomes. Further study based on our findings of this class of vExosomes can help isolate cell signaling triggers for immune modulation, metabolic pathways, disease pathogenesis and homeostasis. These exomeres may constitute a breakthrough discovery and offers a promising avenue for identifying new biomarkers. We continue to profile their proteomes and investigate their downstream effects. Additional research may provide valuable insights into the complex interplay between cells and their control over their extracellular environment. Funding: UC‐CCC keywords: Extracellular vesicles, biomarkers, exosomes, exomeres, vExosomes, cell signaling, proteomics, and imaging informatics. LB3.6. Does a vesicle‐like structure at the tip of airinemes a type of extracellular vesicle? Guan‐Yin Stevens^1; Dae Seok Eom ^2 ^1UC Irvine, USA; ^2UC Irvine, Irvine, USA Introduction: Intercellular communication is critical for development and homeostasis in multicellular organisms. Over the years, various means of cell‐to‐cell signaling mechanisms have been discovered, including extracellular vesicles (EVs) and signaling cellular protrusions such as airinemes, cytonemes, and tunneling nanotubes. Among them, airinemes are unique in that they possess vesicle‐like membranous structures at their tips, which are tethered in filaments. In vivo time‐lapse imaging has revealed that airineme vesicles arise from the membrane of signal‐sending cells. They are believed to form through membrane bulging, leading to a bleb‐like shape. Tissue‐resident macrophages “nibble” on the blebs, drag and deposit them on the target cell surface, where they activate downstream signaling pathways. Since airineme blebs are precursors of airineme vesicles that carry signaling molecules and potentially others, it is crucial to understand the mechanisms of airineme vesicle (bleb) formation. Methods: EVs and airineme vesicles are similar in size and morphology, and both are used for long‐distance intercellular communication. Microvesicles are a type of extracellular vesicle shed by outward blebbing of the plasma membrane. Therefore, we hypothesized that the mechanism of microvesicular bleb formation is shared with airineme blebs. It is known that microvesicle shedding is regulated by ARF6 (ADP‐ribosylation factor 6). We tested whether ARF6 is required for airineme bleb formation by treating airineme bleb‐forming xanthoblasts with an ARF6 inhibitor (NAV‐2729). Results: The ARF6 inhibitor‐treated cells showed a significantly smaller number of blebs than the controls. Summary/Conclusion: The result suggests ARF6 plays a role in airineme bleb formation. We plan to further investigate the requirement of ARF6 in airineme bleb formation with a cell type‐specific manipulation, as well as the dynamics of bleb formation with live imaging. Additionally, we will study the functional consequences of ARF6 inhibition on airineme extension and signaling. Funding: NIH R35GM142791 LB3.7. High‐yield immunomagnetic isolation of EV sub‐populations for downstream applications Susana Vagueiro^1; Ricardo Figueiras^1; Elnaz Persia^2; Tomas Dias ^2 ^1Mursla Ltd., Milton, United Kingdom; ^2Mursla Ltd., Cambridge, United Kingdom Introduction: Although EV mixtures are routinely isolated by size or density, the isolation of EV sub‐populations based on specific markers is challenging and existing solutions lack versatility and have poorly validated specificity. Here we introduce NEXPLOR (Novel EXosome PopuLatiOn Revealer), a versatile immunomagnetic method for the capture and isolation of intact EV sub‐populations, with high‐yield and suitable for downstream applications. Methods: To validate NEXPLOR, we isolated CD9+, CD63+, HER2+, ASGR1+ and CD9+CD81+ EV sub‐populations from three different cell lines (MCF‐7, BT‐474, and HepG2). The eluted EV sub‐populations were characterized using NTA, Optical‐Nanoparticle EXOsome Sensing (O‐NEXOS) and Mass Spectrometry (MS) to confirm their suitability for downstream applications. We also compared the performance of NEXPLOR with a commercial kit, EXoFlowTM. Results: We observed the percentage of CD9+, CD63+, HER2+, ASGR1+, and CD9+CD81+ EVs was significantly dependent on the cell line of origin but consistent across EV isolations. We also determined a capture rate efficiency of 97 % for the method after two cycles of capture and elution with NEXPLOR. To further confirm the enrichment of EV‐specific cargo by NEXPLOR, we compared EVs processed by differential centrifugation, ultrafiltration and SEC with NEXPLOR‐derived EVs with O‐NEXOS and MS. O‐NEXOS detected a significant signal increase of EV sub‐population markers for the NEXPLOR‐derived EVs. Equally, MS identified an enrichment of proteins associated with exosome biogenesis, vesicles and secretion pathways, such as CD63, CD81, TSG101, ALIX, and Syntenin‐1, and a substantial decrease in extracellular proteins not associated with EVs, such as Albumin, Lipoproteins, Fibronectin and others. By comparing NEXPLOR with ExoFlow, NEXPLOR captured twice as many CD9+ EVs and eluted 33x more intact EVs, making it more suitable for downstream applications. Summary/Conclusion: In conclusion, we present a new methodology and workflow for the isolation of EV sub‐populations, which can be crucial for understanding their specific roles in various biological processes and downstream applications, such as in diagnostics and therapeutics. NEXPLOR offers a promising alternative to current isolation methods, with high yield and specificity for functional EVs. keywords: Immunoaffinity, Immunomagnetic, Mass Spectrometry, EV sub‐population isolation, Tissue‐specific EVs LB4: Late Breaking Oral Symposia: Cancer Chairs: Suresh Mathivanan, Andreas Moeller Location: Ballroom 6BC 10:15 ‐ 11:45 LB4.1. Varying contribution of viral particles from endogenous retrovirus in tumor‐derived extracellular vesicles isolated from several mouse tumor cell lines Federico Cocozza^1; lorena martin‐jaular^1; aurelie di cicco^2; daniel levy^2; nicolas Ansart^3; louise merle^3; george kassiotis^4; alain joliot^3; mercedes tkach^3; Clotilde Thery ^1 ^1INSERMU932, Institut Curie, PSL University, Paris, France; ^2CNRS UMR168, CNRS UMR144, Institut Curie, PSL University, Paris, France; ^3INSERMU932, Institut Curie, PSL University, France; ^4Francis Crick Institute, Imperial college, London, United Kingdom Introduction: Tumor cells secrete extracellular vesicles (EVs) and nanoparticles (ENPs) that have been associated with a plethora of immune regulatory functions. However, the EV composition and their cargo can be remarkably diverse, and different EVs and ENPs may have varying abilities to interact and modify recipient cells. We thus recently set out to characterize the heterogeneity of the secreted EVs of EO771 cells, a murine mammary adenocarcinoma. Unexpectedly, we found viral‐like particles (VLPs) of infectious endogenous murine leukemia virus (MLV) in the bulk isolates of EVs/ENPs. A preprint of these results can be found in BioRxiv. We now explored the relevance of this observation to a range of mouse tumor cell lines. Methods: We cultured several murine tumor cell lines in complete culture medium, and prepared total cell lysates, or switched them to serum‐free medium for 24h before collecting the conditioned medium to isolate crude EV pellets by successive centrifugations. We also set up a density gradient‐based protocol to separate small EVs from VLPs. We analysed these samples by Western blot with antibodies recognizing the envelop or the gag proteins of MLV, and by single EV‐analyses methods. Results: We analysed 13 commonly used murine tumor cell lines, from either C57Bl/6 or Balb/c origin, from different tissues (mammary, lung, bladder, colon, skin) or cellular origins (epithelial cells, macrophages, T cells, dendritic cells), as well as primary cells or non‐tumoral fibroblasts. We found different patterns of expression of env and gag in the cells, and different patterns of VLP presence in their EVs. We will describe these results and our attempts to discriminate VLPs and EVs with single vesicle approaches. Summary/Conclusion: ‐EVs isolated by regular protocols from several mouse tumor cells contain VLPs coming from endogenous MLV. ‐ Combination of filter concentration, centrifugation and density gradient allows to separate small EVs and VLPs. ‐ These results call for re‐evaluation of previous functional studies of mouse tumor‐derived EVs to decipher the respective contribution of non‐viral EVs and VLPs to the observed functions. Funding: “TRAIN‐EV” a MSCA ITN, grant agreement No 722148. Fondation Chercher‐Trouver. French funding sources: ARC, FRM, INCa, ANR. LB4.2. Analysis of extracellular nucleic acids in plasma of glioblastoma patients by flow cytometry Elisabeth Rackles ^1; Elena Zaccheroni^2; Giuliana Pelicci^2; Juan Manuel M. Falcon^1 ^1CIC bioGUNE‐BRTA, Bizkaia Technology Park, Derio, 48160 Bizkaia, Spain., Derio, Spain; ^2European Institute of Oncology (IEO), Milan, Italy, Milan, Italy Introduction: Circulating cell free nucleic acids are considered as a promising source for biomarkers for cancer. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Application of biomarkers in the clinics requires their easy and cost‐effective measurement. We aimed to establish a method to analyze EV‐associated nucleic acids by conventional flow cytometry. Methods: EVs were isolated by size exclusion chromatography from U‐87 MG glioblastoma cell conditioned media and human plasma samples from healthy donors and glioblastoma patients. The fractions were analyzed by electron microscopy, NTA, total protein content, and western blotting. Nucleic acids were stained by Pyronin Y and analyzed by flow cytometry. Results: Fluorescent staining of EVs isolated from cell conditioned media was suitable for DNA and RNA detection by flow cytometry. The nucleic acids were partially protected from degradation by the EV's membrane. Additionally, we applied our method to the nucleic acid content analysis of plasma. We were able to stain DNA and RNA in plasma samples and plasma derived EVs. Staining of plasma from patients and healthy individuals revealed a difference in their nucleic acid profiles. Summary/Conclusion: Taken together our method is a promising tool for nucleic acid analysis of plasma samples by flow cytometry. This technique has potential to be used for the diagnostic analysis of extracellular nucleic acids in cancer. Funding: ERAPerMed EU project (AECC‐ PERME20733FALC; ISCIII‐AC20/00024), Deutsche Forschungsgemeinschaft (DFG; 471840646), Alzheimer's Association Grant (AARG‐NTF‐22‐968911). LB4.3. Roles of extracellular vesicles in the aging microenvironment and epithelial ovarian cancer progression: cell adhesion and proteome profiling Reihaneh Safavisohi ^1; Rebecca Whelan^2; Sharon Stack^3 ^1University of Notre Dame, USA; ^2University of Kansas, USA; ^3Unversity of Notre Dame, USA Introduction: Epithelial Ovarian Cancer (EOC) is the most fatal gynecological cancer. Because the early stage symptoms of EOC are mild or can be similar to those from other benign sources, EOC is usually diagnosed as advanced disease (stage III or IV). The majority of cases diagnosed with the disease are over 60 years of age, and 90% are over 40. Extracellular vesicles (EVs) have emerged as critical regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. In our lab, we aimed to identify aging‐related changes in the peritoneal proteome and assess the impact of EVs transfer on tumor‐cell and mesothelial‐cell behavior in Young (Y) and Aged (A) mice. Methods: To determine whether there is differential EVs‐mediated communication with aging, we isolated EVs from peritoneal lavage obtained from aged (22 months) and young (24 weeks) healthy (tumor naïve) mice using a microfluidic device. Isolated EVs were characterized based on their size distribution (Nanoparticle Tracker Analysis (NTA)) and the presence of common EV markers (TSG101, CD9, CD63) (Western Blot). We then treated ovarian cancer (OVCAR5) cells with these EVs and tested the ability of these cells to adhere to a layer of LP9 (peritoneal mesothelial) cells and to invade in a Boyden chamber containing a 3‐dimensional collagen gel, to model early adhesive and invasive events in EOC metastasis. Results: We found that cells treated with EVs isolated from aged mice were more adhesive and more invasive than cells treated with EVs isolated from young mice. To identify EV‐associated proteins that may differentially impact metastatic success in the aged host, we performed a proteomic analysis. EV samples were lysed and their proteome content was analyzed by high‐resolution mass spectrometry. To develop tools for EV proteome analysis in biological samples, we optimized the critical parameters in bottom‐up proteomics workflows. Our results for Label‐Free Quantification Analysis and Gene Ontology analysis for A versus Y samples indicate vesicular proteins that are overexpressed in aged samples that may play a significant role in cell adhesion. Summary/Conclusion: Furthermore, our data highlight a list of proteins as potential candidates for EV proteins in the aged host, which provide valuable insights into extracellular vesicular pathways and contribute to the identification of host factors that may impact EOC progression. LB4.4. YBX1 containing small extracellular vesicles derived from acute myeloid leukemia influences mesenchymal stromal cells differentiation in the bone marrow microenvironment Venkatesh Kumar Chetty ^1; Jamal Ghanam^2; Dirk Reinhardt^1; Basant Kumar K. Thakur^1 ^1Department of Pediatrics III, University Hospital Essen, Essen, Germany, Essen, Germany; ^2Department of Pediatrics III, University Hospital Essen, Essen, Germany, Essen, USA Introduction: Bone marrow microenvironment (BMM) containing various hematopoietic and non‐hematopoietic cells is known to play a key role in acute myeloid leukemia (AML) progression and contributes to treatment failure or success. Only recently, it is shown that small extracellular vesicles (sEVs) released by AML cells induce molecular changes in the BMM and transform the BMM into a leukemia‐permissive microenvironment. However, it remains unclear which active biological cargo from AML‐derived sEVs acts as a functional mediator in BMM. Methods: sEVs were isolated from AML cell line (MV4‐11) cell‐conditioned media using a combination of tangential flow filtration (TFF), size exclusion chromatography (SEC), and ultrafiltration (UF), collectively named as TSU. sEVs were also isolated from plasma of AML patients and healthy controls using SEC and UF. sEVs were characterized according to MISEV2018 guidelines using NTA, micro‐BCA, TEM, western blot, and beads‐based flow cytometry. Results: We found that MV4‐11 sEVs downregulated the key proteins (CxCl2, Scf, Col1A1, and Angpt1) in healthy bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) that are important for normal hematopoietic function in BMM. Additionally, CFU and MSC differentiation assays confirmed that MV4‐11 sEVs lead to increased proliferation and decreased differentiation in healthy BM‐MSCs. Next, LC‐MS proteomics analysis revealed that many proteins including YBX1 are upregulated in both untreated MV4‐11 sEVs and healthy BM‐MSCs treated with MV4‐11 sEVs. Supporting this fact, we found that YBX1 is significantly upregulated in AML patients‐derived sEVs compared to healthy controls. Interestingly, incubation of healthy BM‐MSCs with sEVs isolated from MV4‐11 cells with the downregulation of YBX1 using pharmacological YBX1 inhibitor or siRNA‐YBX1 significantly rescued the observed effect of proliferation and differentiation. Summary/Conclusion: Altogether, our research revealed that YBX1 is a novel protein in AML sEVs, which disrupts normal hematopoiesis in BMM by influencing the differentiation of BM‐MSCs. Targeting YBX1‐containing sEVs in AML patients could serve as a potential therapeutic approach to improve their survival outcomes. LB4.5. CD147 defines miRNA‐enriched extracellular vesicles that derive from cancer cells Song Yi Ko^1; WonJae Lee^1; Melanie Weigert^2; Eric Jonasch^1; Ernst Lengyel^3; Honami Naora ^1 ^1University of Texas MD Anderson Cancer Center, Houston, USA; ^2University of Chicago, USA; ^3University of Chicago, Chicago, USA Introduction: Extracellular vesicles (EVs) are ideal for liquid biopsy because EVs can be detected in body fluids of cancer patients and protect their cargo from degradation. MiRNAs contained in EVs have attracted substantial attention as cancer biomarkers, but the miRNA content of EVs has been contentious. Furthermore, almost all types of cells release EVs and there are currently no well‐defined methods that can identify cancer cell‐derived EVs in body fluids. Methods: EVs were isolated by ultrafiltration, fractionation based on buoyant density and immunocapture, and analyzed by flow cytometry, electron microscopy and nanoparticle tracking analysis. MiRNAs were analyzed by RT‐qPCR and a microfluidics‐based platform. Animal and human subject studies were approved by local ethics committees and with informed consent. Results: We identified that the glycoprotein CD147 is predominantly expressed in EVs that lack tetraspanins. As compared to healthy individuals and patients with benign diseases, ovarian and renal cancer patients had increased prevalence of circulating CD147+ EVs. Studies of mouse xenograft models revealed that CD147+ EVs predominantly derive from cancer cells. We found that CD147+ EVs are selectively enriched in miRNA through the interaction of CD147 with heterogeneous nuclear ribonucleoprotein A2/B1. Circulating miRNAs isolated by CD147 immunocapture more closely reflected the tumor miRNA signature than circulating miRNAs isolated by conventional methods, and isolating circulating miRNAs by CD147 immunocapture increased the diagnostic performance of cancer‐associated miRNAs. Summary/Conclusion: CD147 defines a subpopulation of EVs that is miRNA‐enriched and derives from cancer cells. Because CD147 is overexpressed in >20 types of cancers, CD147 immunocapture could be potentially used to isolate cancer‐derived circulating miRNAs for liquid biopsy in multiple disease sites and various clinical settings. Funding: NIH grants CA270508, [31]CA207034, [32]CA217931 (Naora) LB4.6. Tumor‐derived extracellular vesicles and particles induce liver metabolic dysregulation Gang Wang ^1; Jianlong Li^2; Linda Bojmar^2; Haiyan Chen^2; Zhong Li^3; Gabriel C. Tobias^2; Mengying Hu^2; Edwin A. Homan^4; Serena Lucotti^5; James C. Lo^4; Valentina Posada^6; Christin E. Burd^6; Jonathan Hernandez^7; Constantinos P. Zambirinis^8; Jacqueline Bromberg^9; David Kelsen^10; Vinagolu K. Rajasekhar^11; John H. Healey^11; Irina Matei^1; William Jarnagin^10; Robert E. Schwartz^12; Haiying Zhang^13; David C. Lyden^5 ^1Weill Cornell Medicine, New York, USA; ^2Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA, USA; ^3Duke Proteomics and Metabolomics Shared Resource, Duke University School of Medicine, Durham, NC, USA, USA; ^4Cardiovascular Research Institute and Department of Medicine, Weill Cornell Medicine, New York, NY, USA, USA; ^5Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA, New York, USA; ^6Departments of Molecular Genetics, Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA, USA; ^7Thoracic and Gastrointestinal Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA, USA; ^8Division of Surgical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA, USA; ^9Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA, USA; ^10Gastrointestinal Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA, USA; ^11Orthopedic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA, USA; ^12Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA, USA; ^13Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, USA Introduction: Cancer is a systemic disease, whereby primary tumors can disrupt homeostasis not only by metastasizing, but also by releasing soluble factors, including extracellular vesicles and particles (EVPs) into the circulation. Tumor EVPs establish pre‐metastatic niches at future sites of metastasis and systemically reprogram cell function in multiple organs that are otherwise metastasis‐free. The function of one such organ, the liver, may be impacted by tumor‐secreted factors. Although the mechanisms underlying cancer metastasis to the liver have been studied to some extent, the functional reprogramming of the liver during cancer progression without liver metastasis has been overlooked. Methods: Liver tissues from tumor‐bearing mice, cancer patients, tumor EVPs educated mice were analyzed using RNA‐seq, metabolomic and lipidomic mass spectrometry techniques. Precision‐cut liver slices from tumor‐bearing mice were subjected to drug‐metabolizing assay. Tumor EVPs educated mice were treated with chemotherapy, and the resulting chemotoxicity was evaluated. Results: We found that inflammation, fatty liver, and dysregulated metabolism are hallmarks of systemically affected livers in murine models and patients with extrahepatic metastasis. We identified tumor‐derived EVPs as crucial mediators of cancer‐induced hepatic reprogramming, as these alterations were diminished when primary tumor EVP secretion was reduced by Rab27 loss. Importantly, all EVP subpopulations, small and large exosomes, and principally exomeres, could dysregulate hepatic function. Tumor EVP‐packaged fatty acid cargo, particularly palmitic acid, induced tumor necrosis factor alpha (TNFα) secretion by Kupffer cells, generating a pro‐inflammatory microenvironment and promoting fatty liver formation. Tumor implantation or pre‐treatment with tumor EVPs attenuated drug metabolism and enhanced chemotoxicities in murine models. Decreased cytochrome P450 expression were also observed, at diagnosis, in tumor‐free livers of pancreatic cancer patients, highlighting the clinical relevance of our findings. Summary/Conclusion: Our results revealed how tumor‐derived EVPs dysregulate hepatic function and their targetable potential, along with TNFα inhibition, in preventing fatty liver formation and enhancing chemotherapy efficacy. Funding: National Cancer Institute ([33]CA232093, [34]CA163117 and [35]CA207983 to D.L., and [36]CA218513), the Thompson Family Foundation, the Tortolani Foundation, the Pediatric Oncology Experimental Therapeutics Investigator's Consortium, the Malcolm Hewitt Weiner Foundation, the Manning Foundation, the Sohn Foundation, the AHEPA Vth District Cancer Research Foundation, the Children's Cancer and Blood Foundation, the Hartwell Foundation. keywords: Extracellular vesicles and particles, fatty liver, lipid metabolism, drug metabolism LB5: Late Breaking Oral Symposia: Function Chairs: Muller Fabbri, Lata Adnani Location: Ballroom 6A 11:50 ‐ 13:20 LB5.1. Extracellular vesicle‐mediated interorgan communication from heart David W. Greening La Trobe University, University of Melbourne, Baker Heart & Diabetes Institute, Melbourne, Australia Introduction: Cell‐cell communication roles for EVs have been mostly studied in ex vivo and in vitro cell models. The heart is a dynamic signaling organ known to influence systemic organ cross‐talk. While extracellular vesicles (EVs) have emerged as mediators of intercellular signaling, including their description from the heart itself, understanding how they impact systemic signaling – specifically inter‐organ signaling remains unknown. Using a transgenic mouse model to investigate estrogen receptor alpha deficiency in cardiomyocytes, we describe specific composition and function of endogenous EVs from heart as systemic regulators to skeletal muscle and adipose. Methods: Transcriptomic, proteomic, lipidomic and metabolomic analyses in heart following cardiomyocyte‐specific knockdown of estrogen receptor alpha (ERa‐KO), in addition to skeletal muscle and white adipose tissue. Heart‐derived extracellular vesicles (EVs) were obtained from perfusate from optimized Langendorff‐isolated heart approach with MISEV guidelines adhered (yield, size, particle enumeration, morphology, molecular marker composition, surface composition, low plasma protein/cell damage marker presence). Subsequent multi‐omic analyses using state‐of‐the‐art mass spectrometry, in addition to morphological, molecular, lipidomic and bioinformatic analyses performed. Results: We show that endogenous heart EV proteome composition is dynamically regulated ‐ both cargo and surface expression to impart tropism of EVs in response to changes in cardiac phenotype (following ERa‐KO). Further, we show metabolic shift in cardiac landscape directly impacts signaling capacity of EVs to target and metabolically reprogram skeletal myocytes and functionally regulate glycolytic capacity and reserve in a sex‐specific manner. Summary/Conclusion: Our findings show that fat deposition is mediated by circulating EVs from the heart, leading to systemic adiposity, metabolic and lipidomic dysregulation in skeletal muscle. This integrated multi‐omics study uncovers a cardiac‐initiated and sex‐specific metabolic phenotype regulated by ERα, and provides new insights into cardiovascular biology and EV‐mediated interorgan communication. Our findings will facilitate knowledge translatability of EVs as signaling regulators between organs and streamline development of proteomics as a platform to decipher EV‐mediated composition and signaling. keywords: extracellular vesicles; heart; organ; proteomics; signaling, multi‐omics LB5.2. Hematopoietic stem and progenitor cell regulation by blood plasma extracellular vesicles at different stages of adulthood Isabelle J. Grenier‐Pleau ^1; Mykhaylo Slobodyanyuk^2; Pierre Thibault^3; Eric Bonneil^3; John Rudan^4; Kathrin Tyryshkin^5; Christine Hall^6; Juri Reimand^7; Sheela Abraham^5 ^1Queen's University, Kingston, ON, Canada, Kingston, Canada; ^2Ontario Institute for Cancer Research, Computational Biology Program, Toronto, Canada, Canada; ^3Institute for Research in Immunology and Cancer (IRIC), Montreal, QC, Canada, Montreal, USA; ^4Queen's University Department of Surgery, Kingston, ON, Canada, USA; ^5Queen's University, Kingston, ON, Canada, USA; ^6Queen's University, Canada; ^7Ontario Institute for Cancer Research, Canada Introduction: Hematopoietic stem and progenitor cells (HSPCs) sustain the production of blood and immune cells and reside in the bone marrow niche, connected with the hematopoietic system through a network of blood vessels. We have previously shown that plasma derived extracellular vesicles (EVs) play a role in HSPC regulation during aging, however EV‐stimulated signaling pathways that function in hematopoietic stem cell regulation have yet to be defined. Methods: Enrichment of blood plasma EVs was performed using a two‐step iodixanol density cushion and size‐exclusion chromatography protocol. HSPCs were incubated for 48h with or without EVs from various age groups (PBS control, young 20–39yrs, middle‐aged 40–59yrs, older 60–85yrs). Proteomic profiling of HSPCs post EV incubation was performed by LC‐MS/MS. The differentially expressed protein P‐values from the three EV incubation conditions, independently generated by comparison to control, were provided as input to ActivePathways to perform integrative pathway enrichment analysis. ActivePathways identified enriched biological processes of Gene Ontology and molecular pathways of Reactome present in each EV incubation condition compared to control. Results: Pathway enrichment analyses identified several signaling hubs exclusively enriched from HSPCs stimulated by older EVs, including: ribosome biogenesis, ribosomal RNA processing, non‐coding RNA processing, translation, signaling by roundabout (ROBO) receptors, cellular response to stress/stimuli, and non‐sense mediated decay. Highlighted pathways support our previous results demonstrating proliferation effects of EVs enriched from middle‐aged and older individuals on HSPCs. Summary/Conclusion: In this study, we highlight blood EVs as a potential age‐dependent regulatory factor of hematopoiesis as seen by distinct pathways enriched in hematopoietic stem and progenitor cells. Funding: This work was supported by the Canadian Institutes for Health Research, Canadian Foundation for Innovation, and the Bickell Foundation. keywords: Hematopoietic stem and progenitor cell (HSPC) regulation, ActivePathways, omics, aging LB5.3. Halting type 1 diabetes progression: use of regulatory T cell‐derived extracellular vesicles Clorinda Fusco^1; Claudio Procaccini^2; Giusy De Rosa^1; Claudia Russo^3; Elisabetta Vitiello^1; Alessandra Colamatteo^4; Mario Galgani^1; Giuseppe Matarese^5; Paola de Candia ^6 ^1Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Italy, Italy; ^2Immunology Lab, Institute for the Endocrinology and Experimental Oncology, National Research Council, (IEOS‐CNR), Naples, Italy; Neuroimmunology Unit, IRCCS Foundation Santa Lucia, Rome, Italy, Naples, Italy; ^3Neuroimmunology Unit, IRCCS Foundation Santa Lucia, Rome, Italy, Italy; ^4Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Italy, Naples, Italy; ^5Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Italy; Immunology Lab, Institute for the Endocrinology and Experimental Oncology, National Research Council, (IEOS‐CNR), Naples, Italy, Naples, USA; ^6University of Naples Federico II, Naples, Italy Introduction: Type 1 diabetes (T1D) is an autoimmune disease of the childhood in which immune cells invade the pancreatic islets causing β‐cell destruction and insulin loss. Defective CD4+ regulatory T (Treg) cells have been reported in T1D individuals and while much effort is devoted to expand/activate them for slowing disease progression, these “living drugs” are prone to cell plasticity and loss of immune suppressive capacity in vivo. We here aim at analyzing whether the extracellular vesicles released by Treg cells (Treg‐EVs) may stably mediate the immune protective effects of the originating cells. Methods: Human Treg cells were isolated from peripheral blood, stimulated in vitro and released EVs were isolated by size exclusion chromatography and characterized through nanoparticle tracking analysis and ExoView (EV‐TRACK: [37]EV210300). For in vitro experiments, human heterologous CD4+ T cells were treated with purified Treg‐EVs; for the in vivo functional studies, Treg‐EVs were also intraperitoneally injected into experimental autoimmune encephalomyelitis (EAE, as an additional model of human autoimmunity) and non‐obese diabetic (NOD) mice (modeling type 1 diabetes). Results: CD4+ T cells treated with Treg‐EVs in vitro showed reduced cell activation, proliferation, and cytokine secretion, relatively to controls. Treg shuttling of EV‐miR‐142‐3p was pinpointed as a molecular determinant for the down‐regulation of mRNAs necessary to engage active proliferation in EV‐target cells, including the cystine carrier SLC7A11, crucial to protect cells from oxidative stress during cell growth. In vivo, the injection of Treg‐EVs into EAE mice slowed disease progression, by hampering lymphocytic infiltration in the central nervous system and we are now testing the capacity of Treg‐EVs to similarly curb diabetic pathology in NOD mice. Summary/Conclusion: Based on the above results, we believe Treg‐EVs have to potential to be developed as novel cell‐free therapeutics in autoimmune diabetes. Funding: Funding: Juvenile Diabetes Research Foundation (JDRF, grant 2‐SRA‐2022‐1192‐S‐B) to P.d.C. keywords: Type 1 Diabetes, Regulatory T cells LB5.4. DNA‐protein complex from AML‐derived extracellular vesicles alters bone marrow mesenchymal stem cells behavior by inducing non‐mutational dysfunction of p53 Jamal Ghanam ^1; Venkatesh Kumar Chetty^2; Xiaomin Liu^3; Dirk Reinhardt^2; Basant Kumar K. Thakur^2 ^1Department of Pediatrics III, University Hospital Essen, Essen, Germany, Essen, USA; ^2Department of Pediatrics III, University Hospital Essen, Essen, Germany, Essen, Germany; ^3Max Planck Institute for Polymer Research, Mainz, Germany, Germany Introduction: Despite the growing interest in Extracellular vesicles DNA (EV‐DNA), many secrets related to its nature, loading mechanism, localization, and post‐shedding function(s) remain unrevealed. Exploring the DNA fraction of sEVs would help the rapid expansion of the field of EV biology. Here, We have identified a chromatin‐like structure we termed EV‐chromatin as a novel component of small EVs. We found that EV‐chromatin derived from leukemic blasts modifies the proliferation behavior of bone marrow mesenchymal stem cells (BM‐MSCs). Methods: EV isolation was done based on size exclusion chromatography. The EV quality check was performed according to MISEV2018 guidelines. EV‐chromatin was Sheared by sonication and EV‐DNA was pulled down using an anti‐dsDNA antibody. Next, DNA‐associated proteins were identifies by LC‐MS analysis. EV‐chromatin was imaged using AFM and cryo‐EM. Both EV‐DNA and EV‐chromatin were formulated in artificial polymersomes to perform functional studies. Results: EV‐chromatin released from acute myeloid leukemia blasts (AML) represents a mixture of DNA and proteins, such as histones and S100 proteins (S100A4, A8, A9, and A16). EV‐chromatin isolated from leukemic cells can alter the proliferation of BM‐MSCs. Mechanically, our data suggest that EV‐chromatin downregulates the p53‐mediated transcription of p21. This was accompanied by a significant increase in MDM2 levels, a negative regulator of p53. Conversely, treatment of BM‐MSC with either the MDM‐2 inhibitor Siremadlin or siRNA against MDM‐2 rescued the p53 transcriptional activity. S100A4 and S100B, the primary proteins of EV‐chromatin, can bind to the tetramerization domain of p53 and prevent its nuclear translocation. The EV‐chromatin‐mediated degradation of p53 could culminate in the synergic activity of S100 proteins that restricts p53 nuclear translocation and MDM2‐mediated degradation. Summary/Conclusion: EVs have recently emerged as a major player that promotes leukemogenesis in the bone marrow niche. Here, we have shown that AML‐EV‐chromatin induce non‐mutational dysfunction of p53 in BM‐MSCs. We also emphasized the importance of targeting the interaction between E3 ligase MDM2 and p53 as a promising treatment strategy in cancer with wild‐type/functional p53. LB5.5. Calcified apoptotic bodies promote the immune‐osteogenic cascade of reactive heterotopic ossification through inducing the mineralization in the early stages of injury Jianfei Yan ^1; Weicheng Lu^1; Xiaoxiao Han^1; Changhe Gao^2; wenpin Qin^1; Haoqin Xv^3; Weiwei Zhu^4; kai jiao^1 ^1School of Stomatology, The Fourth Military Medical University, Xian, China (People's Republic); ^2FMMU, USA; ^3School of Stomatology, The Fourth Military Medical University, China (People's Republic); ^4School of Stomatology, The Fourth Military Medical University, USA Introduction: Heterotopic ossification (HO) is a disease comprising abnormal formation of ectopic bone in extraskeletal soft tissue, but its initiating factor is still unclear. Apoptosis has been reported to involved in the occurrence and progression of many calcification‐related diseases, such as osteoarthritis and atherosclerosis. Nevertheless, the effects of apoptosis in pathological calcification of HO remain unknown. Methods: Achilles tenotomy was performed to establish the HO model, and the stage‐specific and location‐specific pathology of HO calcification and the calcified apoptotic bodies were examined dynamically. The first single‐cell atlas for HO tendon cells at different stages were built and the occurrence mechanism of calcified apoptotic bodies were analyzed. Calcified apoptotic bodies from HO tendon were collected and in situ injected to evaluate their effect on calcification. The mechanisms of calcified apoptotic bodies‐driven calcification were also investigated by molecular dynamics simulation. Results: Microcalcification of Achilles tendon leads to increased stiffness of extracellular matrix in the early stage of injury, and were tightly related to the calcified apoptotic bodies by a location‐ and time‐dependent manner. Most fibroblasts undergo apoptosis in the early stage of Achilles tendon injury, resulting the release of calcified apoptotic bodies. In addition, the calcified apoptotic bodies in situ injected aggregated to produce calcifying nodules with high concentrations of calcium and phosphate, resembling those observed in HO calcified tendon. Mechanism wise, apoptotic bodies continuously enrich calcium and phosphorus through Annexin A2 and phosphate carrier protein and are adsorbed to the collagen I by electrostatic interaction. Increased stiffness of extracellular matrix induced by calcified apoptotic bodies also promotes the transformation of macrophages from pro‐inflammatory to anti‐inflammatory, forming a local microenvironment for osteogenesis. Apoptosis inhibition with z‐VAD‐fmk significantly decrease the microcalcification in the early stage of injury and restrain the HO at injured Achilles tendons. Summary/Conclusion: Calcified apoptotic bodies from fibroblasts induce the microcalcification of Achilles tendon in the early stage of injury. Increased stiffness of extracellular matrix caused by the microcalcification promotes the formation of a local microenvironment for osteogenesis, resulting the heterotopic ossification. Funding: NSFC 82170978 keywords: apoptotic bodies;heterotopic ossification LB5.6. Characterization of large‐oncosome encapsulated mitochondria released by prostate cancer cells Jamelle Brown ^1; Krizia Sagini^2; Diana Kitka^3; Taylon Silva^4; Tatyana Vagner^5; Wei Yang^5; Elizabeth Hutchins^6; Anja Karlstaedt^7; Kendall Van Keuren‐Jensen^8; Rioberta Gottlieb^9; Neil Bhowmick^7; Harry Rossiter^10; Dolores Di Vizio^11 ^1Cedars‐Sinai Medical Center, Los angeles, USA; ^2OUH ‐ Institute for Cancer Research, Oslo, Norway; ^3Department of Surgery, Cedars‐Sinai Biomedical Sciences and Translational Medicine, Los Angeles, CA US, Los Angeles, USA; ^4Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, USA; ^5Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, Los Angeles, USA; ^6Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, U, Phoenix, USA; ^7Cedars‐Sinai Medical Center, USA; ^8Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA; ^9Cedars‐Sinai Medical Center, Los Angeles, USA; ^10The Lundquist Institute for Biomedical Innovation at Harbor‐UCLA Medical Center, USA; ^11Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles CA, USA, Los Angeles, USA Introduction: Extracellular vesicles (EVs) are highly heterogeneous and include a subset of EVs released by cancer cells termed large oncosomes (LO) for their atypically large size (1‐10mm). LO are enriched in prostate cancer patients with high Gleason scores and metastasis and in tumor vs benign prostatic tissue. They have been recognized as key players in oncogenesis enhancing the metastatic potential of the primary tumor creating a tumor supportive phenotype. As the main function of EVs is intercellular communication, understanding the EV cargo will provide insight into their role in cancer and metastasis. Methods: Differential ultracentrifugation, density gradient centrifugation, tunable resistive pulse sensing (qNano), flow cytometry, western blot, 3D cell culture, confocal microscopy, high‐resolution respirometry, targeted metabolomics, single EV RNA‐Seq. Results: When comparing LO and small EVs, LO are the EV population containing mitochondria and mitochondrial material. Confocal microscopy shows increased MitoTracker Red staining within LO. LO are enriched in mitochondrial proteins (i.e., SOD2) and mitochondrial transcripts associated with oxidative phosphorylation (i.e., MT‐ND1, MT‐CYB, MT‐CO1, and MT‐ATP6) whereas small EVs are deficient. Additionally, mass spectrometry and next‐generation sequencing show that proteins and transcripts associated with mitochondrial dysfunction are enriched in large EVs as compared to small EVs. Preliminary assessment of EV bioenergetics suggests mitochondrial respiration in LO. Summary/Conclusion: Large, but not small EVs are enriched in mitochondria and mitochondrial material including proteins and transcripts associated with oxidative phosphorylation and mitochondrial dysfunction, thereby potentially altering the metabolic profile of the recipient cell. Funding: 5T32CA240172‐04 R01CA223819 keywords: Mitochondria, oxidative phosphorylation LB6: Late Breaking Oral Symposia: Cargo Delivery Chairs: Dave Carter, Daniel Humphreys Location: Room 606/607 11:50 ‐ 13:20 LB6.1. Study of sEV internalization by antigen‐presenting cells Valeria Barreca ^1; Lorenzo Galli^1; Deborah Polignano^1; Valentina Tirelli^1; Giancarlo Poiana^2; Massimo Sargiacomo^1; Maria Luisa L. Fiani^1 ^1Istituto Superiore di Sanità, Rome, Italy; ^2“Sapienza” University of Rome, Italy, Italy Introduction: Small extracellular vesicles (sEV), which are released by both normal and tumor cells, are emerging as key mediators of intercellular communication with an importat role in modulating immune responses. However, it remains unclear whether sEV secreted by donor cells are selectively or non‐selectively incorporated into recipient cells. Recent studies have demonstrated that sEVs of endosomal origin are enriched with high mannose glycans on their surface. The aim of this study is to analyze the mechanisms of sEV internalization by antigen‐presenting cells, specifically immature dendritic cells that express the mannose receptor (MR) on their surface. Methods: Fluorescent sEV (Bodipy sEV) were obtained by pulsing melanoma cells with BODIPY FL C16, a fluorescent palmitic acid analogue, isolated through differential ultracentrifugation, and quantified by Flow Cytometry (FC). Bodipy sEV were also characterized for typical exosomal markers. Monocyte derived immature Dendritic Cells (iDC) were used to determine the specific uptake of Bodipy sEV. Uptake and induction of phenotypic changes and proliferation in DCs and T‐cells was evaluated by FC. Results: The dose and time‐dependent kinetics of internalization of Bodipy sEV was determined at 4°C and 37°C. Bodipy sEV were specifically taken up by iDC but not by PBMC or other cell lines. Furthermore Bodipy sEV were found to induce phenotypic changes in primary DC. Summary/Conclusion: These findings provide evidence of the specific internalization via MR of sEV by iDCs, capable of inducing phenotypic changes in DC. These results provide insights into the immunomodulatory role of sEV in the innate immune system. Further research is needed to fully understand the underlying mechanisms and explore the therapeutic potential of these findings. Funding: This work was supported by the Italian Ministry of Health (grant RF‐2019‐12369719) keywords: exosome, immature dendritic cells, Mannose receptor/CD206, uptake, Flow cytometry, LB6.2. Specific integrin (LFA‐1; αLβ2) overexpressing extracellular vesicles (EV) ‐ increased cargo delivery to ICAM‐1 expressing endothelial cells Markus Bergqvist ^1; Kyong‐Su Park^2; Nasibeh Karimi^3; Cecilia Lässer^4; Jan Lötvall^2 ^1Krefting Research Centre, Institute of Medicine at Sahlgrenska Acadamy, University of Gothenburg, Gothenburg, Sweden, Göteborg, Sweden; ^2Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, USA; ^3Krefting Research Centre, Institute of Medicine at Sahlgrenska Acadamy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, USA; ^4Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, Sweden Introduction: The integrin lymphocyte function‐associated antigen 1 (LFA‐1) is present on lymphocytes and other leukocytes and its ligands include different adhesion ligands, including ICAM‐1. Furthermore, ICAM‐1 is highly overexpressed on inflamed cells, including endothelium. We aimed to preferentially target ICAM‐1 by developing Extracellular Vesicles (EV) that are overexpressing LFA‐1 (integrin αLβ2; CD11a and CD18). An anti‐inflammatory peptide was loaded into these EVs by an open‐and‐close procedure. Methods: Stable overexpression of CD11a and CD18 was induced in HEK293f via sequential transfections, and EVs were isolated. Furthermore, the EVs underwent an open‐and‐close procedure using anionic shock, which opens membranes and allows for loading of an anti‐inflammatory peptide. The expression and functionality of LFA‐1 on the EV was determined by proteomics and binding assays. An ICAM‐1 expressing endothelial cell line was used to assess binding, uptake and the anti‐inflammatory potential of the peptide loaded LFA‐1 EVs. In the anti‐inflammation assay, the cells were treated with the loaded LFA‐1 EVs, after which IL‐8 release was quantified with ELISA. Results: The EVs were highly overexpressing LFA‐1 and the proteomics and binding assays showed that the expression and functionality of LFA‐1 on the EVs were essentially unchanged after the open‐and‐close procedure. The LFA‐1 expressing EVs rapidly bind to ICAM‐1 positive cells, and this binding was blocked by a neutralising antibody. Treating the cells with the peptide loaded into the LFA‐1 expressing EVs, increased the anti‐inflammatory function with approximately 200%. Summary/Conclusion: In summary, expressing LFA‐1 on EVs increase their binding to and uptake into cells expressing ICAM‐1. The open‐and‐close procedure had little to no negative effect on the expression and functionality of the LFA‐1 expressed on the EVs, but allowed for efficient loading of the peptide into the EVs. The anti‐inflammatory peptide loaded LFA‐1 EVs successfully reduced the IL‐8 expression, thus reducing the cytokine release from the treated cells, which was more potent than the peptide alone. Utilizing specific integrin overexpression on EVs may result in improved delivery of cargo into specific cells, and may be useful in EV‐based drug development. Funding: Heart and Lung Foundation LB6.3. The use of labelled‐cholesterol demonstrates effective EV‐mediated metabolite transfer in a prostate cancer model Guillermo Bordanaba‐Florit ^1; Sebastiaan van Liempd^2; Diana Cabrera^3; Mickael Couty^4; Oihane Elena Albóniga Díez^5; Félix Royo^6; Guillaume van Niel^4; Juan Manuel M. Falcon^7 ^1Exosomes Laboratory, CIC bioGUNE‐BRTA, Bizkaia Technology Park, Derio, 48160 Bizkaia, Spain, Bilbao, Spain; ^2Metabolomics Platform, CICbioGUNE‐BRTA, Technology Park, Derio, 48160 Bizkaia, Spain, Derio, USA; ^3Metabolomics Platform, CICbioGUNE‐BRTA, Technology Park, Derio, 48160 Bizkaia, Spain, Bilbao, Spain; ^4Paris Institute for Psychiatry and Neuroscience, Inserm, Paris, France; ^5Metabolomics Platform, CICbioGUNE‐BRTA, Technology Park, Derio, 48160 Bizkaia, Spain, USA; ^6Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Madrid, Spain, Bilbao, Spain; ^7CIC bioGUNE‐BRTA, Bizkaia Technology Park, Derio, 48160 Bizkaia, Spain., Derio, Spain Introduction: Lipids and specifically cholesterol play a pivotal role in prostate cancer progression. Moreover, cholesterol can serve as signal for protumorigenic metabolic pathways and it is the main precursor of steroid hormone metabolism. Intriguingly, it starts by translocating cholesterol into mitochondria and producing pregnenolone. Internalization of cholesterol is typically done by extracellular LDL uptake; however, extracellular vesicles could also be a source of cholesterol. The effective transfer of EV‐associated metabolites to recipient cells has not been explored yet. Methods: Prostate cancer cell cultures were treated with labelled cholesterol in lipid‐depleted (LPDS) media and treated or not with U‐Drug; a cell‐permeable, amphiphilic amino‐steroid that alters intracellular membrane protein trafficking by impairing intracellular biosynthesis and transport of cholesterol. Then, EVs were isolated by Size Exclusion chromatography (SEC), characterised by Western blotting, NTA, protein and cholesterol content and further fed to non‐labelled recipient cells to trace EV‐associated internalisation of cholesterol and its further metabolisation. Internalisation of cholesterol was detected by confocal microscopy using fluorescently labelled cholesterol and its metabolisation was traced using a UPLC‐MS assay developed for steroid hormones. Results: EV‐associated cholesterol was detected in the recipient prostate cancer cells. Cholesterol was accumulated in different compartments upon treatment with or without U‐Drug, which impairs cholesterol trafficking. This hints cholesterol associated to EVs is internalised but it can also be used physiologically. Furthermore, stable isotope labelling was detected in recipient cells upon treatment with cholesterol labelled EVs. Summary/Conclusion: In summary, we demonstrated that a targeted metabolomics combined to a molecular biology approach is useful to study EV internalisation and effective transfer of cholesterol to recipient cells carried by EVs. We also present an optimised and rapid UPLC‐MS assay for detection of steroid‐related metabolites from cells and extracellular vesicles. Funding: European Union (Horizon 2020) with proEVLifeCycle programme under grant agreement no. 860303 and EMBO SEG‐9685 to G. B.‐F. keywords: UPLC‐MS; Prostate Cancer; Metabolomics; EV uptake LB6.4. Integrin beta 1 mediates extracellular vesicle uptake and RNA delivery Omnia M. M. Elsharkasy ^1; Willemijn S. de Voogt^1; Leanne Van der Werff^1; Raymond M. M. Schiffelers^1; Olivier G de Jong^2; Pieter Vader^3 ^1University Medical Centre Utrecht, Utrecht, Netherlands; ^2Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, Netherlands; ^3CDL Research, Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht, Netherlands Introduction: Extensive research has been conducted to study extracellular vesicle (EV) uptake pathways. Nonetheless, little is known about genes responsible for EV‐mediated functional RNA delivery in the recipient cells. Here, we show a novel role for integrin beta 1 (ITGB1) protein on recipient cells in EV uptake and RNA delivery. Methods: In order to detect functional RNA delivery, a fluorescent reporter system that is activated following sgRNA delivery was utilized (De Jong et al., Nat Commun. 2020). EVs were isolated from sgRNA‐expressing MDA‐MB‐231 cells by tangential flow filtration before further purification by size exclusion chromatography. Isolated EVs were then added to HEK293T reporter cells that were treated with ITGB1 siRNA or control siRNA. Flow cytometry was used to measure functional RNA delivery. For uptake experiments, EVs were labelled using MemGlow640. Additionally, intracellular EV trafficking was tracked in live cells using a Nanoimager‐S (ONI). A role for different potential ITGB1 binding partners on EVs such as laminin, collagen and fibronectin was screened using blocking antibodies or peptides. Results: Reduced expression of ITGB1 in recipient cells resulted in a significant reduction in functional RNA transfer, both in co‐culture and after the addition of isolated EVs. Furthermore, a substantial decrease in labelled EV uptake was observed when recipient cells were treated with ITGB1 siRNA or ITGB1 blocking antibodies. Using live‐cell imaging, we observed co‐localization of MDA‐MB‐231 EVs with ITGB1 and their trafficking together inside recipient cells. Initial screening for ITGB1 binding partners showed that blocking fibronectin using blocking antibodies resulted in a decrease in RNA transfer in a co‐culture setting. Summary/Conclusion: ITGB1 plays a crucial role in EV uptake leading to functional RNA delivery in recipient cells. These findings may help us to better understand the factors that regulate RNA transfer through EVs. Funding: The work of O.M.E, R.M.S. and P.V. is supported by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 825828. W.S.d.V. and P.V. are supported by the European Research Council (ERC) Starting grant OBSERVE (# 851936). O.G.d.J. is supported by a VENI Fellowship from the Netherlands Organisation for Scientific Research (NWO) VI.Veni.192.174. keywords: RNA delivery, EV uptake, drug delivery LB6.5. Bioengineered small extracellular vesicles as a vaccine platform can simultaneously deliver multiple antigens to drive multiple immune responses Hannah K. Jackson^1; Heather M. Long^2; Graham S. Taylor^2; Juan Yam‐Puc^3; Lesley Loung^4; John Trowsdale^4; Roberta Palmulli^5; Mathew Lo^6; James Thaventhiran^7; James R. Edgar ^5 ^1University of Cambridge, Cambridge, USA; ^2University of Birmingham, Birmingham, United Kingdom; ^3University of Cambridge, United Kingdom; ^4Exosis Inc, Palm Beach, United Kingdom; ^5University of Cambridge, Cambridge, United Kingdom; ^6Exosis Inc, Palm Beach, USA; ^7University of Cambridge, USA Introduction: The COVID‐19 pandemic highlighted the clear risk that zoonotic viruses pose to global health and economies. The scientific community responded by developing several efficacious vaccines which were expedited by the global need for next‐generation vaccine platforms. Here we report the design and preclinical testing of small extracellular vesicles (sEVs) a multi‐subunit vaccine. We have engineered cell lines to generate small extracellular vesicles containing one or two subunits of SARS‐CoV‐2. We demonstrate that these small extracellular vesicles can be used to simultaneously deliver distinct antigens to cells. Methods: Cell lines were engineered to produce sEVs containing either the SARS‐CoV‐2 Spike receptor‐binding domain, or an antigenic fragment of SARS‐CoV‐2 Nucleocapsid, or both, and we tested their ability to evoke immune responses in vitro and in vivo. Antigen presenting cells were primed with peptides, proteins or bioengineered sEVs, and these APCs were assessed for their ability to activated specific T cell clones. Mice were also immunized with sEVs containing both sRBD and N antigenic fragments and serum analysed for the presence of specific IgGs. Results: B cells primed with bioengineered sEVs were able to activate antigen‐restricted T cell clones. Only APCs primed with sEVs corresponding to the analogous T cell clones evoked responses. Mice immunized with sEVs containing both sRBD and antigenic fragments of Nucleocapsid elicited both anti‐sRBD IgGs and anti‐N IgGs. Neutralisiation assays determined that the antibodies generated by immunzation with sEVs were protective from SARS‐CoV‐2 infection. Summary/Conclusion: We have demonstrated that sEVs can be bioengineered to deliver multiple immunogens at once, and can drive multiple immune responses. We demonstrate that sEVs are endocyosed by APCs and their antigens processed and presented by MHC‐II, irrespective of whether antigenic fragments are positioned on the inside or outside of sEVs. sEV‐based vaccines allow multiple antigens to be delivered simultaneously resulting in potent, broad immunity and provide a quick, cheap and reliable method to test vaccine candidates. Funding: Wellcome Trust and the Royal Society (216370/Z/19/Z) Exosis Inc. keywords: vaccine, antigen presentation, SARS‐CoV‐2, extracellular vesicles LB7: Late Breaking Oral Symposia: Biogenesis & Cargo Chairs: Dylan Burger, Metka Lenassi Location: Room 608/609 11:50 ‐ 13:20 LB7.1. Assessment of long RNAs in CSF and plasma from healthy donors or individuals with Parkinson's disease Kendall Van Keuren‐Jensen ^1; Elizabeth Hutchins^2; Rebecca Reiman^3; Joanna Palade^4; Bessie Meechoovet^3; Cherae Bilagody^3; Megan Hall^4; Mark Frasier^5; Bradford Casey^5 ^1Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA; ^2Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, U, Phoenix, USA; ^3TGen, Phoenix, USA; ^4TGen, USA; ^5Michael J Fox Foundation, USA Introduction: For patients with Parkinson's disease (PD), progression does not follow a straight line. A significant clinical challenge is the heterogeneity observed across PD patients. As PD pathology varies across patients, there is an unmet need to develop biomarkers that correlate with disease detection, progression, and therapeutic actionability. Extracellular RNA (exRNA) cargos hold promise as biomarkers for PD. ExRNA can be carried in extracellular vesicles, released from cells in the CNS into biofluids. ExRNA signatures can be captured, quantified, and provide some information about the CNS. We provide simultaneous assessment of two clinically relevant biofluids (CSF and plasma). CSF is a more central biofluid to disease, and plasma is an easier biofluid to obtain. Methods: We sequenced the total exRNAs from 374 samples from the Michael J Fox Foundation's BioFIND cohort; matched plasma and CSF samples from 187 individuals. We isolated RNA from 1 mL of CSF (Qiagen miRNeasy plasma/serum kit) and 500 ul of plasma (Norgen plasma/serum kit) for total cell‐free RNA and we DNase treated all samples. RNA was sequenced using the Takara SMARTSeq stranded assay. RNA was aligned using STAR (v2.6+) to GRCh38 and to additional lncRNAs from the highly curated LNCpedia/GENCODE datasets. Count data from featureCounts, as well as transcripts per million abundance using Salmon was created. Results: We describe the number of transcripts for both CSF and plasma, the number of brain‐enriched transcripts CSF and plasma, the coverage of the transcripts from the 5’ to 3’ end in each biofluid, and identify a number of differentially expressed transcripts associated with Parkinson's disease. Summary/Conclusion: While we isolated total exRNA from each biofluid, we focused on the long RNA contents which may be representative of EV‐protected RNAs, as long RNAs would typically be degraded. We also focused on transcripts that are enriched in brain tissue. We find that more transcripts can be detected in plasma, and that plasma contains more brain‐enriched transcripts that are more fully covered than in CSF, using the starting volumes in this project. It is possible that CSF would still yield more information if larger volumes were used. Funding: Data used in the preparation of this article were obtained from the Accelerating Medicine Partnership® (AMP®) Parkinson's Disease (AMP PD) Knowledge Platform. For information on the study, visit [38]https://www.amp‐pd.org. Clinical data and biosamples were obtained from the (i) Michael J. Fox Foundation for Parkinson's Research (MJFF) and National Institutes of Neurological Disorders and Stroke (NINDS) BioFIND study. BioFIND is sponsored by MJFF with support from the NINDS. The BioFIND Investigators have not participated in reviewing the data analysis or content of the manuscript. For up‐to‐date information on the study, visit michaeljfox.org/news/biofind. LB7.2. Tug‐of‐war between senescence and Wnt signaling: implications in the biogenesis and secretion of small extracellular vesicles Pamali Fonseka ^1; Taeyoung Kang^2; Suresh Mathivanan^3 ^1La Trobe University, Doncastter East, Australia; ^2La Trobe University, USA; ^3La Trobe University, Australia, Melbourne, USA Introduction: It has been well established that a tug‐of‐war exists between the tumor suppression barrier senescence and the tumor promoting Wnt signaling pathway. However, the implications of this complex feedback loop in the context of small extracellular vesicles (sEV) biogenesis and secretion are unclear. Methods: sEVs were isolated using differential centrifugation coupled with ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy and western blotting analysis. To investigate the role of Wnt signaling and senescence in sEV secretion, CRISPR/Cas9‐based knockout, luciferase assay, quantitative proteomic analysis, co‐localization assays, immunofluorescence, and RNAi‐based knockdown and cellular survivability assays were performed using isogenic cells that are both addicted and non‐addicted to Wnt. Results: CRISPR/Cas9‐based knockout of β‐catenin attenuated Wnt signalling activity, reduced proliferation and induced senescence via AKT signalling pathway in colorectal cancer cells. Induction of senescence increased the secretion of sEVs and altered their protein cargo. Confocal microscopy, proteomic analysis and Western blotting highlighted the perturbation of several EV regulators including CD63, Rab27a and Cortactin upon induction of senescence. RNAi‐based knockdown of multiple regulators rescued the secretion of sEVs. Utility of P53 and P21 knockout and exogenous expression of P21 or P16 highlighted the role of senescence in sEV biogenesis and secretion in Wnt addicted cells. Summary/Conclusion: Overall, this study, for the first time, reveals the mechanism by which senescent cells regulate the increased secretion of sEVs in Wnt regulated manner. Funding: Pamali Fonseka is supported by the National Health and Medical Research Council (NHMRC) Australia, CASS Foundation Medicine/Science Grant and Jack Brockhoff Foundation Early Career Medical Research Grant program. This project was not funded by the above funders. keywords: senescence, Wnt signaling, sEV biogenesis, sEV secretion LB7.3. Ubiquitination of extracellular proteins is specific for tetraspanin‐enriched small extracellular vesicles Dennis K. Jeppesen ^1; Qin Zhang^2; James N. Higginbotham^2; Robert J. Coffey^3 ^1Department of Medicine, Vanderbiltu University Medical Center, Nashville, USA; ^2Department of Medicine, Vanderbilt University Medical Center, Nashville, USA; ^3Department of Medicine, Vanderbilt University Medical Center; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, USA Introduction: Extracellular vesicles (EVs) are a heterogeneous group of lipid bilayer membrane vesicles released by cells to the extracellular space. They range in size from 50‐10,000 nm but are often categorized as small EVs (sEVs) and large EVs (lEVs) based on diameters smaller or larger than 200 nm, respectively. Exosomes are a type of sEV that form as intraluminal vesicles (ILVs) in multivesicular endosomes (MVEs) and are highly enriched in specific tetraspanin proteins, including CD63 and CD81. In contrast, microvesicles are generated by direct outward budding of the plasma membrane and span the sEV and lEV size ranges. Non‐vesicular extracellular nanoparticles (NVEPs) are amembraneous particles that include the newly discovered exomeres and supermeres, and they also appear to be released by many cell types.The post‐translational modification of proteins by ubiquitin regulates many processes in eukaryotic cells. Methods: We have used ultracentrifugation, high‐resolution density gradient fractionation, transmission electron microcopy, direct immunoaffinity capture (DIC), fluorescence‐activated flow sorting (FAVS) and immunoblotting to investigate ubiquitination and neddylation of EVs and NVEPs. Results: We have investigated the presence of proteins post‐translationally modified by ubiquitin (Ub) in the extracellular space and find that they are specifically enriched in sEVs. Strikingly, the enrichment of Ub in sEVs was as dramatic as the established exosome marker tetraspanin, CD63. In contrast, proteins in microvesicles and exomeres are not associated with Ub modification. Isolation of sEVs by DIC of CD63/CD81/CD9‐enriched vesicles indicated that tetraspanin‐enriched sEVs account for most of the secreted Ub‐modified proteins. Likewise, CD63/CD81 double‐positive exosomes, sorted by FAVS, account for the majority of the extracellular ubiquitin signal. Neddylation, post‐translational modification by NEDD8, has been linked to negative regulation of exosome secretion, and, in contrast to ubiquitination, we find that neddylation is more associated with exomeres than with sEVs. Summary/Conclusion: Modification of extracellular proteins with ubiquitin is enriched, and specific, for tetraspanin‐enriched sEVs and not other types of EVs. In contrast, neddylation of extracellular proteins is a predominant feature of exomeres and not sEVs. keywords: Ubiquitin, NEDD8, microvesicles, exosomes, exomeres, supermeres, tetraspanin LB7.4. EGFR inhibition prevents calcifying CAV1‐dependent extracellular vesicle biogenesis in vitro Sophie K. Ashbrook ^1; Amirala Bakhshian Nik^2; Joshua Hutcheson^3 ^1Florida International University, Miami, USA; ^2Medical College of Wisconsin, USA; ^3Florida International University, USA Introduction: Vascular calcification significantly predicts cardiovascular events with no current therapeutic options for prevention or treatment. Osteogenically‐differentiated vascular smooth muscle cells (VSMCs) release calcifying extracellular vesicles (EVs) that nucleate nascent minerals. Calcifying EV formation in VSMCs requires caveolin‐1 (CAV1), a membrane scaffolding protein located in caveolar domains. Epidermal growth factor receptor (EGFR) interacts with and modulates the intracellular trafficking of CAV1. Given this, we hypothesized that EGFR inhibition may prevent the biogenesis of calcifying EVs, thus decreasing vascular calcification. Methods: We assessed the potential of EGFR tyrosine kinase inhibition (AG1478 and [39]PD153035, 2.5 uM, N = 3) to prevent calcification in vitro using VSMCs cultured in osteogenic media (OS). Day 14 and 28 media were ultracentrifuged at 100,000×g for 1 hour at 4°C to isolate EVs. Results: EGFR inhibition significantly reduced the release of calcifying EVs in OS cultures (p < 0.05 and p < 0.001) as measured by tissue non‐specific alkaline phosphatase activity, a key enzyme in mineral formation. Of note, the level of CD63, a common exosomal marker enriched in exosomes, was preserved across in vitro groups following 28 days of culture (p = 0.9). In addition, a decrease in RUNX2 (p < 0.01), and bone morphogenetic protein 2 (BMP2) (p < 0.001), both key proteins in the development of bone, was observed within EGFR inhibitor groups compared to OS cultures. Summary/Conclusion: Our results suggest that EGFR inhibition interferes with trafficking mechanisms that lead to calcifying EV biogenesis without altering CD63‐positive EV release. Future studies will test the efficiency of EGFR inhibition in vivo. Given that EGFR inhibitors exhibit clinical safety, the current data show that EGFR may be a propitious target in preventing vascular calcification. Funding: This work was supported by a grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (1R01HL160740). keywords: Cardiovascular disease, calcification, epidermal growth factor receptor, caveolin‐1 LB7.5. Human milk extracellular vesicle cargo is influenced by timing of birth Brett Vahkal ^1; Emanuela Ferretti^1; Jean‐François Beaulieu^2; Illimar Altosaar^1; Marceline Côté^1 ^1University of Ottawa, Ottawa, Canada; ^2University of Sherbrooke, Sherbrooke, Canada Introduction: One of the leading causes of death in premature infants is necrotizing enterocolitis (NEC). Human milk (HM) and its extracellular vesicles (EV) could prevent NEC via EV cargo of mRNA and proteins. Since mothers birth infants at different gestational ages (GA), we hypothesized that EV cargo from term HM is distinct from preterm HM. Methods: EVs were isolated using differential and ultra‐centrifugation from HM following term or preterm birth, and characterized by nanoparticle tracking analysis, surface markers, and electron microscopy (EVTrack ID [40]EV230362). EV peptides were detected by tandem mass spectrometry in SWATH/DIA acquisition mode. Differentially expressed proteins were identified with pOmics R package. EV mRNA was sequenced on Illumina Novaseq 6000, and differential expression was determined on Degust platform. Functional annotations were done using PANTHER and STRING. Results: Over 19000 unique peptides were identified. Term HM EVs contained 1257 and preterm 872 unique proteins. 13 significantly (p< 0.05) upregulated and 26 downregulated proteins were identified in term HM EVs. Cathepsin B and SERPINE2 were the most significantly upregulated. ENSA was most significantly upregulated in preterm EVs. STRING analysis revealed enrichment in immune system processes for both term and preterm HM EVs, with neutrophil degranulation and leukocyte activation in the top 3 with the largest enrichment. From mRNA sequencing, term and preterm HM EVs shared 5522 genes, with additional 1067 and 2912 unique genes respectively. In term HM EVs, unique mRNA with the highest abundance included translation and ribosomal genes (EIF3CL, RPL34) as well as cell death related genes (PDCD5 and CASP4). In unique preterm HM EV mRNA, most abundant genes were zinc‐finger‐related (ZCCHC17, ZCWPW1, ZNF710). Summary/Conclusion: These results shed light on the bioactive components of GA‐specific milk and could provide basis for research in a clinical setting to investigate direct protection by HM EVs against the development of NEC. Funding: Canadian Institutes of Health Research. keywords: human milk, preterm birth, term birth, proteomics, RNA sequencing, characterization. LB7.6. Profiling of Astronaut Plasma Extracellular Vesicles Reveals Increased Oxidative Stress, Coagulation and Inflammatory Responses Following Short‐term Spaceflight Irina Matei ^1; Nadia Houerbi^2; JangKeun Kim^2; Eliah Overbey^1; Laura I. Patras^1; Richa Batra^2; Annalise Schweickart^2; Serena Lucotti^3; Anand Narayanan^4; Lucy Williamson^5; Namita Damle^6; Qiuying Chen^2; Deena Najjar^2; Krista Ryon^2; Braden Tierney^2; Evan Afshin^2; Aaron Gajadhar^5; Susan Bailey^7; Ryan Benz^5; Qiu Zuo Yang^5; Purvi Tandel^5; jessica Chu^2; Daniel Hornburg^5; Asim Siddiqui^5; Sean Mullane^8; Jonathan Foox^2; Doru Paul^9; Cem Meydan^2; Jaime Mateus^8; Steven Gross^2; Jan Krumsiek^2; Christopher Mason^2 ^1Weill Cornell Medicine, New York, USA; ^2Weill Cornell Medicine, USA; ^3Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA, New York, USA; ^4Florida State University, USA; ^5Seer Bio, USA; ^6Oxford Nanopore Technologies, USA; ^7Colorado State University, USA; ^8SpaceX, USA; ^9Department of Medicine, Weill Cornell Medicine, New York, USA, USA Introduction: Human ambition to conquer space is technologically achievable with the advent of commercial space missions, yet our understanding of spaceflight‐induced molecular and cellular changes is limited. The first all‐civilian SpaceX Crew Dragon I4 orbital mission reached 590km elevation and lasted 3 days during which the crew experienced spaceflight hazards similar to months‐long International Space Station missions: radiation and microgravity exposure and hostile space environment confinement. The mission provided a platform to longitudinally study EVPs as biomarkers of acute spaceflight exposures in correlation with the most comprehensive multiomic and clinical data of human response to spaceflight. Methods: Blood was collected in K2 EDTA tubes at 3 pre‐launch (L‐92,‐44,‐3 days), and 4 post‐return (R+1, +45, +82, +194 days) time points. EVPs isolated by sequential ultracentrifugation were validated via transmission electron microscopy, nanoparticle tracking analysis and Western Blot. EVP plasma proteomes, metabolomes were characterized via LC‐MS/MS. Results: As in NASA Twins studies, plasma EVP numbers increased post‐flight and during recovery relative to baseline. Unbiased EVP proteomics across time points revealed 151 proteins dysregulated immediately post‐flight that returned to pre‐flight levels during recovery as well as EVP protein alterations sustained through recovery. The 8 EVP proteins exclusively dysregulated immediately after Earth return (HAGH, NUTF2, ACHE, ART4, PTPA, MPST, RUVBL1 and LXN) function in metabolism, immunity, and inflammation, particularly neuroinflammation, consistent with multiomics, clinical data and previous Twin Studies. GSEA analysis of EVP proteins dysregulated post‐flight show a positive enrichment in brain proteins. Importantly, antioxidant defense enzymes (CAT, BLVRB, GPX1) were enriched in R+1 EVPs, likely a physiological response to oxidative stress induced by spaceflight and radiation, corroborated by plasma proteome and metabolome meta‐analyses, lipid peroxidation and single cell immune transcriptomics. Summary/Conclusion: Short‐term spaceflight induced broad physiological and stress responses, some consistent with long‐term spaceflight, but almost all of which returned to baseline post‐Earth return. The acute and long‐term EVP cargo changes suggest that even short‐term space travel promotes systemic inflammation, oxidative stress, coagulopathies and neuroinflammation. Funding: Children's Cancer and Blood Foundation, the Malcolm Hewitt Weiner Foundation, the Manning Foundation, the Theodore A. Rapp Foundation, STOP Cancer, WorldQuant Foundation, NASA, National Institutes of Health. keywords: spaceflight, neuroinflammation, systemic inflammation, oxidative stress, coagulation, proteomics, multiomics LB8: Late Breaking Oral Symposia: Biomarkers: Isolation & Characterization Chairs: Navneet Dogra, Julie Saugstad Location: Ballroom 6BC 11:50 ‐ 01:20 LB8.1. Single Molecule Localization Microscopy analysis of extracellular vesicles from biofluids Kathleen M. Lennon; Andras G. Miklosi; Chloe Celingant‐Copie; Anna Caballe Oxford NanoImaging, San Diego, USA Introduction: Extracellular vesicles (EVs) are a group of heterogeneous membranous particles ranging in biogenesis, size, and biomarker content. EVs are present in various biofluids, including blood, urine, and milk, and can carry various biomolecules such as proteins, nucleic acids, and lipids. These biofluid EVs have attracted attention in recent years as potential non‐invasive biomarkers for various diseases, including cancer, neurological disorders, and infectious diseases. However, the isolation and characterization of biofluid EVs remain challenging due to the heterogeneity of biofluids and the limited sensitivity and specificity of current EV isolation and detection methods. Methods: Purified EVs from plasma, milk, or urine were immobilized on the surface of coverslips using individual or pooled antibodies against CD9, CD63 and CD81. Affinity isolated EVs were then fluorescently labeled with a pan‐EV marker, antibodies against CD9, CD63, and CD81, and RNA carrier protein Ago2. EVs were imaged with dSTROM on a Nanoimager and vesicle characterization was performed using cluster‐based analysis to extract key features, including circularity, size, density, and biomarker content. Results: We describe a workflow for the robust isolation, detection, and quantification of EVs, which allows us to better understand the complex biodistribution of EVs from blood, urine, and milk. We show that affinity enriched EV subpopulations can be distinguished based on morphological and biomarker content across biofluids, suggesting common biogenesis and functional roles. Summary/Conclusion: A super‐resolution based workflow allows the quantification of individual EVs and subpopulations in heterogeneous EV preparations from multiple biofluids. keywords: super‐resolution, nanoimaging, microscopy, single‐EV analysis, quantitative imaging LB8.2. Development of novel approaches for upscalable chromatographic methods to purify extracellular vesicles from donated blood products Heikki Saari^1; Heli Marttila^2; Reetta Pusa^3; Marjo Yliperttula^2; Saara Laitinen ^1 ^1Finnish Red Cross Blood Service, Helsinki, Finland; ^2University of Helsinki, Helsinki, USA; ^3Finnish Red Cross Blood Service, Helsinki, USA Introduction: Purification of extracellular vesicles (EVs) for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size‐based separation techniques. This is especially case with the complex fluids such as blood. Their major downfalls include isolation method induced EV aggregation, poor scalability of the technology and one‐dimensionality of the separation process. Widely used size exclusion chromatography as an example fails to separate similar size lipoprotein particles from EVs and in concentrating already dilute large volume samples. Methods: Ion exchange chromatography (IEC) is a promising alternative or supplementary method candidate, as it offers a different approach for EV‐separation, which is surface charge. There we have identified two options: anion exchange chromatography (AEC) as it successfully relies on the strongly negative surface charge of EVs and as another cation exchange chromatography (CEC) as EVs are very complex in their structure and due to individual cationic domains on the EV surface. In our study we compared both AEC and different type of CECs for the purification of the platelet derived EVs (pEV). EVs were characterized for their purity, size, and shape as well as surface markers, using nanoparticle tracking analysis, electron microscopy and western blot analysis. Results: Ion exchange chromatography is efficient in isolating EVs from very challenging mixture of contaminant particles and impurities. In contrary to Size Exclusion Chromatography (SEC) IEC also concentrates diluted samples so no additional steps re needed for concentration. We were able to show that when compared AEC to different types of CEC for the purification of platelet derived EV (pEV) samples also containing plasma‐derived impurities CEC can be found to be even superior for AEC when used in combination with conventional and tentacle‐type resin of CECs. Summary/Conclusion: When comparing the yield and purity we were surprised to see the performance of developed CEC approach. From all tested protocols we found tandem CEC to be the best in enriching pure EVs. Overall IEC offers new avenues for gentle and fast method for large scale manufacturing of EVs. Funding: The work conducted at the Finnish Red Cross Blood Service was funded by Business Finland EVE Consortium 2346/31/2019, Academy of Finland project no 315406 (MY) and Business Finland EVE ecosystem 1842/31/2019 (MY). Heli Marttila's work was funded by the Academy of Finland (grant number 331627) and Sigrid Juselius Foundation. keywords: Chromatography; ion exchange; isolation; large scale manufacturing LB8.3. Optimization and Characterization of Track‐Etch Membrane Devices for High‐Throughput Immunomagnetic Isolation of Extracellular Vesicle and Outer Membrane Vesicle Subpopulations Andrew A. Lin ^1; Hanfei Shen^1; Griffin Spychalski^2; Lynn Marie Martin^2; Zhenting Xiang^2; Erica Carpenter^1; Flavia Teles^2; Hyun Koo^2; David Issadore^1 ^1University of Pennsylvania, Philadelphia, USA; ^2University of Pennsylvania, USA Introduction: Extracellular vesicles (EVs) and their protein and nucleic acid cargoes have great potential as biomarkers from bodily fluids such as blood and saliva in multiple diseases. However, existing EV subpopulation isolation platforms are not scaled to EVs’ nanoscale size, lack the throughput for the high circulating concentrations of EVs, and often do not account for the heterogeneity of target vs. background EVs. We present the optimization, characterization, and benchmarking of an inexpensive modular device using track‐etch membranes for rapidly (< 1 hour) isolating both EVs and OMVs (outer‐membrane vesicles) across multiple diseases. Methods: EVs were functionalized with anti‐biotin magnetic nanoparticles (Miltenyi) bound to biotinylated capture antibodies chosen based on the target EV subpopulation before capture on our magnetic track‐etch membrane devices. EVs were then either lysed on‐chip for nucleic acid analysis via PCR or eluted off‐chip for analysis of size ranges and quantification of total protein (Qubit) versus background albumin (ELISA, Thermo Fisher). For the initial parameter optimization of our device, cancer cell culture media (pancreatic, liver, or lung) spiked into fetal bovine serum (FBS) was used. Results: We first titrated parameters including membrane pore size, device flow rate, device cross‐sectional area, and number of membranes in series to optimize track‐etch EV isolation using our device. The resulting scaling laws showed close concordance with finite‐element simulations. We then validated this device in three different models of cancer (pancreatic, liver, and lung) via cell culture media spiked into either FBS or healthy human plasma (ZenBio). We found strong specificity of the antibody labeling in the device versus both isotype controls and sample controls (non‐spiked human plasma). We also validated the performance and specificity of the device in isolating OMVs from P. gingivalis spiked into healthy saliva. Summary/Conclusion: We present a modular EV isolation platform which uses immunomagnetic labeling to perform the sensitive and specific isolation of EVs and OMVs from complex media across four different disease models. This device offers the potential for fast and modular EV and OMV isolation for the discovery and translation of EV and OMV biomarkers towards clinical usage. keywords: Microfluidics, nanofluidics, extracellular vesicles, liquid biopsy, cancer, infectious disease LB8.4. Exobase: Advancing Exosome Research through a Comprehensive Bioinformatics Platform Ahmed Fadiel ^1; Kenneth D. Eichenbaum^2; Kunle Odunsi^3 ^1University of Chicago, Chicago, USA; ^2Oakland University William Beaumont School of Medicine, Royal Oak, USA; ^3University if Chicago, Chicago, USA Introduction: Exosomes are minute vesicles secreted from almost every cell and they play a crucial role in intercellular communication and cell signaling. Increasing research focuses on developing exosomes as drug‐delivery vehicles because of their capacity to transport a diverse range of biomolecules. Leveraging bioinformatics, predictive models can be created to identify exosome cargo and their roles in various biological processes. Establishing a versatile bioinformatics platform can advance our understanding of exosome and secretome biology. Specifically, it can provide access to databases that contain exosome‐associated lipids, nucleic acids, and proteins, as well as analytical tools to identify exosome‐specific pathways and functions. It can also cross‐reference and analyze multi ‘omics data, such as transcriptomics, genomics, proteomics, and metabolomics. A comprehensive bioinformatics platform designed around extracellular vesicles/exosomes will facilitate the discovery of novel biomarkers and therapeutic targets forexosome‐based therapies. Methods: The Exobase Knowledge Base (EKB) is computational platform developed to address the need for a holistic and practical approach to exosome research. This platform combines different ‘omics domains and contains public data gathered from various research and online sources related to exosomes. The EKB will be a global hub for all exosome researchers to mine and store data about exosomes, cargo, and source cells. In addition, analytical tools will be included and can be used to evaluate exosomes in diseases and normal conditions. The goal is to make this knowledge base freely available to researchers worldwide. Results: We present the computational structure and design constructs of the Exobase Knowledge Base bioinformatics platform and its developing applications. With further development of this platform, we aim to advance our understanding of exosomes and advance the development of exosome‐based therapies for various diseases. Summary/Conclusion: The Exobase Knowledge Base provides a comprehensive bioinformatics platform for researchers to understand better exosome biology and its potential applications in drug delivery and disease treatment. The integration of ‘omics data and analytical tools offered by the platform allows researchers to explore exosome‐specific pathways and functions, identify exosome cargo, and develop additional predictive models. This platform can serve as a foundation for analyzing the intercellular communication between exosomes and help furnish data for other cell‐signaling and molecular trafficking pathways. Funding: NA keywords: Bioinformatics, Exosomes, Secretome, Knowledgebase, Informatics and Biomarkers. LB8.5. Optimizing EV‐based Assays for Clinical Biomarker Development Marco Faini Roche, Basel, Switzerland Introduction: Extracellular vesicles hold a great potential to report on organ or cell type‐specific biomarkers reducing the patient burden of direct tissue sampling (liquid biopsy). The Pharmaceutical industry is increasingly interested in exploiting EVs as a possible source of safety and pharmacodynamic biomarkers. Although EVs could revolutionize the access to organ‐specific biomarkers, several biological and technical hurdles hinder the development of reliable biomarker assays that are practically applicable in the context of clinical studies. In this contribution we will detail our findings targeted at the development of EV‐based clinical biomarkers. Methods: We focused our work on blood serum derived EVs and contrasted different enrichment and purification methods to derive EV preparations following MISEV guidelines. First, we verified positive control (cell derived EVs) and negative control matrix reagents with standard EV markers by western blot and LC‐MS. Then, we contrasted precipitation based reagents, size‐exclusion chromatography and centrifugation‐based methods titrating purified EVs in decreasing ratios to a blood‐like matrix. Lastly, we replicated EVs enrichment from blood serum individuals and respective pools. Results: For each method we verified the recovery rate, the associated variance and the level of contamination combining Western blots, LC‐MS and flow cytometry targeting positive and negative EV markers. We also assessed the robustness to variation in biological samples derived by different individuals. Summary/Conclusion: The comparison of different samples and methodologies enabled us to recommend bespoke workflows for different objectives such as purifying organ‐specific EVs vs total EVs, surface marker detection and cargo detection and for different detection means such as ELISA and hybridization. Funding: The work was funded by Roche Pharmaceuticals, informed consent has been collected for all human samples. keywords: biomarkers, clinical assay, blood, purification, enrichment, liquid biopsy LB8.6. Identification and Validation of EV based biomarkers for Inflammatory Bowel Disease Kanchana K. Ayyar ^1; Alan C. Moss^2 ^1Boston Medical Center, East Boston, USA; ^2Boston Medical Center, boston, USA Introduction: Inflammatory Bowel Disease is a chronic illness that severely affects the life of the patients. Current diagnosis of IBD is time consuming and puts the burden of the test on the patient (endoscopy, colonoscopy, CT and MRI). There is an urgent need for novel diagnostic tool that can detect inflammation and disease severity with minimal risk and inconvenience to the patient. This study characterizes the RNA signatures contained in EVs isolated from pre‐fecal fluid. Methods: Pre‐fecal fluid was collected from healthy individuals and IBD patients during colonoscopy. EVs were isolated by differential ultracentrifugation. Isolated EVs were characterized using nanoparticle tracking analysis on the Nanosight NS300 instrument. Tetraspanin markers were identified on isolated EVs using Exoview platform. EVs were treated with proteinase K and RNase A to remove any extra vesicular RNA content before RNA extraction. RNA was extracted from 1011 EVs and converted to cDNA which was used in qPCR. Initial screening of RNA expression was performed using a Taqman array plate containing 96 different genes. Select genes were validated using qPCR. Results: We identified 14 genes that were differentially expressed between healthy and IBD group. Most of these genes were involved in the innate and adaptive immune response. The differentially expressed genes include IL1B, CXCL8, CCL2, CACYBP, C/EBPβ, IFN‐γ and TNF. These genes were validated and their expression was studied in different disease severities. Summary/Conclusion: Intestinal fluid contains abundant EVs with RNA cargo. We have successfully identified and validated genes that are differentially expressed between healthy and IBD patients. Most of the differentially expressed genes are involved in the Th17 pathway and play a role in inflammation. Funding: This study was funded by the Helmsley Charitable Trust. keywords: IBD, Intestinal Fluid, Biomarker LB8.7. SARS‐CoV‐2 infection during pregnancy alters the placental EV fingerprint – a comparative human cohort study Isabel Graf ^1; Dennis Yüzen^2; Anke Diemert^3; Petra Clara Arck^1 ^1Laboratory for Experimental Feto‐Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center of Hamburg‐Eppendorf, Germany, Hamburg, Germany; ^2Laboratory for Experimental Feto‐Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center of Hamburg‐Eppendorf, Germany; Institute of Immunology, University Medical Center of Hamburg‐Eppendorf,, Hamburg, Germany; ^3Department of Obstetrics and Prenatal Medicine, University Medical Center of Hamburg‐Eppendorf, Germany, Hamburg, Germany Introduction: During pregnancy the placenta is essential for the healthy development of the fetus by regulating the transfer of nutrients, oxygen and hormones. External stressors, such as infections or environmental hazards, can easily disrupt placental functionality and promote adverse pregnancy outcomes. To date the early detection of placental inflammation during ongoing pregnancy is limited and mainly relies on ultrasound diagnostics. In this context placenta‐derived extracellular vesicles (EV) might function as mirrors of the placenta and provide insights into the placental functionality. We aim to characterize the placental EV fingerprint in healthy pregnancies and unearth deviations upon SARS‐CoV‐2 infection. Methods: Cushion ultracentrifugation was performed to isolate EV from serum samples of third trimester healthy (n = 40) and SARS‐CoV‐2 infected (n = 25) pregnant women, which were provided by the prospective cohort study “PRINCE” (Prenatal Identification of Children's Health). To assess particle concentration and size‐distribution Nanoparticle‐Tracking Analysis was applied. Subsequent staining with antibodies against CD81, CD63 and CD9 as well as PLAP (placental‐alkaline phosphatase) and CD34 as placental‐endothelial markers allowed EV phenotyping by Imaging Flow Cytometry. Clinical parameters were accessed for correlation. Tandem mass spectrometry will further provide in‐depth information on the EV proteome. Results: In healthy pregnancies the levels of placenta‐derived EV depend on the number of previous pregnancies, but are neither correlated with the placental volume nor the blood flow to the placenta. Infection with SARS‐CoV‐2 decreased overall EV levels and size, but increased the percentage of PLAP+ EV and double‐positive (PLAP+CD34+) EV. Additionally, the levels of CD81+ EV were increased upon infection and positively correlate with the levels of placenta‐derived EV. Summary/Conclusion: Placental EV fingerprints mirror the SARS‐CoV‐2 infection and serve as indicators for placental stress as well as placental‐endothelial dysfunction possibly mediated by upregulation of CD81. In healthy pregnancies placental quality not quantity is decesive for placental EV release. Funding: Isabel Graf was supported by the Else Kröner‐Fresenius‐Stiftung iPRIME Scholarship (2021_EKPK.10), UKE, Hamburg. Funding of this study was made possible by grants provided by the German Research Foundation (KFO296: AR232/25‐2 and DI2103/2‐2) and the Authority for Science, Research and Equality, Hanseatic City of Hamburg, Germany. keywords: pregnancy, infection, placenta‐derived EV, tetraspanins, human cohort Late Breaking Poster PT01: EV Separation/Technology 1 Chairs: Natalie Turner, An Hendrix Location: Hall 4A 16:45 ‐ 18:45 PT01.11. Large capacity extraction of EVs using a positive charge mesh filter in continuous flow: ExoFilter MinJu Bae^1; SEHYUN SHIN ^2 ^1Korea University, Seoul, Republic of Korea; ^2Korea University, Republic of Korea Introduction: As the clinical potential of extracellular vesicles (EVs) as therapeutic agents and drug delivery vehicles becomes more and more evident, related industries are accelerating technology development for high‐quality mass production of extracellular vesicles. Unfortunately, however, most of the exosome isolation technologies developed so far have been customized in small quantities in a laboratory environment. Therefore, there is an urgent need to develop new groundbreaking technologies suitable for large‐scale isolation of exosomes. Methods: Here, we have proposed a novel method for isolating EVs with a charge‐based filter in continuous flow mode. A positively charged protamine salt was bound to a porous nylon mesh (pore size 1 μm) installed on a spin column (d = 6 mm). The number of stacked meshes ranged from 1 to 30. A plasma sample (1 mL) was passed through the filter within 1 minute under a negative pressure of 50 kPa. NTA, RT‐qPCR and Western blot analyses were used to evaluate the efficiency of exosome isolation. Results: SEM image analysis confirmed EVs captured on the mesh surface. EV isolation efficiency increased exponentially as the number of stacked meshes increased. The 30‐layer mesh filter showed the highest yield, slightly higher than the commercially available method (Exoquick), which is known to have the highest yield of exosomes. Summary/Conclusion: These results demonstrated that the positively charged mesh filter can rapidly and effectively capture and extract exosomes from continuously flowing samples. Thus, this study demonstrated a proof‐of‐concept that current continuous EV filter method can be utilized to efficiently isolate exosomes from large‐scale samples (∼300 L) by adjusting the filter size and flow conditions. Funding: This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government, MSIP (2016R1A5A1010148). keywords: EVs, large‐capacity, isolation, positive‐charge, mesh, filter PT01.12. Comparative study of efficient extracellular vesicle isolation method for genetic examination Hyeon Jeong Lee ^1; Gyeong Hwa Kim^2; Jae Young Hur^2; Kye Young Lee^2 ^1Konkuk Medical Center, Seoul, Republic of Korea; ^2Konkuk University Medical Center, Republic of Korea Introduction: Lung cancer is the leading cause of cancer death among Koreans, but with the recent development of targeted therapies, the survival rate improved by about two to three folds. Molecular diagnostics using DNA is especially important for targeted therapy, as it is used to determine a treatment target or confirm therapeutic response and drug resistance. Extracellular vesicles(EVs) well preserve the characteristics of host cells, therefore molecular diagnosis using the EVs DNA has the potential to be an innovative method for cancer diagnosis without an invasive biopsy. Development of a high‐yield extracellular vesicle isolation method for rapid and accurate molecular diagnosis in the clinical field is urgently needed and for this reason, a simpler and time‐saving method was constructed. Methods: The basis of isolation methods was an affinity‐based aggregation of EVs and various polyphenols, antibodies, polymers, and salts were used in the process. EVs from each isolation method were analyzed for size and concentration using nanosight. DNA was extracted from EVs followed by confirmation of DNA concentration and then a PCR test was performed for detection of EGFR mutation. Results were analyzed in comparison to the most wildly used ultra‐centrifugation(UC) method. Results: As a result, it was confirmed that the EV isolation efficiency was the best when polyphenol was used, and the mutation was well detected in the EGFR mutation PCR result using EV DNA. In comparison, other materials decreased the efficiency of EVs isolation and yielded lower DNA concentration, but mutation detection was confirmed by PCR in all cases. Summary/Conclusion: In conclusion, we suggest that the methods used in this experiment would be more efficient in clinical settings where rapid and accurate results are desired, than the existing methods, which require expensive equipment, difficult sample handling, and longer testing time. PT01.13. Electrokinetic‐assisted diffusion‐based extracellular vesicle separation using hundreds of nanometre‐thick silicon nitride nanosieves Gijung Kim; Mingyu Seo; Honggu Chun Interdisciplinary Program in Precision Public Health, Department of Biomedical Engineering, Korea University, Seoul, Korea, Republic of Korea Introduction: There has been increasing interest in the clinical applications of EVs, as they offer a promising alternative to traditional drug delivery systems. Thus, methods to isolate intact EVs have been required. However, conventional EV isolation methods using ultracentrifugation and ultrafiltration apply centrifugal force (∼100,000g) and transmembrane pressure, respectively, which can cause vesicle fusion and rupture. Our preliminary studies showed that intact EVs could be obtained via diffusion‐based separation, but the separation time was long (∼6 hours) due to the large membrane thickness (15 μm) relative to the size of the EVs. To address this issue, we fabricated ultra‐thin silicon nitride nano‐sieves with 200 nm pores and 50 nm pores. Furthermore, to increase the separation rate of diffusion, an electric pulse was applied. The EVs then were concentrated while removing soluble proteins by electroosmotic pumping. Methods: The fabricated ultra‐thin Si3N4 nano‐sieve was placed between the PDMS inlet and outlet chambers, and EVs were isolated from human serum (purchased from Sigma‐Aldrich) with a 300 nm thick ‐ 200 nm pore membrane by applying electric pulses, and soluble protein was removed with a 100 nm thick ‐ 50 nm pore membrane by electroosmotic pump. Results: From 3 mL of human serum (1:1 diluted with PBS containing 0.01% Triton X‐100), a 3.2×10^10 number of EVs could be isolated in 30 min and concentrated in 10 min. After the removal of soluble protein, the EV concentration was increased from 1.2×10^10 to 5×1∙10^10 [particles/ml] and a purity ratio of 17.6 [107 particles/μg protein] was attained. Summary/Conclusion: The EV separation rate was increased more than 10‐fold compared to diffusion‐based separation. The feasibility of using electroosmotic pumps for EV concentration and purification steps on 50 nm pore membranes was also demonstrated. Funding: This work was supported by the National Research Foundation of Korea (NRF) (NRF‐2020R1A2C3010322). keywords: Ultra‐thin, nano‐sieve, electrokinetic, diffusion‐based separation PT01.14. Isolation, Characterization, and Investigation of the Unique Functional Characteristics of Supermeres: New Approaches Oleg S. Tutanov ^1; Clark Massick^1; James N. Higginbotham^1; Mark Castleberry^1; Qin Zhang^1; Dennis K. Jeppesen^2; Jeffrey L. Franklin^3; Kasey Vickers^1; Robert J. Coffey^3 ^1Department of Medicine, Vanderbilt University Medical Center, Nashville, USA; ^2Department of Medicine, Vanderbiltu University Medical Center, Nashville, USA; ^3Department of Medicine, Vanderbilt University Medical Center; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, USA Introduction: Recently, we reported our discovery of supermeres, non‐vesicular extracellular nanoparticles (NVEPs) harboring distinct cargo and functions, separate from EVs (Zhang, Q et al., … Coffey RJ. Nat Cell Biol. 2021;23(12):1240‐54). Supermeres have been shown to contain a large proportion of the extracellular RNA (exRNA) and are enriched with metabolic and matrix interacting proteins. The most abundant protein of supermeres is TGFBI, a secreted protein interacting with collagen and cellular integrins. The biological relevance of supermeres are only now begging to emerge; however, supermeres are likely released from specific solid tumor cancers and may cross the blood‐brain barrier. Methods: We have established protocols for isolating supermeres and exomeres by differential ultracentrifugation (Zhang Q. et al., … Coffey RJ Nat Protoc. 2023). We have now established another method to isolate NVEPs based on Size‐exclusion chromatograpy (SEC). We have produced colorectal cancer cells, DiFi and CC‐CR, expressing TGFBI‐tagged with neon green and a HIS, Nickel binding tag. By growing these cells in a hollow fiber bioreactor ample supermeres containing tagged TGFBI were produced. Using a combination of SEC and a nickel binding column, affinity purified TGFBI‐containing supermeres were produced. NVEPs produced from different fractions and purification strategies were compared using multiple ‐omics strategies. Cytokine expression levels were measured following treatment of microglial cells with different NVEP preparations. Results: SEC‐based purification of supermeres presents a reliable method to quickly purify supermeres from biofluids for down‐stream applications. Supermere treatment of M059K microglial cells lead to a significant increase in expression of pro‐inflammatory cytokines, including IL6, IL1B, and TNF‐alpha. Although UC purified supermeres were found to harbor more cargo than SEC only purified supermeres, SEC‐supermeres showed greater functional capacity towards microglial cells than UC‐supermeres. This, coupled with the supermere's ability to be efficiently taken up by the brain highlights their distinct functional properties compared to sEVs and exomeres, emphasizing their importance in understanding intercellular communication in health and disease. Summary/Conclusion: Supermeres continue to offer great potential in our understanding of the secretome, displaying promising functional characteristics in vitro, different from EVs and exomeres derived from the same starting material. Funding: NIH grants R35CA197570, UH3241685, P01 CA229123 and P50 236733 to R.J.C. We acknowledge the generous support of the Nicholas Tierney GI Cancer Memorial Fund keywords: extracellular vesicles; exomeres; supermeres; colorectal cancer PT01.15. SmartSEC technology: A novel and effective method for isolation of EVs Wei Zheng^1; Suvarna Sathe ^2; Fangting Wu^3 ^1Scientist, R&D, USA; ^2Associate Manager Technical Support, USA; ^3Associate Director, R&D, Palo Alto, USA Introduction: Size exclusion chromatography (SEC) is a traditional technique employed for separating molecules according to their size, conformation, charge, and interaction with stationary phase. This technique can be applied for isolating extracellular vesicles (EVs). The SmartSEC EV isolation system represents a new generation of mixed‐mode chromatography, featuring beads with affinity interaction modes that enables highly efficient purification. In this system, contaminants like IgG and albumin are retained by the resin, while EVs are eluted in a single fraction instead of being partitioned into multiple fractions. Methods: The SmartSEC EV isolation system was assessed for EV isolation from diverse samples. Various methods were used to evaluate yield (BCA Assay and fNTA), purity (western blotting), structural characteristics (TEM), and functionality (cargo loading, labeling, uptake using fluorescence confocal microscopy) of the isolated EVs in numerous downstream applications (RT‐qPCR and mass spectrometry). The performance was compared to EVs isolated using other methods such as ultracentrifugation and conventional SEC. Results: The EVs isolated with SmartSEC show higher yield, higher purity, and low levels of contaminating proteins while maintaining structural and functional characteristics . Summary/Conclusion: SmartSEC is a user‐friendly and powerful tool for EV isolation for a wide array of samples with different origins, numbers and scales. keywords: Size exclusion chromatography exosomes SmartSEC high‐throughput high yield high purity PT01.16. Yield, purity, and throughput of on‐market EV isolation methodologies Amber Murray Exokeryx, Inc, San Diego, USA Introduction: The success of extracellular vesicles (EVs) in diagnostics and therapeutics depends on scalable isolation methods that produce high recovery of highly pure EVs. Current methods for EV isolation are often long, complex, and require considerable hands‐on time. Resulting samples exhibit poor purity (non‐EV marker contamination), poor EV recovery, or both. A reproducible, high‐throughput, automated method to isolate EVs from biological fluids with high recovery and high purity is needed to ensure the integrity of downstream results and to decrease time to discovery. Methods: Specimens were created (1) by isolating plasma from EDTA blood and (2) by clarifying and concentrating conditioned cell culture media via centrifugation. Each specimen was then split, such that EVs from the same specimen could be isolated using size exclusion chromatography, differential ultracentrifugation, density gradient ultracentrifugation, polymer precipitation (ExoQuick Ultra®), or dielectrophoresis (ExoPrep™). Isolated EVs samples were compared for each isolation method for recovery/concentration (tetraspanin‐positive particle counts by ExoView™; EV marker quantification by immunochemistry; exosome visualization by transmission electron microscopy) and for purity (contaminant quantification by silver staining and immunochemistry; contaminant visualization by transmission electron microscopy). Results: EV samples isolated by dielectrophoresis (ExoPrep™) were the only samples that exhibited both high recovery and high purity. Other methods exhibited lower recovery, more contamination with non‐EV markers, or both. The ExoPrep™ isolation method was higher throughput and required less hands‐on time than the other four methods. Summary/Conclusion: There is a critical need for an automated, high recovery, high purity EV isolation platform to fuel EV research and increase the rate of discovery. The ExoPrep™ method, which exploits the dipole moment of EVs in a radio frequency field to separate EVs from other biological structures/molecules (dielectrophoresis), out‐performs all other methods tested. The ExoPrep™ platform is a chip‐based, scalable EV isolation solution that can be incorporated as a high throughput pre‐analytical step for downstream EV‐based tests and therapeutics. PT02: Milk EVs Chairs: Brett Vahkal Location: Hall 4A 16:45 ‐ 18:45 PT02.09. Prenatal SARS‐CoV‐2 infection alters postpartum human milk‐derived extracellular vesicles Somchai Chutipongtanate ^1; Hatice Cetinkaya^2; Xiang Zhang^3; Damaris Kuhnell^4; Desirée Benefield^5; Wendy Haffey^6; Michael Wyder^6; Richa Patel^2; Shannon C. Conrey^2; Allison R. Burrell^7; Scott Langevin^4; David S. Newburg^2; Kenneth D. Greis^6; Mary A. Staat^7; Ardythe L. Morrow^2 ^1Division of Epidemiology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, USA; ^2Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, USA; ^3Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, USA; ^4University of Cincinnati, USA; ^5Department of Molecular Cellular Biosciences, Center for Advanced Structural Biology, University of Cincinnati College of Medicine, USA; ^6Department of Cancer Biology, University of Cincinnati College of Medicine, USA; ^7Department of Infectious Disease, Cincinnati Children's Hospital Medical Center, USA Introduction: Human milk‐derived extracellular vesicles (hMEVs) are crucial functional components in breast milk, contributing to infant health and development. Maternal conditions could affect hMEV cargos. The impact of SARS‐CoV‐2 infection on hMEVs remains unknown. Methods: This study evaluated the influence of SARS‐CoV‐2 infection during pregnancy on postpartum hMEV molecules. Milk samples (9 prenatal COVID‐19 vs. 9 controls) were retrieved from the IMPRINT birth cohort (protocol ID 2019‐0629). After defatting and casein micelle disaggregation, 1 mL milk was subjected to a sequential process of centrifugation, ultrafiltration, and qEV‐size exclusion chromatography. Particle and protein characterizations were performed following the MISEV2018 guidelines. EV lysates were analyzed through proteomics and miRNA sequencing, while the intact EVs were biotinylated for surfaceomic analysis. Multi‐Omic analysis was employed to predict hMEV functions associated with prenatal COVID‐19. Results: Demographic data between COVID‐19 cases and controls were similar. The median duration from maternal SARS‐CoV‐2 test positivity to milk collection was 3 months (range: 1–6 months). Transmission electron microscopy showed the cup‐shaped nanoparticles. Nanoparticle tracking analysis demonstrated particle diameters of < 200 nm and yields of >1e11 particles from 1 mL milk. Western immunoblots detected ALIX, CD9 and HSP70, supporting the presence of hMEVs in the isolates. Thousands of hMEV cargos and hundreds of surface proteins were identified and compared. ACE2‐expressing hMEVs were comparable between groups. Multi‐Omics predicted that prenatal COVID‐19 mothers produced hMEVs with enhanced GNAQ‐driven metabolic reprogramming and RAB11/STK26‐mediated mucosal tissue development, while reducing abilities to trigger pro‐inflammatory responses. Downregulation of the surface adhesion molecules, i.e., ITGB1/4 and LAMB1, together with galectin‐3 binding protein, suggested lower hMEV transmigration potential. Notably, multifunctional breast milk‐derived miR‐148a‐3p, stemness‐related miR‐7641, and cell cycle‐regulated let‐7 constituted >50% of miRNA abundance in the hMEV isolates and were comparable in both groups. Summary/Conclusion: Our findings suggest that SARS‐CoV‐2 infection during pregnancy boosts mucosal site‐specific functions of hMEVs, potentially preparing infants to resist the same illness. Further prospective studies should be pursued to re‐evaluate the short‐ and long‐term benefits of breastfeeding in the COVID‐19 era. Funding: Good Ventures Foundation and NIH/NIAID grant number U01AI144673. keywords: Breastmilk; Extracellular vesicles; Multi‐Omics; Post‐acute SARS‐CoV‐2 infection PT03: EVs and Viral Infection 1 Chairs: Location: Hall 4A 16:45 ‐ 18:45 PT03.09. Identification of MACROH2A1 and CNDP2, extracellular vesicle proteins associated with COVID‐19 refractoriness Takahiro Kawasaki ^1; Yoshito Takeda^2; Ryuya Edahiro^3; Yuya Shirai^3; Atsushi Kumanogoh^2 ^1Osaka University, Japan; ^2Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Japan; ^3Osaka University, Suita, Japan Introduction: The majority of patients with the coronavirus disease 2019 (COVID‐19) recover with mild disease, but some cases become severe and even refractory to therapy. Extracellular vesicles (EVs) in peripheral blood are associated with a variety of pathological conditions and are ideal resources for biomarker exploration. In this study, we combined “next‐generation” proteomics of serum EVs with single cell RNA sequencing (scRNA‐seq) of peripheral blood mononuclear cells (PBMCs) to explore biomarkers which are useful for predicting COVID‐19 refractoriness. Methods: First, non‐targeted proteomics by data independent acquisition was performed on serum EVs collected from 12 COVID‐19 patients, including refractory, non‐ refractory, and moderate cases, and 4 healthy volunteers. EVs were isolated using Tim4‐affinity method. At the same time, scRNA‐seq was performed on PBMCs from patients with the same disease severity. Results: A total of 3046 proteins were identified by proteomics, among which MACROH2A1, a variant of histone H2A, was significantly increased in refractory cases compared to non‐refractory cases, and increased along disease severity. CNDP2, a dipeptidase, was significantly decreased in severe cases compared to moderate cases, and decreased along disease severity. Notably, MACROH2A1 was upregulated in monocytes in peripheral blood in severe cases, while CNDP2 was downregulated in these cells. Molecular network analysis involving MACROH2A1 and CNDP2 showed common pathways in the serum EV proteome, and the transcriptome of monocytes in PBMCs. Summary/Conclusion: These results suggest that MACROH2A1 and CNDP2, which are associated with severe COVID‐19 in peripheral EVs and monocytes, are the potential biomarkers of refractory COVID‐19. Funding: This work was supported by the Japan Society for the Promotion of Science KAKENHI (22K16193 to T. K.) and the JRF (Japanese Respiratory Foundation) Grant (to T.K.). PT05: Musculoskeletal System 1 Chairs: Hua Shen, Wei Seong Toh Location: Hall 4A 16:45 ‐ 18:45 PT05.09. Characterization of plasma – derived small extracellular vesicles from patients with chronic rhinosinusitis Katarzyna Piszczatowska ^1; Katarzyna Czerwaty^2; Karolina Dżaman^2; Mirosław J Szczepański^3 ^11. Department of Biochemistry, Medical University of Warsaw, 02–097 Warsaw, Poland, Poland; ^22. Department of Otolaryngology, The Medical Centre of Postgraduate Education, 01–813 Warsaw, Poland, Poland; ^31. Department of Biochemistry, Medical University of Warsaw, 02–097 Warsaw, Poland; 2. Department of Otolaryngology, The Medical Centre of Postgraduate Education, 01–813 Warsaw, Poland, Poland Introduction: Chronic rhinosinusitis (CRS) is inflammatory disease affecting almost 5–12% of human population and appears in two main subtypes depending on the presence (CRSsNP) or absence of nasal polyps (CRSwNP). CRS relates to the chronic inflammation and tissue remodeling with still elusive molecular background. Currently, extracellular vesicles containing various molecules involved in the CRS pathogenesis are considered as the contributors to its development and potential tools to be use in the diagnostic and therapeutic approaches in the future. The aim of this study was to characterize the quantity and quality of plasma – derived small extracellular vesicles (sEVs) obtained from patients diagnosed with CRSsNP or CRSwNP qualified for Functional Endoscopic Sinus Surgery (FESS) for the first time. Methods: Plasma collected from CRS was filtered, sEVs were isolated using Size Exclusion Chromatography (SEC) and characterized by Nanopartickle Tracking Analysis (NTA), Western Blot (WB) and Cryo‐Electron Microscopy (Cryo‐EM). Results: To evaluate size and concentration of isolated sEVs we applied NTA measurements that estimated sEVs size range from 83 to 94 nm and concentration divergence between 8,20E+09 and 1,30E+11 particles/mL. WB analysis indicated positive expression of CD9, CD63 tetraspanis and negative of Grp94 protein. Cryo‐EM enabled to visualize the morphology of sEVs – characteristic shape, enriched sEVs in lipid bilayer membrane and size comparable with NTA. Summary/Conclusion: In our study, we claimed with the SEC isolation method the presence of sEVs in the plasma obtained from CRS patients. Contribution of plasma‐derived sEVs to the pathogenesis of CRS need further investigation that allow to evaluate their cargo, functions and perhaps reflect the subtype of CRS. Funding: Polish National Agency for Academic Exchange Grant: NAWA BPI/PST/2021/1/00071/U/00001, Young Investigator Grant from Medical University of Warsaw: 1WK/1/M/MBM/N/21 PT06: Cardiovascular EVs 1 Chairs: Bo Li, Claire Crew Location: Hall 4A 16:45 ‐ 18:45 PT06.10. Senescent cardiac fibroblast‐derived extracellular vesicles induce autophagy in cardiac fibroblast Yusei Fujioka ^1; Kosuke Otani^2; Tomoko Kodama^3; Muneyoshi Okada^4; Hideyuki Yamawaki^4 ^1Laboratory Of Veterinary Pharmacology, School Of Veterinary Medicine, Kitasato University, Towada, Japan; ^2School of Veterinary Medicine, Kitasato University, Towada City, Japan; ^3School of Veterinary Medicine, Kitasato University, Japan; ^4School of Veterinary Medicine, Kitasato University, Towada, Japan Introduction: Cellular senescence is termed a cell cycle arrest that is elicited in response to different stresses. The accumulation of senescent cells in tissues drives age‐related diseases. Extracellular vesicles (EV) are lipid‐bilayer‐capsuled particles released by various cells mediating cell‐to‐cell communication. In the present study, we investigated whether senescent cardiac fibroblast‐derived EV play a paracrine effect. Methods: Neonatal rat cardiac fibroblasts (NRCF) and cardiomyocytes (NRCM) were isolated from Wistar rats (1‐3‐day old). NRCF were treated with doxorubicin (DOX) to induce cellular senescence. To determine an induction of cellular senescence, mRNA expression of p16 and p21 was measured by a qPCR. EV were isolated from NRCF culture media by using total exosome isolation reagent. Results: In NRCF, DOX (1000 nM, 24 h) increased mRNA expression of p16 and p21, a cellular senescence‐related gene. Particles isolated from NRCF culture media showed protein expression of Alix and TSG101 and cup‐shaped morphology with a diameter of 100–150 nm as determined by transmission electron microscopy, indicating a successful isolation of small EV (sEV). Vehicle‐treated NRCF‐derived sEV (D0‐sEV, 72 h) significantly increased living cell number in NRCF, which was attenuated by DOX (1000 nM)‐treated NRCF‐derived sEV (D103‐sEV, 72 h). While D0‐EV had no effect on protein concentration in NRCF, D103‐sEV decreased it. Furthermore, D103‐sEV significantly increased LC3‐II to LC3‐I ratio in NRCF, indicating an induction of autophagy. In NRCM, however, NRCF‐derived sEV (72 h) had no effect on living cell number, protein concentration, and LC3‐II to LC3‐I ratio. Alternatively, D103‐sEV (48 h) in the presence of DOX (1000 nM, 24h) decreased living cell number and protein concentration, and increased LC3‐II to LC3‐I ratio in NRCM. Summary/Conclusion: We for the first time revealed that DOX‐induced senescent NRCF‐derived sEV induced autophagy in a paracrine manner. Further researches are required to elucidate the mechanisms and pathological meaning of autophagy induction by NRCF‐derived sEV. keywords: cellular senescence, cardiac fbroblasts, cardiomyocyte, doxorubicin PT08: Immunity, Autoimmunity, and Inflammation 1 Chairs: Marie‐Helene Normand, Sheela Abraham Location: Hall 4A 16:45 ‐ 18:45 PT08.11. Allogeneic extracellular vesicles captured by dendritic cells manipulate T cell responses in colorectal cancer Travis Gates^1; Dechen Wangmo^2; Kevin C. Hicok ^3; Xianda Zhao^2; Subbaya Subramanian^2 ^1University of Minnesota, USA; ^2University of Minnesota, Minneapolis, USA; ^3EV Therapeutics, La Jolla, USA Introduction: Colorectal Cancer (CRC) is the second leading cause of cancer‐related death in the United States. Most CRC patients present with a microsatellite stable (MSS) phenotype and are highly resistant to immunotherapies. Tumor extracellular vesicles (TEVs), secreted by tumor cells, have been implicated as an intrinsic resistance mechanism to immunotherapy in CRC. We previously showed that TEVs in a syngeneic context containing immunosuppressive cargo such as miR‐424 can reduce costimulatory molecule expression (CD28/CD80) and suppress anti‐tumor immune responses. Obtaining sufficient tumor tissue from patients in a syngeneic context hinders translational applications of modified TEVs for treating CRC. We hypothesized that allogeneic modified TEVs depleted of miR‐322 (mouse homolog of human miR‐424) derived from an MC38 background would have shared tumor antigens with CT26 and B16F10 mouse cell lines and would be effective at limiting tumor growth through activation of dendritic cells (DCs) and T lymphocytes (T cells). Methods: Before EVs isolation, cells were cultured in media supplemented with 10% exosome‐depleted FBS (Gibco) for 24h. A standard differential centrifugation protocol was used to purify EVs from the cell culture supernatant. We first imaged the purified EVs using electron microscopy. CD81, CD63, and Alix proteins were analyzed using western blotting. To establish the tumor model, we injected 2*105 CT26 tumor cells resuspended in 100μl matrigel (BD Biosciences) to the right flank of BALB/c mice. To validate that tumor‐derived EVs can be uptaken by dendritic cells (DCs), we performed in vitro uptake assays. Standard tumor growth measurements, IHC, and image analysis were conducted. Results: We observed that prophylactic administration of TEVs derived from MC38 CRC cell lines increased CD8 T cells in CT26 CRC tumors and limited tumor growth but not B16F10 melanoma tumors. We further show that the depletion of CD4 and CD8 T cells eliminated the protective effects of MC38 TEVs. We further mechanistically determined that TEVs can be captured by DCs in vitro, and subsequent prophylactic administration of autologous DCs exposed to MC38 TEVs were capable of suppressing tumor growth and increasing CD8 T cells compared to MC38 wild‐type TEVs exposed to DCs in Balb/c mice bearing CT26 tumors. Summary/Conclusion: These data suggest that allogeneic‐modified TEVs can be captured by dendritic cells, modulate T‐cell responses, and limit tumor growth in vivo. Funding: This project was funded by the Minnesota Colorectal Cancer Foundation, University of Minnesota, ODAT/ CTSI funding, and the Masonic Cancer Center Translational grant award. PT08.12. miR‐223‐transfected macrophages release miRNA‐enriched extracellular vesicles that are taken up by lipotoxic hepatocytes Bootsakorn Boonkaew ^1; Bonita H. H Powell^2; Zhaohao Liao^1; Natthaya Chuaypen^3; Pisit Tangkijvanich^3; Kenneth W. Witwer^1 ^1Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA; ^2Johns Hopkins University School of Medicine, Baltimore, USA; ^3Center of Excellence in Hepatitis and Liver Cancer, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Thailand Introduction: Lipotoxicity and inflammation in the liver play a critical role in the development of nonalcoholic fatty liver disease (NAFLD). Extracellular vesicles (EVs) putatively carry cargo including microRNAs (miRs) to modulate cellular crosstalk, and miR‐223 is thought to be a key regulator of inflammatory response that is highly expressed in immune cells. Here, we investigated whether miR‐223 can be transported from macrophages to lipotoxic hepatocytes via EVs. Methods: To evaluate possible transfer of macrophage EVs containing miR‐223 to hepatocytes, we transfected macrophages with a Cy3‐miR‐223 mimic and dye‐only control. Macrophage‐derived EVs were separated using size exclusion chromatography, and the presence of miR‐223 in EV and non‐EV fractions was measured by quantitative PCR (qPCR). Lipotoxicity was induced in hepatocytes (Huh7) by palmitic acid treatment. Next, co‐culture of transfected macrophages and lipotoxic hepatocytes was done with a transwell system for 24 hours, and hepatocyte miR‐223 levels were measured. EV uptake by hepatocytes was also assessed with MemGlow 488‐labeled EVs. Results: Red fluorescence was observed only in macrophages transfected with Cy3‐miR‐223 mimic, not those exposed to dye alone, and 52‐fold more miR‐223 was released by transfected versus control macrophages. After co‐culturing with miR‐223‐transfected macrophages, lipotoxic hepatocytes had 13‐fold more miR‐223 than controls. Furthermore, the MemGlow dye was transferred to lipotoxic hepatocytes when incubated with labeled EVs. Summary/Conclusion: Our results suggest that macrophages can transfer transfected miR‐223 to lipotoxic hepatocytes. However, we cannot yet rule out non‐cytosolic forms of cell‐EV association. Further investigation of possible effects of miR‐223 on target genes is required. Funding: This work was supported by National Research Council of Thailand (NRCT) (N41A640187), the Second Century Fund (C2F), Chulalongkorn University, Center of Excellence in Hepatitis and Liver Cancer, Faculty of Medicine, Chulalongkorn University, and Thailand‐United States Educational Foundation (Fulbright Thailand). This work was also supported by in part by grants AI144997, [41]MH118164, and UG3CA241694 to Kenneth W. Witwer. keywords: Extracellular vesicles (EVs), MicroRNAs, Nonalcoholic fatty liver disease, Lipotoxicity, PT09: EVs from Microorganism 1 Chairs: Meta Kuehn, Irma Schabussova Location: Hall 4A 16:45 ‐ 18:45 PT09.14. Examining the role of bacterial extracellular vesicles in host blood pressure regulation Feiya Shi^1; Rishi Wagle^1; Sriram Ayyaswamy^1; DAVID DURGAN ^2 ^1Baylor College of Medicine, USA; ^2Baylor College of Medicine, Houston, USA Introduction: We have previously demonstrated that disruption of the normal gut microbiota composition, termed gut dysbiosis, is an underlying cause of hypertension (HT). However, the mechanisms by which the microbiota influences host blood pressure (BP) are largely unknown. We hypothesized that extracellular vesicles released by the gut microbiota gain access to the circulation, and deliver cargo to host tissues involved in BP regulation, inducing inflammation and ultimately HT. Methods: To examine our hypothesis, we used the spontaneously hypertensive stroke prone rat (SHRSP), a well‐known animal model of essential hypertension, and its corresponding normotensive control, the Wistar‐Kyoto rat (WKY). Bacterial EVs (bEVs) were isolated from cecal contents of both strains by size exclusion chromatography and quantitated by nanoparticle tracking analysis. BEV cargo including small RNAs, LPS, 16s rRNA, was assessed. To directly examine the role of bEVs on BP, purified WKY and SHRP bEVs were transplanted to recipient rats of the same and opposite strain by oral gavage (1.5E10 EVs/rat) every other day for 4 weeks. BP was monitored weekly by tail cuff measurements. Following 4 weeks of bEV treatments, the inflammatory status of ileum and brain were assessed by flow cytometry. Results: Small RNA cargo of bEVs was found to be significantly different between WKY and SHRSP (n = 6, p = 0.001). BEVs from SHRSP contained significantly greater LPS as compared to those from WKY (n = 6, p = 0.01). EVs isolated from plasma of both strains, containing host and bacterial EVs, contained bacterial 16s rRNA, demonstrating bEVs gain access to host circulation. Finally, as compared to WKY rats receiving WKY bEVs, WKY rats receiving SHRSP bEVs had 1) significant elevations in BP (176 mmHg vs 150 mmHg, n = 5, p< 0.01), 2) significant reduction of anti‐inflammatory T regulatory cells in ileum and brain (n = 4, p< 0.05), and 3) significant increases in macrophages in ileum and brain (n = 4, p< 0.01). Additionally, SHRSP rats receiving WKY bEVs had significantly lower BP (160 mmHg vs 175 mmHg, n = 5, p< 0.05), and reduced macrophages in ileum and brain (n = 4, p< 0.001 and p< 0.01 respectively), as compared to SHRSP receiving SHRSP bEVs. Summary/Conclusion: BEVs originating from the dysbiotic gut of hypertensive SHRSP rats carry distinct cargo, reach the host circulation, and are involved in host gut‐ and neuroinflammation and HT. These studies identify bEVs as a previously unrecognized mechanism for gut microbiota‐host interaction involved in BP regulation. Funding: NIH‐NHLBI R01HL134838 (DJD) PT09.15. Extracellular vesicles of the commensal intestinal microbiota of newborns and their regulatory effect on immune cells Catalina Adasme‐Vidal^1; Aliosha I. Figueroa‐Valdés^1; Javiera De Solminihac^2; Karina Pino‐Lagos^2; Patricia Valdebenito^3; Sebastián Illanes^3; Jimena Cuenca^1; Francisca Alcayaga‐Miranda ^1 ^1IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Centro de Investigación e Innovación Biomédica (CiiB), Universidad de los Andes, Santiago, Chile, Santiago, Chile; ^2Facultad de Medicina, CiiB, Universidad de los Andes, Santiago, Chile, Santiago, Chile; ^3Laboratory of Reproductive Biology, CiiB, Universidad de los Andes, Santiago, Chile., Santiago, Chile Introduction: Introduction: The type of birth ‐vaginal delivery (PV) or cesarean section (CS)‐ determines the composition of the commensal intestinal microbiota of newborns (CIMN). This CIMN has been associated with pediatric diseases, including immunological ones. How the gut microbiota interacts with the immune system is poorly understood, but it could be mediated by bacterial extracellular vesicles (bEVs) derived from gut microbiota. This study aims to determine if the PV/CS influences the characteristics and immunomodulatory potential of the microbiota‐derived bEVs of newborns. Methods: Methods: bEVs were isolated from feces samples collected from PV/PC newborns and characterized in terms of concentration, size, morphology, and protein markers. bEVs immunomodulatory potential was assessed through a lymphocyte immunosuppression assay. Results: Results: Protocol for bEVs isolation was established. No differences were observed in the concentration and size of the bEVs isolated from the CIMN. However, bEVs shapes vary depending on their PV/CS origin. Likewise, bEVs derived from PV exert more immunomodulation capacity than bEVs obtained from CS. Summary/Conclusion: Summary/Conclusion: The established protocol allowed obtaining a concentrate enriched in bEVs whose morphology and immunosuppressive effect depend on their PV/PC origin, perhaps influenced by the Gram‐positive/negative bacterial composition of CIMN. Funding: Funding: ANID—Basal funding for Scientific and Technological Center of Excellence, IMPACT, #[42]FB210024 & ANID‐FONDECYT #1190411/1210654. keywords: bacterial extracellular vesicles PT09.16. The effect of antibiotics on the release of bacterial extracellular vesicles (bEVs) from Escherichia coli (E. coli) PANTEHA TORABIAN ^1; Lea V. Michel^2; Thomas Gaborski^3; Anthony P. Pietropaoli^4; James Crawford^5; Navraj Singh^5 ^1Rochester institute of technology, USA; ^2Rochester Institute of Technology, Rochester, USA; ^3Rochester institute of technology, Rochester, USA; ^4University of Rochester Medical Center, Rochester, USA; ^5rochester institute of technology, USA Introduction: Sepsis is a life‐threatening inflammatory response to infection leading to severe organ dysfunction. While sepsis can arise from fungal, viral, or parasitic infections, it most commonly results from bacterial infections and is typically treated with broad‐spectrum antibiotics. However, we hypothesize that antibiotic treatment can have the negative side effect of enhancing the release of bEVs from Gram‐negative bacteria, such as E. coli. The study aimed to identify which classes of antibiotics, based on their mechanism of action, could increase bEVs production, which contributes to sepsis‐related inflammation. Methods: After incubation with nine clinically‐relevant antibiotics at twice their minimum inhibitory concentrations, bEVs were collected from a strain of pathogenic E. coli (K1 RS218) through ultracentrifugation. The isolated bEVs were quantified by immunoblot with antibodies to E. coli outer membrane lipoprotein, peptidoglycan associated lipoprotein (Pal). Particle size and concentrations were measured by Nanoparticle Tracking Analysis (NTA), and bEVs were visualized by TEM. Results: The bEV concentrations, determined by NTA, were higher for beta‐lactam antibiotics compared to quinolone and aminoglycoside antibiotics. However, not all beta‐lactam antibiotics enhanced bEV release to the same extent. Relative concentrations determined by NTA and quantitative immunoblotting were generally in agreement. Summary/Conclusion: The results corroborated the hypothesis that different antibiotics enhance bEV release from E. coli to different extents depending on their mechanism of action. Beta‐lactam antibiotics generally resulted in higher levels of bEV production due to destruction of peptidoglycan layer, which destabilizes the cell envelope and leads to higher levels of vesiculation. These findings have important implications for sepsis treatment, since bEVs have been associated with acute and chronic inflammation. Funding: NIH R21AII63782 PT09.17. Using the Virus Counter® 3100 platform for bacterial extracellular vesicles (bEVs) characterization ‐ Comparison with traditional Nanoparticle Tracking Analysis Nadezhda Nikiforova ^1; Mehdi (Aslan) Dehghani^2; Munther Alsudais^1; Anna Kasper^1; Martina Videva^1; Lea V. Michel^1; Thomas Gaborski^3 ^1Rochester Institute of Technology, Rochester, USA; ^2Sartorius Stedim North America, Boston, USA; ^3Rochester institute of technology, Rochester, USA Introduction: Quantification of nanosized particles, such as extracellular vesicles (EVs), remains a challenge due in part to the heterogeneity of size and structure of the particles. Many researchers have used NTA to quantify EV size distribution and concentration. However, this technique does not effectively detect smaller particles, especially in a mixture with larger particles. The VC is a single particle detecting platform designed to characterize nanoparticles, such as viruses. The current study aims to determine whether the VC can detect and characterize bEVs in the same range as NTA. Methods: Escherichia coli (E. coli) (K1 RS218 strain) were grown in lysogeny broth (37°C, 150 rpm) until OD at 600nm was 1.5. The bacterial cells were removed by centrifugation (5000 g) and filtering (PES 0.22μm), and bEVs were isolated by ultracentrifugation (100,000xg, 1 h, +4⁰C). The bEVs were characterized by a protein quantification assay (Bradford), NTA, (scattering mode, camera level 14, detection threshold 5; 5 measurements by 30 sec), TEM, and Western blot. bEVS labeled with CellMask™Orange stain (CMO) were also characterized by the NTA (fluorescent mode, camera level 16, detection threshold 5, 5 measurements by 15 sec, camera level 16, detection threshold 5, 5 measurements by 30 sec, pump flow rate 35) and the Virus Counter® 3100 (nucleic acids channel, 0.8 PMT gain). Results: We estimated 1×1011 particles at a concentration of 7×1010 particles/mL using NTA on our isolated bEV sample, with a size distribution range from 57 nm to 171 nm, determined by ImageJ analysis of our TEM images (Close‐up and Wide‐field). For the same sample, we estimated the protein concentration was 49.6 μg/ml. Western blots showed that three bEV‐specific markers (Pal, TolB, and OmpA) were present in the bEVs, and one apoptotic body marker (LexA) was not present. After staining bEVs with CMO, the concentration of the fluorescent particles, determined by NTA in fluorescent mode, was 10–30% of the concentration, determined by NTA in scattering mode. However, the same samples run using the VC showed dramatically increased staining, with 70–80% CMO‐positive bEVs. Summary/Conclusion: This study shows that the Virus Counter® 3100 platform is more sensitive in the detection of fluorescently labeled bEVs compared to NTA. Funding: Funding: Sartorius Stedim North America, NIH R21 AI163782 keywords: bacterial extracellular vesicles (bEVs), Virus Counter® 3100 platform (VC) PT10: EV Separation from Biological Sources Chairs: Carlos Salomon Location: Hall 4A 16:45 ‐ 18:45 PT10.13. Identification and characterization of extracellular vesicles and nanoparticles derived from mouse tissues and human plasma Qin Zhang ^1; Dennis K. Jeppesen^2; James N. Higginbotham^1; Zheng Cao^3; Ping Zhao^3; Robert J. Coffey^4 ^1Department of Medicine, Vanderbilt University Medical Center, Nashville, USA; ^2Department of Medicine, Vanderbiltu University Medical Center, Nashville, USA; ^3Vanderbilt University Medical Center, USA; ^4Department of Medicine, Vanderbilt University Medical Center; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, USA Introduction: Extracellular vesicles (EVs) and non‐vesicular extracellular nanoparticles (NVEPs) play pivotal roles in both physiological and pathological conditions. However, the field has largely focused on studies related to EVs, while studies on extracellular amembranous NVEPs, including the recently discovered exomeres and supermeres, are limited. We previously reported that EVs derived from colorectal cancer (CRC) cells are highly enriched with exosomal markers and membrane proteins, while supermeres are highly enriched with cargo involved in multiple cancers (TGFBI), Alzheimer's disease (APP) and cardiovascular disease (ACE2). However, it is unknown whether plasma‐ and brain‐derived EVs and NVEPs are also enriched with circulating biomarkers and therapeutic targets. Methods: EVs, exomeres and supermeres were isolated from plasma of healthy individuals, colorectal cancer patients, and mouse tissues by centrifugation, dilution, filtration, and differential ultracentrifugation. Plasma‐derived samples were subjected to albumin depletion. All human studies were approved by the Vanderbilt University Medical Center Institutional Review Board. Results: Consistent with fractions derived from cell line conditioned media, sEVs derived from the plasma of CRC patients contained EV markers and membrane proteins such as FLOT1, Syntenin‐1 and CD73, while supermeres contained shed membrane proteins including ACE and CEACAM5. Furthermore, after albumin depletion, TGFBI, a potential CRC biomarker, was detectable in supermeres derived from CRC plasma. Brain‐specific membrane proteins and EV markers were enriched in sEVs isolated from mouse brain, while supermeres contained high levels of metabolic enzymes. Summary/Conclusion: The sEVs, exomeres and supermere derived from human plasma and mouse tissues are distinctive EVs and particles replete with disease biomarkers and therapeutic targets. PT12: Techniques and Methods Single EV Analysis Chairs: Edwin van der Pol, Eduardo Reategui Location: Hall 4A 16:45 ‐ 18:45 PT12.13. How to image EVs with super‐resolution microscopy? First Abbelight EV day seminar Quentin Lubart ^1; Cataldo Schietroma^1; Caterina Severi^1; Mouna Triki^2; Daniele D'Arrigo^3; Sybille Capelle^4; Ipek Altinoglu^2; Benjamin Compans^2 ^1Abbelight, 191 Avenue Aristide Briand, 94230 Cachan, France, Cachan, France; ^2Abbelight, Cachan, France; ^3MSC lab, Paris, France; ^4Abbelight, France Introduction: On 28th March 2023, Abbelight organized a daily seminar focused on single‐vesicle imaging and EV characterization with super‐resolution microscopy (SRM). The aim of the seminar was to draw attention on the SRM in the EV field and to expand both the theoretical and the practical knowledge of SRM to researchers and EV experts. Methods: During the morning, a comprehensive theoretical overview on different SRM approaches and how they can be applied to the EV field was held by Quentin Lubart, senior scientist at Abbelight. Then, three researchers (Cronemberger Andrade, PhD, Paris University; Prof. Bussolati, Turin University and EVIta society; Olesia A. Gololobova, PhD, Johns Hopkins University) presented practical applications of SRM in the EV research. During the afternoon, the participants actively participated in all the practical sessions of the EV imaging with SRM: sample preparation, imaging and data management. Results: 30 participants (27 in presential, 3 remotely) attended the event. Attenders were internationals from France, Italy, Belgium, England and USA with multiple function represented: PhD students, postdoctoral researcher, professors and industrials. During the morning the participants had an in‐depth overview on the requirements and the state‐of‐the‐art approaches currently in use and the future perspective of the SRM in the EV field. In the afternoon, they extended their expertise on how to perform super‐resolution imaging of the EVs, starting from the sample preparation to the data management. Summary/Conclusion: The theoretical and practical expertise acquired ensured participants to enter the single‐EV imaging or to remain up‐to‐date with the latest advancements. The seminar represented an invaluable opportunity to increase the knowledge of the SRM as powerful tool in the EV characterization, but it also stimulated the scientific exchange and collaborations between researchers. Given the success of this first EV day seminar, we are planning to make the seminar a recurring appointment, increasing progressively the audience. Funding: Organized and supported by Abbelight, Cachan, France keywords: SMLM, EVs, super resolution microscopy, workshop, seminar, sample preparation, imaging EVs, data analysis PT12.14. A proximity tagging approach to catalog microvesicle‐specific protein cargoes Alex Boomgarden ^1; Veronica Dougherty^1; Uma Aryal^2; Crislyn D'Souza‐Schorey^3 ^1Department of Biological Sciences, University of Notre Dame, IN, USA; ^2Bindley Bioscience Center, Purdue University, IN, USA; ^3Department of Biological Sciences, University of Notre Dame, IN, Notre Dame, USA Introduction: Extracellular vesicles (EVs) have been extensively studied in cancer and shown to promote cell‐cell communication in a wide range of processes that underlie disease progression. Furthermore, accruing evidence of their presence in body fluids and potential as disease biomarkers has heightened both interest and need to better understand EV biology. A major challenge in EV research is the diversity of these vesicles, and in particular, the inability to separate and characterize EV subclasses. Microvesicles (MVs) are a distinct EV subtype of relatively larger size and formed by the outward budding and pinching of the plasma membrane. We still lack a full understanding of the mechanisms that underlie MV biogenesis as well as the specific cues that dictate the loading of MVs with molecular cargoes. Given our findings that the ARF6 protein is present in MVs and regulates the recruitment of molecular cargoes for incorporation into nascent microvesicles, we reasoned that a proximity ligase screen for proteins using ARF6 as bait will identify proteins specifically targeted to MVs. Methods: We have utilized pupylation‐based interaction tagging to interrogate the ARF6 interactome in MVs. By fusion of the pafA sequence to the C‐terminus of ARF6 in a lentiviral background, we have generated melanoma cells stably expressing ARF6‐PafA. ARF6‐PafA localization in cells and MVs is indistinguishable from endogenous ARF6. Infection of ARF6‐PafA cells with lentivirus encoding PupE/bio‐DE28 allowed for the use of standard techniques to isolate pupylated proteins. PupE‐tagged MV proteins were trypsin‐digested followed by LC‐MS/MS and searched against a human protein database. Results: PUP‐IT tagging enabled the enrichment and detection of multiple ARF6 interactors in MVs. Gene ontology analysis revealed an enrichment of membrane‐bound and cytoplasmic proteins involved in cytoskeletal remodeling, vesicular transport, nuclear‐cytoplasmic exchange, RNA processing and multiple signaling pathways. Summary/Conclusion: This study comprehensively analyzes the ARF6 interactome in MVs providing rich insight into potential mechanisms of MV biogenesis and function. Funding: NIH/National Cancer Institute ([43]CA273469) keywords: Microvesicles, proximity tagging, proteomics PT13: Cancer Biomarkers 1 Chairs: Serena Lucotti, Fabrice Lucien Location: Hall 4A 16:45 ‐ 18:45 PT13.14. Targeted capture of hepatocyte‐derived extracellular vesicles from plasma and subsequent multi‐omic analysis reveals potential diagnostic markers of hepatocellular carcinoma Ricardo Figueiras^1; Susana Vagueiro^1; Gillian Reynolds^2; Elnaz Persia^3; Raissa Kay ^1; Tomas Dias^3 ^1Mursla Ltd., Milton, United Kingdom; ^2Mursla Ltd., United Kingdom; ^3Mursla Ltd., Cambridge, United Kingdom Introduction: Extracellular vesicles (EVs) are released by every cell in the body and can be found in biofluids such as blood, urine, breast milk, etc. These EVs contain tissue specific information that can be used as helpful biomarkers of disease and dysfunction, but finding tissue‐specific EVs in biofluids,and mining them for information, is a difficult process. We have previously introduced NEXPLOR (Novel EXosome PopuLatiOn Revealer) for the capture and isolation of EV sub‐populations. Herein, we apply NEXPLOR for the isolation of hepatocyte‐derived EVs from the plasma of healthy, cirrhosis and patients with hepatocellular carcinoma (HCC). Methods: We isolated extracellular vesicles (EVs) from human plasma samples with differential centrifugation, ultrafiltration and size exclusion chromatography followed by our patented tissue‐specific immunoaffinity capture technology NEXPLOR tuned to capture hepatocyte‐derived EVs. We applied NEXPLOR to human plasma samples of healthy individuals, patients with liver cirrhosis, or patients with HCC, and compared their mass spectrometry (MS) and small RNAseq profiles with EVs from the same samples but isolated by SEC alone. All plasma collection was approved by the ethical review committee from each source (UCL biobank, Cambridge Bioscience, iMM biobank) and informed consent was obtained in all instances. Results: MS of NEXPLOR EVs revealed that they are enriched for EV (MS:ALIX, CD63, CD9, CD81, ATP1A1, etc.) and liver (F2, AHSG, C9, IGFALS, etc.) specific proteins. Further comparison of SEC‐only isolation and NEXPLOR of patient samples using small RNA‐seq resulted in a higher percentage of the total miRNA found in NEXPLOR captured EVs that can be classified as liver‐related, cirrhosis‐related, HCC‐related, and even EV‐related overall. Summary/Conclusion: We conclude that NEXPLOR technology accurately and precisely captures hepatocyte‐derived EVs from human plasma in healthy individuals and diseased patients, highlighting the potential of NEXPLOR for biomarker discovery in HCC and potentially other applications. keywords: HCC, Cirrhosis, Mass Spectrometry, Immunoaffinity, small‐RNAseq, Diagnostics, Liquid Biopsy PT13.16. Are PD‐L1 extracellular vesicles biomarkers of immunotherapy response? A cohort study and review of the literature pinpoints improvements towards clinical translation Cherie Blenkiron ^1; Marcella Flinterman^2; Sandra Fitzgerald^1; Cris Print^1; Anastasiia Artuyants^1 ^1The University of Auckland, Auckland, New Zealand; ^2The University of Auckland, New Zealand Introduction: Immunotherapies, such as PD‐1 inhibitors, have revolutionised the treatment of people diagnosed with metastatic melanoma. However, over half of patients do not respond to what can be an expensive and toxic therapy. Many studies have proposed the levels of PD‐L1 on EVs as promising biomarkers for cancer diagnosis, prognosis and importantly, as predictors of immunotherapy response. We aimed to recapitulate those findings in a small New Zealand based cohort. Methods: EV isolation using sequential (ultra)centrifugation was compared to Size Exclusion Chromatography (SEC) for particle and protein yield and various markers using western blotting. SEC was selected to isolate EVs from 2mL plasma taken at two timepoints before (P1) and during (P2; 6 weeks) immunotherapy for 19 consented patients (approval #16/NTA/180). EV PD‐L1 was detected using an optimised ELISA. Results: Quantities of PD‐L1 in SEC EVs were limited, near the lower detection limit of the ELISA, thus requiring larger volumes of plasma for quantitation. In our small cohort, the quantity of PD‐L1 normalised to particle count at P1 (higher or lower than the mean) was associated with disease specific survival (P = 0.035), but not at P2, nor the change between P1 and P2. This was highly dependent on the method used for normalisation of PD‐L1 abundance, whether to plasma volume, particle or protein yields. In contrast to prior studies, EV‐PD‐L1 was not associated with RECIST response to immunotherapy. A review of 13 studies highlighted variations in applied methods and data analyses which likely underly the discordance seen between papers regarding for example the overall yields of PD‐L1 on EVs (2.5pg/mL to 5500ng/mL) and whether they do or do not have biomarker utility. Summary/Conclusion: Although promising, current studies using PD‐L1 EVs as biomarkers for immunotherapy response are widely discordant. Significant standardisation of protocols and cohort expansion are required to answer the question of whether PD‐L1 EVs are truly clinically applicable biomarkers. Funding: JI Sutherland Award, Auckland Medical Research Foundation National Science Challenges, Healthier Lives Programme PT14: Biomarkers from DNA to RNA Chairs: Nicole Noren Hooten, Maija Puhka Location: Hall 4A 16:45 ‐ 18:45 PT14.16. Predicting non‐small‐cell lung cancer patient responses to immunotherapy with tumor‐derived extracellular‐vesicle‐derived miRNA biomarkers Hanfei Shen ^1; Andrew A. Lin^1; Jacob Till^1; Charu Aggarwal^2; Jeffrey Thompson^1; Erica Carpenter^1; David Issadore^1 ^1University of Pennsylvania, Philadelphia, USA; ^2University of Pennsylvania, USA Introduction: Immune checkpoint blockade has transformed the treatment landscape for patients with Non‐Small‐Cell Lung Cancer (NSCLC), however less than 50% of patients respond to these therapies. Given the potential for severe toxicity, high cost of immunotherapy, and lack of accurate biomarkers, NSCLC patients could greatly benefit from a non‐invasive, plasma‐based biomarker that can accurately predict immunotherapy response prior to treatment. Methods: We collected pre‐therapy blood samples from patients with metastatic NSCLC who went on to receive pembrolizumab with or without chemotherapy to develop and validate a tumor‐derived EV (TDE) miRNA‐based algorithm to predict response to therapy. We applied a microfluidic platform to isolate NSCLC TDEs based on the EV surface markers EPCAM, EGFR, CD151 and Tspan8. We first applied miRNA sequencing to a discovery set (n = 20) of 10 known responders and 10 non‐responders to identify candidate miRNA biomarkers. We are in the process of using PCR to measure these miRNAs in a training set (n = 40), followed by validation in a blinded test set (n = 40). Results: miRNA sequencing results from the discovery set yielded a large number (n = 62) of TDE miRNAs for which there are both statistically‐significant differences in expression and mean fold changes >2 or < 0.5 between responders and non‐responders. Among these candidate miRNA biomarkers, many have been validated by existing research to be directly relevant to (lung) cancer or immunotherapy. Summary/Conclusion: This proof‐of‐concept data suggests prediction of metastatic NSCLC patients’ immunotherapy response with TDE miRNA from blood may be possible. Multiple TDE miRNAs are shown to associate with immunotherapy response, which also suggests the possibility of a multi‐feature prediction model for immunotherapy response, if validated in an independent cohort. keywords: Lung cancer, biomarker, blood, plasma, Extracellular Vesicles, miRNA, EV isolation, prognosis, immunotherapy PT14.17. A blood‐based extracellular vesicles DNA methylation signature for colorectal cancer screening Hongzhang He ^1; Sheng Zhang^2; Faming Wang^2; Mingxi Chen^2; Jeongyun Kim^2; Mengrou Lu^2; SiYang Zheng^2 ^1Carnegie Mellon University, pittsburgh, USA; ^2Carnegie Mellon University, USA Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer related deaths, accounting for an estimated 1.8 million new cancer diagnoses and over 880,000 deaths in 20184. Furthermore, CRC patients who are diagnosed at the most advanced (metastatic) stage is estimated to have the 5‐year survival rate of 14%14. Dramatically, studies shows that if it can be detected at an early stage when surgical removal is feasible, the 5‐year survival rate significantly increases to 90%14. Therefore, early detection for CRC with curative treatment intent is crucially important to significantly reduce the mortality and morbidity and even cure CRC. Methods: Extracellular vesicles (EVs) are lipid‐bilayer‐enclosed vesicles of sub‐micrometer size that are secreted by virtually all cell types. They contain hundreds of different proteins, thousands of intact RNA species, and double‐stranded DNA (dsDNA) fragments that sample the entire human genome. Recent findings further corroborate the potential role of EVs in early cancer screening, cancer diagnosis, treatment selection and monitoring. In this study, we used lipid nanoprobe (LNP) for EV isolation and methylation‐specific qPCR assay for detecting SEPT9 methylation to develop a minimal invasive CRC screening test. Results: Firstly, we have successfully detected promoter methylation of cancer specific genes SEPT9 in EVs derived from both genomic DNA and EV DNA of the CRC cell line. Secondly, we isolated EV using LNP from plasma samples in a cohort of 19 CRC patients and 9 healthy donor controls. In parallel, we isolated circulating free DNA (cfDNA) from the same cohort of the plasma samples using commercially cfDNA isolation Kit. Then, SEPT9 methylation signature was analyzed by our quantitative real‐time PCR assay in these 28 EV DNA samples and 28 cfDNA samples, respectively. Summary/Conclusion: In the 19 CRC samples, we identified 10 SEPT9 gene methylation samples from LNP‐isolated EV DNA and cfDNA, respectively. In 9 healthy control samples, no SEPT9 methylation was detection in the EV‐DNA samples, while one false‐positive was detected with cfDNA. Thus, the sensitivity and specificity of EV‐DNA in the detection of SEPT9 gene methylation from CRC were determined to be 53% and 100% respectively. In comparison, the sensitivity and specificity of cfDNA are 53% and 89%, respectively from this preliminary pilot study. This indicated that EV‐DNA might be superior to cfDNA in detecting SEPT9 gene methylation for CRC screening. PT14.18. Acute traumatic stressor modulates vesicular and free circulating immunomodulatory miRNA and inflammatory proteins Tel Kelley ^1; Shelby Hopkins^2; Lida Beninson^3; Monika Fleshner^2 ^1University Of Colorado, Boulder, USA; ^2University of Colorado, Boulder, USA; ^3National Academy of Sciences, USA Introduction: The stress response promotes host survival by initiating dynamic changes to a broad spectrum of physiological systems. Exposure to acute traumatic stressors impacts circulating levels of cellular stress and inflammatory proteins, a process known as sterile inflammation. There is emerging evidence that microRNAs (miRNAs) are impacted by stressor exposure and may modulate features of the stress response. miRNA are short (∼22 nucleotides) non‐coding strands that post‐transcriptionally regulate gene expression. miRNAs are detectable in the blood, and most are either bound to carrier proteins or are packaged into exosomes and microvesicles (EMVs). Using a preclinical model, we tested the hypothesis that exposure to an acute traumatic stressor is sufficient to change circulating miRNA composition and concentration measured in whole plasma and plasma derived‐EMVs. Methods: Given that our preclinical stress model increases inflammatory protein concentration in blood and tissues, a panel of immunomodulatory miRNA were measured using particle‐based multiplex followed with RT‐PCR and GeNorm algorithm normalization. miRNA‐gene interactions were established through miRDB. Predicted genes were input into GeneCards V. 5.12 to investigate related pathways of affected genes. Least absolute shrinkage and selection operator (LASSO) regression was used to select contributing features in a regression model that determine which fraction (EMV‐enriched or plasma) and miRNA related most with stress‐evoked changes in inflammatory proteins and hormonal indices of the stress response Results: The results were that acute stressor exposure impacted miRNA expression in both EMV‐enriched and plasma fractions. While most miRNA expression changes were shared between fractions, there were several unique miRNA changes within both fractions. Most immunomodulatory miRNA concentrations in both plasma and EMVs were reduced. Gene prediction revealed that miRNA changes impacted genes that are related to inflammatory, metabolic, and disease pathways. The EMV‐enriched fraction was most highly predictive of circulating IL‐1β levels. Feature selection revealed that miRNA related to the inflammasome (NLRP3) contributed the most to predictive capability of the final model. Summary/Conclusion: Our results support the hypothesis that acute down‐regulation of immunosuppressive miRNA may play an important role in releasing inflammatory protein gene transcription from miRNA inhibition. Understanding the dynamics of stress‐induced changes in EMV cargo may also have implications when identifying EMV‐associated miRNAs as biomarkers of disease. keywords: extracellular vesicles, miRNA, acute stress, sterile inflammation, inflammatory proteins PT14.19. Comparison of exosomes from tumor tissues and tumor cells for potential therapeutic applications Maryam Firouzi ^1; Changsun Kang^2; Xiaoyu Ren^2; Dongin Kim^2 ^1College of Pharmacy, OUHSC, Oklahoma City, USA; ^2University of Oklahoma Health Sciences Center, USA Introduction: One of the major problems of cancer therapy is due to tumor heterogeneity. Therefore, developing tumor‐specific antibodies is of vital importance in tackling this problem. Currently, our lab has developed a monoclonal antibody production method using cancer cell‐derived exosome as an antigen. This antibody showed specific targeting efficacy to the parent cancer cell. However, because of the patient's tumor heterogeneity, cancer cell‐derived exosomes do not serve as personalized antigens for monoclonal antibody production. In this regard, exosome as an antigen must be obtained from the patient's tumor tissue. Simultaneously, to use our antibody production method using tumor tissues, exosome composition must also be similar between exosomes from the tumor tissues and the cells. In this study, we used exosomes from both the OVCAR8 cells and tumor tissues of OVCAR8 tumor‐bearing mice and compared their membrane composition. We hypothesize that the exosomes derived from the OVCAR8 tumor tissues of the mice are similar to the ones from the OVCAR8 cells in lipid and protein composition. Methods: To prove our hypothesis, we used OVCAR8 ovarian cancer cells transfected with mkate‐2 fluorescent protein (mkate‐2 OVCAR8). The exosomes from mkate‐2 OVCAR8 tumor‐bearing mice were compared to the ones from mkate‐2 OVCAR8 cells. The fluorescent intensity of the two exosome samples was measured. Western blot analysis characterized exosome membrane proteins. Lipidomics also quantified membrane lipids and proteins. Results: Our results confirmed the similarity of exosome membrane composition between mkate‐2 OVCAR 8 cells and mkate‐2 OVCAR 8 tumor tissues. Summary/Conclusion: In conclusion, since the exosomes released from the OVCAR8 cells and OVCAR8 tumor tissues are significantly similar, tumor‐tissue‐derived exosomes can also be used for producing monoclonal antibodies. Therefore, we can develop personalized monoclonal antibodies in clinical situations using the patient's tumor tissue exosomes. PT14.20. Computational meta‐analysis of public extracellular RNA cancer cell line profiles reveals the potential of the combination of extracellular RNA binding proteins and their cargo as cancer biomarkers Emmanuel Esquivel ^1; Emily LaPlante^1; Alessandra Stürchler^2; Robert Fullem^1; Matthew Roth^1; Bogdan Mateescu^3; Aleksandar Milosavljevic^4 ^1Baylor College of Medicine, Houston, USA, Houston, USA; ^2ETH Zürich, Zürich, Switzerland, USA; ^3ETH Zürich, Zürich, Switzerland, Switzerland; ^4Baylor College of Medicine, Houston, TX, Houston, USA Introduction: The presence of specific extracellular RNA binding proteins (exRBPs) in biofluids and their extracellular RNA (exRNA) cargo is an emerging area of research. Although their potential role as biomarkers is not yet clear, the exRBP/exRNA combination has potentially superior biomarker characteristics due to the specificity that can be achieved when combining hundreds of thousands of distinct exRNA fragments and their exRBP carriers. To investigate the potential of this new biomarker class, we performed computational meta‐analysis of public exRNA cancer cell line profiles to identify cell‐line specific exRBP/exRNA combinations. Methods: Our analysis was based on an analysis we previously developed to map the cargo of exRBPs across biofluids and exRNA carriers. Here, we extend the baseline analysis by performing a meta‐analysis of 60 previously published small exRNA‐seq profiles of breast, head and neck, and cervical cancer cell lines. As in the baseline study, we performed correlation footprinting using 168 RBPs, whereby correlation between RNAs bound by an RBP is used as evidence of RBP presence, to identify exRBP/exRNA combinations for each cell line. We established significance of exRBP/exRNA combinations by permutation testing. Results: We predicted 8–22 exRBPs per cell line with a total of 40 unique exRBPs across cell media of 22 cancer cell lines (breast, head and neck, cervical), and the associated exRNA fragments represented 8,863 protein coding genes. We associate the exRBPs with specific vesicular and non‐vesicular exRNA carriers. Summary/Conclusion: Meta‐analysis of 60 previously published small exRNA‐seq profiles of breast, head and neck, and cervical cancer cell lines yields exRBP/exRNA combinations that are potentially informative as biomarkers. Our analysis suggests utility of the correlation footprinting method and the baseline map of extracellular RNA binding proteins and associated exRNAs to guide discovery of a novel class of biomarkers. PT15: EV Sizing and Counting Chairs: Tanina Arab, John Nolan Location: Hall 4A 16:45 ‐ 18:45 PT15.12. It is the frequency that matters HaiLong Wang; YiHua Wang; Gregory Worrell Mayo Foundation, Rochester, USA Introduction: All living cells release extracellular vesicles (EVs) as a means of intercellular signaling to achieve diverse biological functions. The potential advantages of utilizing EVs in diagnostic and therapeutic applications are widely recognized and rapidly expanding, although the molecular mechanisms underlying their release remain elusive. Four years ago, we proposed a hypothesis in a preprint paper (bioRxiv‐566448) that exposure to electromagnetic waves affects cells releasing EVs and their cargos. As proof of concept, we demonstrated how low‐frequency electrical stimulation could alter the size distribution of EVs released from cultured rat astrocytes. The major technical difficulty at that time was the lack of a stimulation device that uniformly distribute electric fields. Methods: Since our initial report, we have modified our experimental protocol in several ways: cultivating cells in the fibercell bioreactor so that cells grow in a near‐natural 3D environment; designing a novel device that fits the bioreactor; avoiding the use of the ultracentrifugation method that often damages EVs; and conducting proteomics studies on EVs as a reference in selecting target proteins. Results: Here, we present our latest work showing that a low‐frequency at 2 Hz electromagnetic waves enhance the release of larger‐sized micro‐vesicles. Summary/Conclusion: These results may have significant implications for fundamental biology and medical applications. First, they raise intriguing questions regarding how endogenous electrical activity might modulate EVs. Second, they suggest a novel mechanism for tuning therapeutic electrical stimulation for the treatment of human brain disorders. Third, they provide a new approach for generating EVs with desired cargos by adjusting stimulation parameters. Unlike other chemical methods used to generate EVs, electrical stimulation is a clean method with a broad range of adjustable parameters, including stimulation frequency, field strength, and waveform morphology. Funding: Brain Initiative NIH NS 112144 keywords: electrical stimulation, frequency, TNFRSF10B, Fibercell system PT16: Immunity, Autoimmunity & Inflammation 2 Chairs: Loren Erickson, Paola Decandia Location: Hall 4A 16:45 ‐ 18:45 PT16.12. Alteration of protein cargo in Lacticaseibacillus rhamnosus‐derived membrane vesicles population during the growth of bacterial culture Kristyna Turkova ^1; Martin Sindelar^2; Miriam Sandanusova^3; Jan Kotoucek^4; Gabriela Ambrozova^5; Lukas Kubala^5 ^1International Clinical Research Center, St. Anne's University Hospital Brno, Czechia, Brno, Czech Republic; ^2Faculty of Science, Masaryk University, Brno, Czechia, Czech Republic; ^3Faculty of Science, Masaryk University, Brno, Czechia, Brno, Czech Republic; ^4Department of Pharmacology and Toxicology, Veterinary Research Institute, Brno, Czechia, Brno, Czech Republic; ^5Institute of Biophysics of the Czech Academy of Sciences, Brno, Czechia, Brno, Czech Republic Introduction: Gram‐positive bacteria produce membrane vesicles (MV) as a crucial intercellular communication tool. MV are often isolated and characterized in just one selected time point of the growth phase. We hypothesize that protein cargo associated with purified Lacticaseibacillus rhamnosus‐derived MV is altered during the growth. Methods: Lacticaseibacillus rhamnosus CCM7091 was cultivated aerobically in MRS broth at 37°C. MVs were harvested at different time points of the growth curve ‐ early exponential (6h), late exponential (12h), stationary (24h) and death phase (48h) ‐ and isolated by ultracentrifugation‐based floatation within the sucrose cushion, followed by the characterization using MADLS, cryo‐EM, and WB (LTA marker). The protein cargo was determined by BCA, proteomics and bioinformatics analyses (PSORTb, KEGG Mapper, ggVennDiagram and ComplexHeatmap in R). Results: The morphologies of Lacticaseibacillus rhamnosus‐derived MVs harvested in four different time points were observed by cryo‐EM, indicating a bi‐lipid layer surrounding vesicles. All samples contain approx.10^13 particles per ml, two populations of particles (peak maxima around 30nm and 100 nm), and a protein concentration of around 300ug per ml. Proteomic analyses reveal that subcellular protein localization and functional protein families did not indicate qualitative differences between the samples isolated from different time points. Nevertheless, the heat map visualizing protein intensities shows significant quantitative differences between samples in selected protein clusters (f.e. genetic information processing proteins). Summary/Conclusion: Lacticaseibacillus rhamnosus‐derived MV harvested at different time points of growth showed similar qualitative protein cargo regarding subcellular localization and functional protein families. Interestingly, MV harvested during bacterial culture growth showed significant quantitative differences in protein cargo, indicating that the harvesting time of bacterial MV could be essential for function studies and application of MV. Funding: Supported by the European Regional Development Fund‐Project INBIO‐No. CZ.02.1.01/0.0/0.0/16_026/0008451. keywords: Lacticaseibaciillus rhamnosus, protein cargo, proteomics PT17: Cancer Microenvironment 1 Chairs: Laura Hueser, Muller Fabbri Location: Hall 4A 16:45 ‐ 18:45 PT17.12. Engineered extracellular vesicles drive immunomodulation and hinder tumor progression in a murine model of breast cancer Ana I. Salazar Puerta ^1; Silvia Duarte‐Sanmiguel^2; Ana Panic^2; Daniel Dodd^2; Diego Alzate‐Correa^2; Lilibeth Ortega Pineda^2; Luke Lemmerman^2; María A. Rincon‐Benavides^3; William Lawrence^2; Sandra Paola Santander^4; Binbin Deng^2; David W. McComb^2; William E. Carson^2; Natalia Higuita‐Castro^5; Daniel Gallego‐Perez^6 ^1The Ohio State University, COLUMBUS, USA; ^2The Ohio State University, USA; ^3Biophysics program and Biomedical Engineering Department, The Ohio State University, USA; ^4Juan N. Corpas University Foundation, Colombia; ^5Department of Biomedical Engineering and Department of Surgery, The Ohio State University, USA; ^6Biomedical Engineering and General Surgery, The Ohio State University, USA Introduction: Myeloid‐derived suppressor cells (MDSCs) are immature innate immune cells that infiltrate the tumor to exert immunosuppressive activity and protect cancer cells from the host's immune system and/or cancer‐specific immunotherapies. There is a need for approaches that selectively target MDSCs, to effectively eliminate their “tumor‐protecting” activity and/or drive an “anti‐tumor” phenotype. Here we report the development of engineered extracellular vesicles (EVs) with the potential to target MDSCs to promote “anti‐tumor” responses in a murine model of breast cancer. Methods: EVs decorated with ICAM‐1 ligand and loaded with miR‐146a and Glut1 (engineered EVs), were biosynthesized in vitro, after the transfection of embryonic fibroblasts, or in vivo after the transfection of the skin surrounding the tumor using Tissue‐Nanotransfection (TNT) that mediate the release of engineered EVs. Isolation of EVs was performed using centrifugation and Total exosome reagent. Their content and surface decoration were characterized using qRT‐PCR and Western Blot and size distribution were obtained via NanoSight and Cryo‐EM. Engineered EVs were administered to tumor‐bearing mice weekly for up to 5 weeks and tumor progression was evaluated via qRT‐PCR, flow cytometry, and histology. Results: Our results indicate that ICAM‐1 decorated EVs target MDSCs. Weekly administration of the engineered EVs (via injection or TNT) hampered tumor progression compared to ICAM‐1 decorated EVs with no cargo. Flow cytometry analyses of the tumors indicated a shift in the phenotype of the immune cell population toward a more pro‐inflammatory state, which appeared to have facilitated the infiltration of tumor‐targeting T cells and was associated with a reduction in tumor size and decreased metastatic burden. Summary/Conclusion: This approach shows the potential of using engineered EVs to selectively drive the activity of tumor‐resident myeloid cells from an anti‐inflammatory to a pro‐inflammatory (“anti‐tumor”) phenotype that can help reactivate the immunosurveillance in the tumor niche. Altogether, our findings suggest that ICAM‐1‐decorated engineered EVs could be a powerful platform nanotechnology for the deployment of immune cell‐targeting therapies to solid tumors. Funding: Funding was partly provided by NIH grants DP1DK126199, and DP2 [44]EB028110‐01 awarded to DGP keywords: Engineered EVs, immunomodulation, solid tumors, nonviral gene delivery, tissue nanotransfection PT17.13. Isolation of neuron‐derived extracellular vesicles from biological fluids for the diagnosis of neurological disorders Igor V. Kurochkin; Lausonia Ramaswamy Sysmex Co., Kobe, Japan Introduction: The molecular content of extracellular vesicles (EVs) largely reflects the content of cells of origin and thus analysis of neuron‐derived EVs (NDEs) present in plasma offers a possibility of minimally invasive diagnosis of neurological disorders. Here we tested several membrane proteins as potential targets for the selective isolation of NDEs from blood plasma and CSF. Methods: Isolation of NDEs was performed from plasma, CSF and conditioned cell culture media using various combinations of ExoQuick® Exosome Isolation Kit, ultrafiltration, size‐exclusion chromatography (SEC) and immunocapture using antibodies against surface markers. Quantification and size determination of EVs was performed using nanoparticle tracking analysis (NTA). Detection of proteins was done using Western blot. Results: First, we tested the utility of neuronal marker L1CAM, the most popular target for NDE isolation. Direct immunocapture of L1CAM from total plasma produced significantly higher amounts of L1CAM compared to ExoQuick‐purified EVs as a starting material. We could not precipitate any L1CAM from the SEC‐purified EVs. Immunocaptured plasma L1CAM material did not contain EV (CD9, flotillin‐1) and neuronal (NF‐M, APP) markers as assessed by Western blot. To elucidate the molecular origin of L1CAM, plasma and media conditioned by various cell lines were fractionated by SEC or dual functionality chromatography on Capto™ Core 700. The vast majority of L1CAM was found to be present in a free protein fraction. The protein had the size indistinguishable from the cell‐bound L1CAM and reacted with antibodies specific for the extra‐ and intracellular parts of the protein. It represents likely, therefore, a product of the recently described splicing isoform of secreted full‐length L1CAM lacking the transmembrane domain. We devised a computational workflow to produce a list of additional potential targets for NDE isolation. Here we validated one such target encoded by ATP2B2 gene. Summary/Conclusion: Here we provide the list of surface proteins, potential targets for NDE isolation. Immunocapture with anti‐ATP2B2 could be used for NDE isolation. Funding: Sysmex Co. (Japan) keywords: neuro‐derived extracellular vesicles; isolation; plasma; CSF Friday 19 May 2023 Late Breaking Poster PF03: Cancer EV Pathogenesis Chairs: Sameh Almousa, Janusz Rak Location: Hall 4A 16:00 ‐ 18:00 PF03.12. CXCR4 is a novel crosslinker to control ciliogenesis and cancer cell death Tae‐Kyu Jang; Eun‐Yi Moon Sejong University, Republic of Korea Introduction: The primary cilia (PC) are microtubule‐based organelles that help detect and transmit environmental signals. Ciliogenesis is influenced by Ciliary extracellular vesicle (EV) formation. Chemokine receptor CXCR4 is required for ciliogenesis in Kupffer's vesicle. Methods: Here, we investigated whether CXCR4 could affect cancer cell growth and migration via ciliogenesis enhanced by hedgehog signaling using HeLa human cervical cancer cells and anticancer drug like vinblastine (VBL). Results: Cell growth inhibition by VBL was reduced under serum‐deficiency (SD) condition which increase ciliogenesis. VBL enhanced CXCR4 expression, which was more increased under SD condition to activate hedgehog signaling compare to 5% FBS. CXCR4 expression was increased by the treatment with smoothened agonist (SAG). Cell death by VBL was decreased by the overexpression of pEGFP‐SMO or SAG treatment increasing PC formation. In addition, SDF‐1 (CXCL12), CXCR4 ligand, increased PC formation, leading to the decrease in cell death by VBL. Increased cell migration was also observed by the treatment with SAG or SDF‐1. Survived cells from VBL treatment repeatedly migrate much rapidly compared to wildtype cells. PC formation and cell death by VBL or cell migration were attenuated by the inhibition of CXCR4 expression with siRNA. Summary/Conclusion: Taken together, data demonstrate that VBL‐mediated cancer cell death could be regulated by ciliogenesis via hedgehog signaling and CXCR4 expression. It suggests that CXCR4 could be a novel crosslinker to control ciliogenesis and cancer cell death. Funding: This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (grant number 2021R1A4A5033289). PF03.13. EMT‐promoter sEV‐miRs enriched in sEV‐MDA231 increase the migration capacity of human normal breast epithelial cells and non‐metastatic breast cancer cells Eduardo F. Durán‐Jara ^1; Nicole Farfán^2; Marcelo Ezquer^1; Lorena Lobos‐González^1 ^1Universidad del Desarrollo, Las Condes, Chile; ^2Universidad del Desarrollo, Chile Introduction: Tumor‐derived sEVs acts as vehicles for several oncogenic miRNAs (sEV‐miRs) that can regulate gene expression when incorporated by recipient cells. Delivery of sEV‐miRs and dysregulation of the miRNA profile of recipient cells can promote tumorigenic and pro‐metastatic processes such as migration and epithelial‐to‐mesenchymal transition (EMT). However, little is known about the specific sEV‐miRs responsible for the promotion of these processes in breast cancer (BC). Methods: We isolated sEVs from different BC cells lines and characterized their miRNA profile through small RNA‐seq. EMT‐promoter candidate sEV‐miRs were identified in sEVs secreted by the triple‐negative, metastatic and highly mesenchymal MDA‐MB‐231 cells (sEV‐MDA231) and validated through miQPCR. MCF10A mammary epithelial and T47D BC cells were co‐transfected with the EMT‐promoter sEV‐miRs mimics or incubated with sEV‐MDA231. EMT‐related markers were measured through qPCR and western blot. Finally, the migration capacity of transfected/recipient cells was assessed through a transwell migration assay. Results: Co‐transfection of the validated EMT‐promoter sEV‐miRs mimics in MCF10A cells, but not in T47D BC cells, increased gene expression and protein levels of some EMT‐related markers such as Vimentin, N‐Cadherin and Fibronectin. Treatment with sEV‐MDA231 promoted similar changes. Interestingly, co‐transfection of sEV‐miRs mimics increased the migration capacity of both, MCF10A “normal” and T47D BC cells; increment even higher than the achieved by treatment with sEV‐MDA231. Summary/Conclusion: Our results shows that specific sEV‐miRs can mediate the promotion of cell migration and EMT in non‐tumorigenic cells. This suggest that sEV‐miRs or their targets can be used as therapeutic options/biomarkers in BC. Funding: Fondap‐15130011, Fondecyt‐1211223, Fondef‐ID21I10210, Fondequip‐EQM160157 and ‐EQM190110, Basal Funding CMR‐UDD, Internal Grant UDD‐23400174, PhD Scholarship‐UDD, National PhD Scholarship‐ANID. keywords: BC: breast cancer; sEV: small extracellular vesicles; miR: microRNA; EMT: epithelial‐mesenchymal transition PF03.15. Extracellular vesicular MiR‐155‐5p‐mediated ibrutinib resistance in B‐cell lymphoma Bon Park ^1; Myung Eun Choi^2; Kyung Ju Ryu^3; Jung Yong Hong^4; Won Seog kim^5; Chaehwa Park^5; Seok Jin kim^5 ^1Sungkyunkwan University, Republic of Korea; ^2Sungkyunkwan university, Seoul, Republic of Korea; ^3Sungkyunkwan University, Seoul, Republic of Korea; ^4Sungkyunkwan university, Samsung medical center, USA; ^5Sungkyunkwan University, Samsung medical center, Seoul, Republic of Korea Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common aggressive B‐cell lymphoma, and ibrutinib, an orally administered BTK inhibitor targeting B‐cell has been tried as a treatment for DLBCL. However, ibrutinib resistance is a problem because patients become resistant to ibrutinib during continuous administration. Methods: To explore the underlying mechanism for ibrutinib‐resistance, we analyzed extracellular vesicles (EVs) derived from ibrutinib‐resistant DLBCL cell lines (OCI‐Ly1‐IbtR) because EVs might contribute to ibrutinib resistance via cell‐to‐cell communication. We established ibrutinib‐resistant DLBCL cell lines continuously exposed to 1uM, and 2uM ibrutinib and isolated EVs from OCI‐Ly1‐IbtR through ultracentrifugation. The analysis of miRNA profiles in OCI‐Ly1‐IbtR cell and EVs were done by Nanostring. The signaling pathways and relative mRNA levels of OCI‐Ly1‐IbtR cell and EV were analyzed by Western blot and qRT‐PCR. Results: The OCI‐Ly1‐IbtR‐derived miRNA profiles showed 118 up‐regulated and 36 down‐regulated miRNAs compared to ibrutinib‐sensitive DLBCL (OCI‐Ly1). Among them, miR‐155‐5p increased in OCI‐Ly1‐IbtR and EVs, and intracellular levels were also higher than in OCI‐Ly1. The analysis of cellular signaling also showed significant activation of PI3K‐AKT, P70S6K, MAPK, and NF‐κB signaling pathways in OCI‐Ly1‐IbtR and EVs. In addition, the PI3K‐AKT, P70S6K, MAPK, and NF‐κB signaling pathways also increased in miR‐155‐5p over‐expressed cells. We found target mRNAs of miR‐155‐5p, KDM5B and DEPTOR, and confirmed that the mRNAs were the actual target of miR‐155‐5p through Ago2 IP and Luciferase. Thus, the EV miR‐155‐5p may be related to ibrutinib resistance by targeting mRNAs. Summary/Conclusion: EV miR‐155‐5p might have a role in the acquaintance of ibrutinib‐resistance in patients with DLBCL. Funding: This research was supported by a National Research Foundation of Korea grant funded by the Korean government (2021R1A2C1007531) PF03.16. Uncovering the Pro‐Tumorigenic role of Extracellular vesicle‐associated miR‐183‐5p in Ovarian Cancer Metastasis Shikha Rani^1; Shayna Sharma^2; Priyakshi Kalita ‐ de Croft^2; Dominic Guanzon^2; Lewis Perrin^3; Tanja Pejovic^4; Terry K. Morgan^5; John Hooper^6; yaowu He^3; Andrew Lai ^2; Carlos Salomon^2 ^1Translational Extracellular Vesicles in Obstetrics and Gynae‐Oncology Group, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4029, Australia, Herston, Australia; ^2Translational Extracellular Vesicles in Obstetrics and Gynae‐Oncology Group, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4029, Australia, Brisbane, Australia; ^3Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia., Brisbane, Australia; ^4Departments of Obstetrics and Gynecology and Pathology, OHSU, Portland, USA; ^5Department of Obstetrics and Gynecology, OHSU, Portland, OR, USA., Oregon, USA; ^6Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia., Brisbane, USA Introduction: Small extracellular vesicles (sEVs) are crucial in intercellular communication and signalling pathways that drive the progression of ovarian cancer. Unfortunately, despite being a highly lethal gynaecological cancer, the underlying mechanisms behind ovarian cancer remain poorly understood, and many patients are diagnosed only in advanced stages. However, studies have highlighted the potential role of sEVs in promoting tumour progression by transferring miRNAs to target cells and triggering tumorigenic behaviours. As such, our research proposed that sEVs derived from the plasma of ovarian cancer patients may activate EMT, promote cell migration, and ultimately lead to cancer metastasis. Methods: sEVs were isolated from the plasma of women with benign ovarian disease and epithelial ovarian cancer, and we analysed their size, levels of sEV‐associated proteins, and morphology using various techniques, including nanoparticle tracking analysis, Western Blot, and electron microscopy. We then examined changes in the sEV miRNA and protein profile by conducting small RNA sequencing using the Illumina NextSeq 500 and quantitative proteomic analysis. To further investigate miRNA expression, we quantitatively analysed tissue microarrays (TMAs) of ovarian cancer patients using in situ hybridization (ISH). Tumor growth in control and exosomal miR‐183‐5p‐treated mice was monitored weekly using bioluminescence imaging on an IVIS spectrum. Results:: A specific set of miRNAs with differing expression levels between cancer‐derived sEVs and benign‐derived sEVs were identified by small RNA sequencing. In vitro studies have demonstrated that cancer‐derived sEVs can alter the expression of genes associated with EMT in target cells, as detected by the EMT RT^2 Profiler PCR Array. Further analysis revealed that miR‐183‐5p overexpression and quantitative proteomics studies indicate association between Wnt5a signalling and EMT. This association led to upregulation of STAT1 and downregulation of EPCAM, resulting in enhanced cell migration. Tissue microarray analysis has also demonstrated higher expression levels of miR‐183‐5p and Wnt5a in ovarian cancer cells compared to benign controls, underscoring their potential significance. The tumor burden quantification in mice demonstrated an increase in tumor size when treated with exosomal miR‐183‐5p. Summary/Conclusion: Our research has revealed that sEVs found in the plasma of ovarian cancer patients carry bioactive substances that promote pro‐tumorigenic changes in target cells. Specifically, miR‐183‐5p encapsulated sEVs has a targeted effect on cancer cells, increasing their migratory capacity and potential for metastasis. Therefore, targeting miR‐183‐5p in sEVs may prove effective in preventing ovarian cancer cell spread. PF04: Therapy Adipose and MSC EVs 1 Chairs: Vera Tscherrig, Yu Fujita Location: Hall 4A 16:00 ‐ 18:00 PF04.09. Local administration of extracellular vesicles from bone marrow‐derived mesenchymal stem cells restores homeostatic communication pathways and slows the progression of retinal degeneration Yvette S M Wooff^1; Xenia Sango^2; Smriti Krishna^2; Adrian Cioanca^1; Nathan Reynolds^3; David N. Haylock ^4; Riccardo Natoli^1 ^1The Australian National University, Eccles Institute of Neuroscience, Canberra, USA; ^2VivaZome Therapeutics, USA; ^3The Australian National University, Eccles Institute of Neuroscience, USA; ^4VivaZome Therapeutics, MELBOURNE, Australia Introduction: Retinal degenerations, including Age‐related macular degeneration (AMD), are a group of neurodegenerative diseases characterised by irreversible vision loss. In the degenerating retina, we have previously shown that a loss of extracellular vesicle (EV) bioavailability is correlated with key pathological features of AMD including photoreceptor cell death and large‐scale inflammatory cascades. We therefore hypothesise that replenishment of EV from cells shown previously to have therapeutic benefits to the eye, such as bone marrow‐derived mesenchymal stem cell EV (BM‐MSC‐EV), and their molecular cargo, may restore these lost EV homeostatic communication pathways and slow the progression of degeneration. Methods: Human BM‐MSC‐EV (RoosterBio) were isolated using tangential flow filtration (TFF) and size exclusion chromatography (SEC) and characterised using Nanotracking Analysis (NTA; ZetaView QUATT). BM‐MSC‐EV or vehicle (PBS) were administered to the retina via intravitreal injection at a dose of 2.0×109 EV/μL (1μL) prior to degeneration using a well‐established photo‐oxidative damage model of retinal degeneration (100k lux bright white‐light for 5 days). Electroretinography and optical coherence tomography were used to measure retinal function and morphology respectively following degeneration, while TUNEL and IBA‐1+ immunohistochemistry was conducted on retinal cryosections to determine levels of cell death and inflammation in the retina. Results: Compared to PBS‐injected controls, mice injected with BM‐MSC‐EV had significantly higher retinal function, reduced presence of infiltration and activated microglia/macrophages as a measure of inflammation, and decreased levels of retinal cell death. Summary/Conclusion: Taken together, our data supports BM‐MSC‐EV as a potential therapeutic EV source to slow the progression of retinal degeneration, possibly providing a novel EV source for delivery of current and future therapeutics. keywords: retinal degeneration, BM‐MSC PF04.10. Superiority of umbilical cord‐derived mesenchymal stem cell extracellular vesicles: A comparison of yield, immunosuppression, regeneration, and SARS‐CoV‐2 inhibition with adipose and bone marrow sources Somchai Chutipongtanate ^1; Nutkridta Pongsakul^2; Jirawan Panachan^2; Supasek Kongsomros^3; Pukkavadee Netsirisawan^2; Wararat Chiangjong^2; Ladawan Khowawisetsut^4; Primana Punnakitikashem^5; Arunee Thitithanyanont^6; Kovit Pattanapanyasat^7; Suradej Hongeng^2 ^1Division of Epidemiology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, USA; ^2Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, Thailand; ^3Department of Pediatrics and Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, Thailand; ^4Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Thailand; ^5Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Thailand; ^6Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand, USA; ^7Excellence for Microparticle and Exosome in Diseases, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Thailand Introduction: Current evidence supports the therapeutic effects of mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) on immunomodulation, regeneration, and SARS‐CoV‐2 infection. However, the most suitable MSC source for MSC‐EV therapy remains unknown. Methods: This study systematically compared MSC‐EVs from three commercial MSC sources (ATCC): adipose (AD), bone marrow (BM), and umbilical cord (UC) tissues. MSC‐EVs were isolated from 100 mL culture media (supplemented with exosome‐free FBS) using a combination of centrifugation, ultrafiltration, and qEV size exclusion chromatography, yielding a final volume of 500 μL. Particle and protein characterizations followed MISEV2018 guidelines. Yield was determined by vesicle count per 100 mL culture media. Cellular assays evaluated MSC‐EV activities, including immunosuppression via CD4+CD25+CD127dimFoxP3+ T regulatory cell (Treg) differentiation from human peripheral blood mononuclear cells (protocol ID COA. MURA2020/1277), regenerative activity through wound healing‐scratch assays on human fibroblasts, and anti‐SARS‐CoV‐2 effects by measuring nucleoprotein expression reduction in infected human respiratory epithelial cells. Results: Transmission electron microscopy, nanoparticle tracking analysis, and Western immunoblotting confirmed the presence of a small subpopulation of MSC‐EVs in the isolates. Yield – UC‐derived MSCs provided two‐to‐four times higher EV production compared to AD‐ and BM‐derived MSCs. MSC‐EV activities were measured at concentrations of 100, 1,000, or 10,000 particles per cell. Immunosuppression – UC‐derived MSC‐EVs demonstrated a higher Treg induction capability than AD‐ and BM‐derived MSC‐EVs. Regeneration – MSC‐EVs from AD, BM, and UC sources exhibited comparable wound healing capabilities. SARS‐CoV‐2 inhibition – UC‐derived MSC‐EVs displayed the strongest anti‐SARS‐CoV‐2 effect. Summary/Conclusion: Our findings suggest that UC‐derived MSCs are the optimal source for MSC‐EV production. With their superior immunosuppressive and anti‐SARS‐CoV‐2 properties, as well as regenerative capabilities comparable to other sources, UC‐derived MSC‐EVs demonstrate considerable potential for clinical applications in immune‐mediated diseases, regenerative medicine, and COVID‐19 treatment. Funding: The Office of National Higher Education Science Research and Innovation Policy Council (PMU‐B), Thailand, grant numbers B17F640004, B05F630082, and B17F640005. National Research Council of Thailand (NRCT): High‐Potential Research Team Grant Program, grant number N42A650870. keywords: COVID‐19; MSC‐EVs; Stem cell‐free therapy; Treg immunomodulation; Wound healing PF04.11. The therapeutic efficacy of exosomes derived from mesenchymal stem cell in a murine model of asthma Jin Yoon ^1; Arum park^1; Eom Ji Choi^1; Eun Young Paek^1; Eonyong Han^2; Ji Youn Lim^1; Seong‐Jin Kim^1; Inuk Jung^2; Gyeong Joon Moon^1; Jinho Yu^3 ^1Asan Medical Center, Republic of Korea; ^2Kyungpook National University, Republic of Korea; ^3Asan Medical Center, University of Ulsan College of Medicine, Republic of Korea Introduction: Asthma is a chronic inflammatory airway disease. In recent years, asthma therapy targeted to a specific signaling molecule has shown promise as a novel strategy to treat asthma, however, there are some limitations because asthma is a heterogeneous disease. Mesenchymal stem cells‐derived extracellular vesicles (MSC‐EVs) can modulate the immune response through multiple molecular pathways. Here, we examined the effects of MSC‐derived exosomes in a murine model of asthma and peripheral blood mononuclear cells (PBMCs) from children with asthma. Methods: To evaluate the effects exosome have in an allergic asthma model, BALB/c female mice were sensitized and challenged with ovalbumin (OVA), with MSC‐EVs being administered intratracheally. The animals were then euthanized, and their lungs and bronchoalveolar lavage fluid (BALF) were collected. Inflammatory cells were assessed via quantification of differential cells counts in the BALF and lung tissues triply stained with hematoxylin and eosin, periodic acid‐Schiff, and Masson's trichrome staining. PBMCs of 6 children with asthma were stimulated with PHA, and were treated with MSC‐EVs for 48 hours. Results: In OVA‐induced animals, MSC‐EVs administration significantly reduced the number eosinophils and neutrophils in BALF. Histological studies showed that exosome treatment markedly decreased inflammatory infiltration, mucus secretion, and collagen deposition in the lung tissues. In addition, we discovered novel miRNAs from the exosomes that associated with the change of mRNA and protein in lung tissues of asthma mouse model. Furthermore, we show that the levels of IFN‐γ and IL‐13 were dose dependently regulated by MSC‐EVs in PBMCs from children with asthma. Summary/Conclusion: Our findings in a mouse model of asthma suggest that MSC‐EVs alleviate airway inflammation and may potentially represent a novel therapeutic strategy for the treatment of asthma. keywords: Asthma, Mesenchymal stem cell, exosome, miRNA, PBMC PF05: Kidney and Binary EVs 2 Chairs: Wouter Woud, Laura Perin Location: Hall 4A 16:00 ‐ 18:00 PF05.09. Technical and clinical assessment of a practical urine EV method to measure complement‐mediated kidney damage Ellen E. Millman ^1; Elena Tarabra^2; Tobin J Cammett^2 ^1Alexion RDU of Astrazeneca, New Haven, USA; ^2Alexion RDU, New Haven, USA Introduction: Introduction: Acute kidney injury (AKI) leading to chronic organ damage can be triggered by dysregulation of the immune system's complement cascade. Kidney biopsy is currently the only method to assess tissue damage due to complement pathway activation but cannot be used to monitor disease progression or local tissue response to treatment. To address this serious unmet clinical need, we have developed a relatively simple, scalable method for high‐throughput testing of patient‐derived urine extracellular vesicles (uEV) to non‐invasively monitor complement‐mediated renal injury before, during, and after therapeutic intervention. Methods: Method: Patient urine is collected, cleared by low speed centrifugation (900 x g 15 min 4°C) and frozen at < ‐70°C. For testing, the thawed urine undergoes simultaneous immunoprecipitation/immunoanalysis to enrich for tissue‐specific uEVs using the Luminex® xMAP® Technology platform. We use an array of xMAP beads coated with antibodies against tetraspanins CD9, CD63, CD81 and against defined regions of the nephron: podocalyxin (PODXL) for the glomerular podocytes or aquaporin 2 (AQP2) for the convoluted tubules. We then probe each bead‐bound EV for deposition of key proteins in the complement cascade shed from the plasma membrane (PM) of tissue‐specific regions of the kidney. Results: Results: Compared to normal controls, patients with renal disease due to complement dysregulation have elevated deposition of Terminal Complement Complex (TCC or C5b‐9 composed of C5b, C6, C7, C8, and multiple C9) on their PODXL+ EV and for some patients also on AQP2+ EV. Summary/Conclusion: Conclusion: We can leverage uEVs as a patient‐friendly, rapid biomarker method to monitor localized complement activity in the nephron for prognostic and repeated longitudinal assessment of response to various therapies including the many novel complement inhibitor treatments being developed. Funding: Funding: This project was funded by Alexion Pharmaceuticals, AstraZeneca Rare Disease. keywords: Complement, TCC, CM‐TMA, Renal Disease PF06: Outreach and Knowledge Synthesis Chairs: Marta Monguio Tortajada, Roger Alexander Location: Hall 4A 16:00 ‐ 18:00 PF06.05. Using a scientometric approach to define a quantitative understanding of the EV field Liam P. Hourigan ^1; William Phillips^2; Amirmohammad Nasiri Kenari^3; Krishna Chaitanya Pavani4^4; An Hendrix^5; Andrew F. Hill^6 ^1La Trobe University, Brunswick, Australia; ^2La Trobe Institute for Molecular Science, Bundoora, Australia; ^3The University of Tokyo, USA; ^4University of Ghent, USA; ^5Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Ghent, Belgium; ^6Institute for Health and Sport, Victoria University, Melbourne, Australia Introduction: The extracellular vesicle (EV) field has seen explosive growth over the last decade, sprawling outwards into ever‐diversifying subfields and research interests. Thus, maintaining a broad and generalized knowledge of the state of the field is a challenge for researchers. Conventional methods of knowledge consolidation, particularly narrative reviews, are ill‐suited towards capturing such a breadth of newly generated knowledge. Applying a quantitative methodology, namely scientometric analysis, allows for a wide‐ranging overview of the EV field, defining its intellectual structure. Methods: A dataset of 52,286 articles published between 1987–2023 and relating to EVs was downloaded from Web of Science, then enriched with additional data from Dimensions and the EV‐Track knowledgebase. This dataset was analysed using the programs CiteSpace, Excel and Bibliometrix. This involved generating a co‐citation network and performing ‘clustering’ using CiteSpace, then analysing clusters and the entire dataset with Excel and Bibliometrix before synthesizing the data qualitatively. Results: Co‐citation analysis paired with clustering using publications between 2015–2023 derived 17 ‘research focuses’, providing a recent snapshot of the field. Across these research focuses and the data set as a whole, a mixture of quantitative and qualitative analyses identified research fronts, or ‘hot topics’, highlighting specific and recent research interests across clusters. Nomenclature usage was examined, evidencing an overall upwards trend in compliance with MISEV recommendations (i.e. generally using EV over biogenesis terms), however certain clusters appear less compliant. Trends in translational vs basic research were identified, finding certain clusters to be more advanced towards translational ends. Overall, the number of articles with EV‐Track data was low (2‐30%) across clusters, with a significant degree of variability. Summary/Conclusion: Scientometric methodology has thus far been underutilized as a tool for understanding the EV field as a whole. Researchers can gain a better understanding of wide‐reaching trends by supplementing their perspectives with a quantitative lens. These analyses are of particular value to overarching organizations such as ISEV which effortfully shape the intellectual structure of a field. PF06.06. Bibliometric analysis of extracellular vesicles research in lung cancer management Adeel Khan ^1; Tauseef Ahmad^2; Haroon Khan^3; Nongyue He^1; Zhiyang Li^4 ^1State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China., China (People's Republic); ^2Vanke school of public health, Tsinghu University, Beijing 10084, China,, Pakistan; ^3Med‐X research institute, School of Biomedical engineering, Shanghai Jiao Tong University, Shanghai 200240, China (People's Republic); ^4Department of Clinical Laboratory, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, P. R. China., Nanjing, China (People's Republic) Introduction: Extracellular vesicles (EVs) are nanosized vesicles with a phospholipid bilayer membrane loaded with physiologically and pathologically active cargo such as nucleic acids, protein, metabolites and lipids. The majority of cell types release EVs. However, EVs released during specific pathological conditions can be vital from the diagnostic and therapeutic points of view. EVs are researched extensively in oncology for various cancers, including lung cancer. Data are generated in the form of hundreds and thousands of articles. It is pertinent to analyze the data subjectively and objectively. To accomplish this task, the most reliable and widely used technique is Bibliometric analysis. This technique provides a better understanding of the huge scientific data gathered over the years. We can track the advances in a particular research area through this technique and predate the crucial gaps and important research directions. Methods: To date, no bibliometric analysis has been performed to specifically analyze EVs‐based research advances for lung cancer management. To fulfill this gap, we selected web of science core collection (WoSCC), using search terms such as “Extracellular Vesicles” OR “Exosomes” OR “Microvesicles” and “Lung Cancer”. To further perform Bibliometric analysis such as authors, journals, citations, affiliations, keywords visualization and mapping, we used VOSviewer version 1.6.0 and R studio version 3.6.2. Results: A total of 1095 publications on EVs were analyzed and characterized. These publications were written by 5801 authors and published in 408 peer reviewed journals between 2004 and 2021. Generally, the number of publications has increased over the years. The annual scientific production growth was found to be 41.83%. The leading author, journal, institution and country of origin based on number of publications in the area of EVs and lung cancer were Li J (n = 22), Cancer (n = 46), Nanjing Medical University (n = 38), and China (n = 472), respectively. Important findings of the analysis suggest cancer biomarker, early detection, microRNA, EGFR (epidermal growth factor receptor), drug delivery, personalized medicine,immunotherapy, brain metastasis as keywords in EVs based research advances in lung cancer management, interestingly the new keywords that appear post‐2020 is gene therapy, and PD‐L1 (programmed death ligand 1). PD‐L1 is an important tumor marker and a focus of attention for Anti‐PD‐1/PD‐L1 immunotherapy. Summary/Conclusion: We are optimistic that our analysis will fill the gap of bibliometric analysis of EVs based research advances for lung cancer management and can be valuable for making targeted EVs based research advances aiming at diagnosis or treatment of lung cancer. Funding: This study received no external funding keywords: Lung cancer, Extracellular vesicles, Bibliometric Analysis, diagnosis, treatment. PF06.07. A new collaborative data analysis platform for exosome research: exosome.ai Tae Hyun Yoon ^1; Sook Jin Kwon^2; Eun yong Ha^3; Haribalan Perumalsamy^3; Zayakhuu GERELKHUU^3; Sneha Sunderraj^3; Seung Guen Park^3 ^1Hanyang University, Republic of Korea; ^2Yoon Idea Lab Co. Ltd., Republic of Korea; ^3Hanyang University, Republic of Korea Introduction: Exosomes are a group of extracellular vesicles that are released by various cell types and are present in the majority of biological fluids, such as saliva, blood, and urine. Due to their roles in intercellular communication, and potentials as a tool for drug delivery and disease biomarkers, there has been a great deal of interest, leading to a plethora of studies that generated huge amount of experimental data. Methods: Particularly. as the developments and applications of novel analytical approaches (e.g., transcriptomics, proteomics and metabolomics), high‐dimensional and multiparametric datasets are continuously produced and become available in public databases. Results: However, as the number and size of experimental datasets grows, it becomes more difficult to analyze, interpret, and understand the true meaning of the observations. Therefore, it is necessary to adapt novel tools for data visualization, analysis, and interpretation. Summary/Conclusion: Here, we present a new collaborative data analysis platform for exosome, where dataset curation, exploratory data analysis as well as predictive modelling can be performed. Details of the available exosome datasets, various tools for exploratory data analysis and interpretations, and algorithms for biomarker discovery will be discussed in this presentation. PF07: Cancer Biomarkers 2 Chairs: Nikki Salmond, Caterina Nardella Location: Hall 4A 16:00 ‐ 18:00 PF07.14. Cellular and extracellular microRNA profiles are associated with PD‐L1 expression of extranodal NK/T‐cell lymphoma Kyung Ju Ryu ^1; Bon Park^2; Sang Eun Yoon^3; Won Seog kim^4; Chaehwa Park^4; Seok Jin kim^4 ^1Sungkyunkwan University, Seoul, Republic of Korea; ^2Sungkyunkwan University, Republic of Korea; ^3Samsung Medical Center, Republic of Korea; ^4Sungkyunkwan University, Samsung medical center, Seoul, Republic of Korea Introduction: Extracellular vesicles (EVs) play an important role in cancer progression, including tumor metastasis, drug resistance, and immune system evasion. Extranodal natural killer /T‐cell lymphoma (ENKTL) is an aggressive malignant tumor with a poor prognosis. Immune checkpoint inhibitors, PD‐1/PD‐L1, have been proposed as an alternative treatment option for relapsed or refractory ENKTL. However, some patients do not respond to immune checkpoint inhibitors. In this study, we investigate cellular and EV miRNAs that regulate PD‐L1 and provide for the potential use of EV miRNAs as a predictor for checkpoint inhibitors therapy in ENKTL. Methods: EVs were isolated from drug‐resistant ENKTL cell lines and patient serum by ultracentrifugation and analyzed by transmission electron microscopy, nanoparticle tracking analysis, RT‐qPCR, and Western blot. To identify miRNAs that regulate PD‐L1 expression, we performed Nanostring nCounter® microRNA arrays and miRNA target prediction database, argonaute2 immunoprecipitation, luciferase reporter assay, and immunohistochemistry. Results: Among the significantly down‐regulated miRNAs, miR‐340‐5p was predicted as a possible target of PD‐L1. RT‐qPCR and western blot analysis revealed that miR‐340‐5p expression was deceased, and PD‐L1 expression was increased in etoposide‐resistant cells compared to control cells. Ago2 IP and luciferase assay showed that miR‐340‐5p was directly bound to the 3′UTR of PD‐L1 mRNA. Overexpression of miR‐340‐5p decreased PD‐L1 expression, and inhibition of miRNA‐340‐5p increased PD‐L1 expression. Additionally, ENKTL patients with higher PD‐L1 expression had lower miR‐340‐5p expression and shorter survival time. Summary/Conclusion: Our results demonstrated that miR‐340‐5p could regulate PD‐L1 expression by targeting PD‐L1 mRNA. These findings suggest that a combination of miR‐340‐5p regulation and PD‐L1 blockade can overcome acquired resistance to immune checkpoint inhibitors. Funding: This research was supported by a National Research Foundation of Korea grant funded by the Korean government (2021R1A2C1007531) and the by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2022R1I1A1A01065286) PF07.15. Discovery of biomarkers of tumor‐derived extracellular vesicles for breast cancer diagnosis Juyong Hyon ^1; Kyung‐A Hyun^2; Min Woo Kim^3; Yeji Yang^1; Seung Il Kim^4; Hyo‐Il Jung^5; Young‐Ho Chung^6 ^1Research Center For Bioconvergence Analysis / Korea Basic Science Institute, Cheongju‐si, Republic of Korea; ^2School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea, Seoul, Republic of Korea; ^3Department of Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea; ^4Department of Surgery, Yonsei Unversity College of Medicine, Seoul, Republic of Korea; ^5Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea; ^6Research Center for Bioconvergence Analysis/ Korea Basic Science Institute, Republic of Korea Introduction: Tumor‐derived extracellular vesicles (tdEVs) have been emerging as potential biomarkers for cancer diagnosis because the tdEVs precisely reflect tumor cell alterations with significantly increased production. The proteomic profiling study of tdEVs represents a promising approach in a non‐invasive manner to novel biomarker discovery for early detection and targeted therapy of cancer. Methods: Previously, we have developed a novel microfluidic chip for rapid and selective isolation of tdEVs. This microfluidic chip enables the selection of two types of EVs by using breast tumor‐derived proteins (EpCAM & CD49f) within two minutes. In this study, we compared and analyzed proteomics of EVs isolated from several breast cancer cell lines by using a commercialized EV isolation kit based on the CD63 marker and our microfluidic chip which can selectively isolate tdEVs with the cancer cell‐specific markers EpCAM & CD49f. Results: A total of 3,700 proteins were identified, and as a result of GO analysis of 474 unique proteins from EVs isolated by CD63, the proteins were related to EV production and transport such as exocytosis and endocytosis. On the other hand, 389 proteins expressed only in tdEVs by GO analysis were found to be related to signal transduction and RNA metabolism. This can be considered to be closely related to the generation of miRNA in EV, which shows a specific expression difference in breast cancer patients. Based on this result, we explored the proteome of tdEVs extracted from the plasma of breast cancer patients and normal persons. As a result of comparative analysis, 179 out of 389 proteins that appear only in tdEVs were identified, and it was confirmed that 103 proteins (about 57.5%) showed significant differences. Summary/Conclusion: In particular, five potential EV markers contributing to the progression and relapse of breast cancer were relatively high in EVs isolated from TNBC patients compared to the normal control group. The best EV protein marker showed excellent performance for discriminating patients with early‐ and advanced‐stage breast cancer from normal control with 83% sensitivity, 80% specificity, and over 0.8 area under the receiver operating characteristic curve; Moreover, several other EV protein markers had similar diagnostic utility. In the future, proposed proteomic biomarkers from tdEVs are expected to serve as a novel assay for diagnosis and management of breast cancer patients. Funding: Korea Basic Science Institute (C270100) funded by the Ministry of Science and ICT keywords: Tumor‐derived extracellular vesicle, Microfluidic chip, Selective isolation, Proteomics, Cancer diagnostics PF07.16. Expression of 4‐1BBL on extracellular vesicles was associated with inhibition of glioma growth in mice Taral R. Lunavat ^1; Lisa Nieland^2; Xandra O Breakefield^1; Koen Breyne^3 ^1Massachussetts General Hospital and Harvard Medical School, Charlestown, USA; ^2Massachusetts General Hospital and Harvard Medical School, USA; ^3Massachusetts General Hospital and Harvard Medical School, Charlestown, USA Introduction: Glioblastoma (GB) is a prototypic solid and antigenically “cold” tumor. GB is largely devoid of tumor‐infiltrating cytotoxic T cell lymphocytes (CTLs) within an immunosuppressive microenvironment in the brain. This renders immune checkpoint inhibitors and gene‐based cytokine treatment ineffective. 4‐1BBL is a membrane‐bound immunostimulant that is normally expressed by tumor‐associated antigen presenting cells (APCs), including dendritic cells (DCs) and B cells, and promotes the activity of CD8+T cell‐mediated immune responses. 4‐1BBL binds to its receptor 4‐1BB, a glycoprotein member of the tumor necrosis factor receptor superfamily. Here, we show that mice implanted with tumors which were then treated with AAV‐F encoding 4‐1BBL or tumor cells overexpressing 4‐1BBL had enriched 4‐1BBL protein on extracellular vesicles (EV) and that expression of 4‐1BBL slowed tumor growth in a murine syngenic glioma model. Methods: Mouse glioma cells (CT2a) cells were stably transduced with a lentiviral vector to express Fluc and used for all subsequent in vivo experiments. To study the effect of 4‐1BBL, in one modality cells were stably transduced with a lentiviral vector encoding 4‐1BBL tagged with fluorescent label ‐ mCherry or the control vector lacking 4‐1BBL. CT2a‐Fluc cells and CT2a‐Fluc‐m41BBL cells were implanted into the brains of syngeneic C57BL/6 mice. In another modality CT2a‐Fluc implanted cells were injected intratumorally with an AAV‐F vector encoding 4‐1BBL. Bioluminescence measuring tumor growth was monitored over time. Brains harvested in the terminal stages were dissociated enzymatically and EVs were isolated by SEC and quantified by NTA. Collected EVs were used to determine the levels of 4‐1BBL, and other EV characterization markers such as CD9, CD81 and Alix by immunoblotting. Results: Orthotopic transplantation of CT2a‐ Fluc and CT2a‐Fluc‐m4‐1BBL cells in syngeneic mouse brains led to tumor growth as detected by in vivo bioluminescence imaging. Brains were harvested from terminally ill mice, the EVs were isolated from either over‐expressing 4‐1BBL tumor cells or from AAVF‐m41BBL treated mice and showed 4‐1BBL enrichment in EVs. These EVs also confirmed the presence of CD81, CD9, and Alix, and were devoid of calnexin, a negative marker for EVs. Summary/Conclusion: Boosting 4‐1BBL expression in glioma cells or tumor‐activated astrocytes through transduction with virus vectors was found on EVs and thus could potentially lead to trans‐presentation to the 4‐1BB receptor on cytotoxic T lymphocytes (CTLs). This could supports the hypothesis of improved survival of glioma‐bearing mice by increased levels of 4‐1BBL in the tumor microenvironment. Funding: Funding: NCI [45]CA232103 (XOB) and Norwegian Research Council ‐ 315566 (TRL) keywords: glioblastoma, tumor microenvironment, immunomodulation, 4‐1BBL, AAV PF07.17. Methionine restriction results in an increase in extracellular vesicles (EVs) secretion and altered packaging of EVs markers in cancer cells Sachiko Inubushi ^1; Tomonari Kunihisa^2; Jun Yamamoto^3; Yoshihiko Tashiro^3; Norihiko Sugisawa^3; Sachiko Mizumoto^2; Shotaro Inoue^2; Mayuko Miki^2; Hirokazu Tanino^2; Robert Hoffman^3 ^1Division of Breast and Endocrine Surgery, Graduate School of Medicine, Kobe University, KOBE, Japan; ^2Division of Breast and Endocrine Surgery, Graduate School of Medicine, Kobe University, Japan; ^3AntiCancer Inc,, USA Introduction: Methionine addiction is the elevated requirement for exogenous methionine for growth by cancer cells even though the methionine‐addicted cancer cells synthesize normal or excess amounts of methionine. Methionine restriction (MR) by either a methionine‐free medium or a low‐methionine diet in vivo or by methioninase selectively arrests cancer cells in the late S/G2 cell cycle phase, but not normal cells, where the cancer cells become selectively sensitive to cytotoxic chemotherapy. The present study focuses on the secretion and packaging of extracellular vesicles (EVs) by cancer cells under methionine restriction. Methods: MDA‐MB‐231 cells (triple negative breast cancer [TNBC]) expressing EV‐specific CD63‐GFP were cultured in methionine‐containing DMEM. Methionine‐free DMEM, MET(−), was used for MR to closely match the methionine‐containing medium. EVs were isolated by ultracentrifugation and were used for NTA analysis (Nanosight NS500) and Western blotting. EVs were labeled with an Exo Sparkler Exosome Membrane labeling kit and the labeling was detected with a fluorescence microscope (BZ‐700X, Keyence). An ExoCounter was used to detect EVs in cell culture by sandwiching EVs with antibodies on discs and nanobeads on a surface antigen. Results: When MDA‐MB‐231 was cultured under MR conditions, CD63‐GFP was found to accumulate in the cells. MR resulted in about a 2‐fold increase in EV secretion per cell even though the cell number decreased. There was a decrease in EVs co‐expressing both CD9 and CD63, with many EVs expressing only one of either of these markers. Summary/Conclusion: Methionine restriction resulted in elevated EVs secretion per surviving cell and abnormal packaging of EVs markers. Funding: PI Training Program for International Collaborative Research, Kobe University, 2021 keywords: Methionine restriction, PF07.18. Protein arginine methylation patterns in plasma small EVs are potential indicators of pancreatic ductal adenocarcinoma Wei‐Qun Ding; Kritisha Bhandari; Chao Xu; Katherine Morris University of Oklahoma Health Sciences Center, Oklahoma City, USA Introduction: Early detection is key to improving the overall survival rate of pancreatic ductal adenocarcinoma (PDAC), yet there are no sensitive and specific biomarkers currently available for early detection of PDAC. Protein arginine methylation is a newly identified molecular feature of PDAC and its potential as diagnostic markers for PDAC remains unexplored. Methods: Patient plasma [early stage PDAC (n = 16), colon cancer (n = 16), late stage PDAC (n = 12), chronic pancreatitis (n = 9)], was obtained from the Stephenson Cancer Center and the Cooperative Human Tissue Network under an approved IRB protocol. Matched healthy plasma (n = 16) was provided by the Oklahoma Blood Institute. Plasma small EVs were isolated by double filtration and polymer precipitation. Arginine methylation was detected using commercially available antibodies and by LC/ESI/MS/MS. Results: The SDMA level of plasma small EVs was significantly reduced in patients with early and late stage PDAC but remains unchanged in patients with chronic pancreatitis as detected by western blot analysis. Both the MMA and SDMA levels in plasma small EVs were lower in patients with colon cancer. Proteomic analysis identified complement C3 and alpha‐2‐macroglobulin as arginine methylated proteins that were detected in plasma small EVs using the SDMA antibody. Further proteomic profiling indicated that some of the arginine methylated proteins are exclusively detected in patients with early stage PDAC. Summary/Conclusion: Protein arginine methylation patterns in plasma small EVs differ among patients with PDAC, colon cancer, chronic pancreatitis, and healthy subjects. These findings support the notion that arginine methylation patterns in plasma small EVs are potential biomarkers for early detection of PDAC. Funding: Presbyterian Health Foundation, National Institute of General Medical Sciences (P20GM103640 and P30GM145423), Stephenson Cancer Center keywords: Plasma small EVs, arginine methylation, pancreatic ductal adenocarcinoma, early detection PF09: EV Biogenesis and Bioengineering Chairs: Shannon Stott, Houjian Cai Location: Hall 4A 16:00 ‐ 18:00 PF09.02. Src family kinases engage differential biogenesis pathways for encapsulation into extracellular vesicles Houjian Cai; Chenming Ye University of Georgia, Athens, USA Introduction: Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. EVs contain proteins, DNA, mRNA, microRNAs, and other biological components. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity and molecular mechanisms of EVs biogenesis. Src family kinases including Src and Fyn are a group of tyrosine kinases with distinct difference in the fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N‐terminus. This study intends to study how different fatty acylations contribute to the enrichment of these proteins in EVs. Methods: Prostate cancer cell lines expressing high levels of Src and Fyn kinase were examined for their enrichment in EVs. EVs were isolated by sequential ultracentrifugation and were characterized by the size distribution, concentration, morphology, and EVs protein biomarkers. Src and Fyn mutants were generated by point mutations to remove myristylation or palmitoylation. Myristoylation or palmitoylation were determination by Click chemistry. Lipid raft was disrupted by treatment of filipin III. TSG101 was knocked down by shRNA. Xenograft tumors was implanted sub‐renally in mice by survival surgery. Results: Src and Fyn kinases were preferentially encapsulated into EVs and fatty acylation including myristoylation and palmitoylation regulated their encapsulation process. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis, led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src‐mediated prostate tumors in vivo. Summary/Conclusion: Protein fatty acylations regulate the EVs biogenesis pathways and the enrichment of proteins into EVs. This study provides an understanding of the EVs heterogeneity and an approach to regulate protein encapsulation into EVs. Funding: U01 [46]CA225784‐01; DOD W81XWH‐22‐PCRP‐EHDA; R21 AI157831‐01A1 and [47]AI171944‐01A1; The American Institute for Cancer Research; Georgia Research Alliance; and the Seed funding from the Georgia Regenerative Engineering & Medicine Center keywords: Myristoylation; palmitoylation; Src family kinases; extracellular vesicles; heterogeneity. PF09.17. Effects of intracellular pathway inhibitors on the secretion, protein, and lipid composition of fluorescent Bodipy sEV Deborah Polignano^1; Valeria Barreca ^1; Lorenzo Galli^1; Valentina Tirelli^1; Giancarlo Poiana^2; Massimo Sargiacomo^1; Maria Luisa L. Fiani^1 ^1Istituto Superiore di Sanità, Rome, Italy; ^2“Sapienza” University of Rome, Italy, Italy Introduction: Exosomes are small extracellular vesicles (sEV) formed within late endocytic compartments/multivesicular bodies (MVB). Various machineries have been described to regulate their biogenesis, including the ESCRT machinery, syntenin–alix pathway, tetraspanins and lipids, but several aspects of these processes have not been fully elucidated. We developed a methodology to obtain fluorescent exosomes (Bodipy sEV) of endosomal origin by using fluorescent palmitic acid Bodipy FL C16. To gain insight of exosome biogenesis we combined this approach with the use of a panel of inhibitors of lipid metabolism and vesicular trafficking. Methods: Bodipy sEV secreted by melanoma cells pulsed with BODIPY FL C16 and treated with inhibitors of cellular pathways were isolated by differential ultracentrifugation and quantified by Flow Cytometry (FC) and Nanoparticle Tracking Analysis (NTA). Characterization of Bodipy sEV lipid and protein content was performed by Thin Layer Chromatography (TLC) and Western Blot analysis for exosomal markers. Colocalization between Bodipy sEV and tetraspanins was determined by FC. Results: Upon treatment of melanoma cells with selected inhibitors of cellular pathways, significant differences were observed in the secretion of Bodipy sEV as evaluated by FC and NTA, compared to the control cells. Interestingly, under all conditions, Bodipy sEV had the same relative distribution of tetraspanins (CD63, CD81, and CD9) as assessed by colocalization analysis. However, Western Blot analysis of sEV markers, including tetraspanins, Alix, TSG101, and Syntenin, highlighted significant differences in protein marker secretion. Additionally, phospholipid analysis revealed differences that could be attributed to the different metabolism of Bodipy lipids. Summary/Conclusion: In summary, our results indicate that the use of inhibitors of intracellular pathways not only affects the secretion of sEV but also their protein and lipid composition. This suggests that this approach has the potential to provide further insights into the process of sEV biogenesis. Funding: This work was supported by the Italian Ministry of Health (grant RF‐2019‐12369719) keywords: exosome, sEV, exosome biogenesis, inhibitors PF09.18. The BCAT1 CXXC motif modulates extracellular vesicle release in acute myeloid leukaemia cell line U937 James Hillier ^1; Mathieu Y. Brunet^2; Rebecca Terry^3; Steven Coles^3; Ivan Wall^4 ^1University of Birmingham, Solihull, United Kingdom; ^2School of Chemical Engineering, University of Birmingham, Birmingham, UK, Birmingham, United Kingdom; ^3University of Worcester, United Kingdom; ^4University of Birmingham, London, United Kingdom Introduction: Extracellular Vesicle (EV) release is altered by oxidative stress which is also a feature of AML pathogenesis. The redox active protein tyrosine phosphatase 2 (SHP2) negatively regulates the biogenesis of EVs through dephosphorylation of CD63 associated syntenin. In turn, SHP2 is positively regulated by CXXC‐motif containing oxidoreductase Thioredoxin. Our recent work has identified cytosolic branched‐chain amino transferase (BCAT1) as a novel oxidoreductase through a redox active CXXC motif which decreases intracellular reactive oxygen species in myeloid cell line U937. Given these data we investigated the effect of the BCAT1‐CXXC motif in syntenin modulated EV release in U937 cells. Methods: Overexpression of vector control, BCAT1‐WT and BCAT1‐CXXS mutant protein in U937 cells was confirmed by western‐blot and qPCR. Cells were cultured in EV‐depleted Foetal Bovine Serum, conditioned media was collected and EVs were isolated via differential ultracentrifugation at 2000 xg for 20 min, 10,000 xg for 30 min and 120,000 xg for 70 min. EV concentration was determined by Nanoparticle Tracking Analysis (NTA). The presence of CD81, CD63, CD9 and syntenin at the single vesicle level was assessed by immunofluorescent staining using the Exoview platform. Results: NTA revealed BCAT1‐WT overexpression resulted in a 5.16‐fold increase in EV release compared to the control and a 1.80‐fold increase compared to BCAT1‐CXXS (p< 0.0001). Size distribution analysis showed smaller average size of BCAT1‐WT (145.5± 3.4nm) and BCAT1‐ CXXS (147.9± 15.75nm) EVs compared to control (202.7 ± 8.2nm). Immunofluorescent staining showed a 1.76‐fold increase in CD63 positive EVs derived from BCAT1‐WT cells compared to control and a 1.94‐fold increase compared to BCAT1‐CXXS. Conversely, BCAT1‐WT CD63 positive EVs displayed a 0.62‐fold decrease in syntenin compared to control and a 0.83‐fold decrease compared to BCAT1‐CXXS cells. Summary/Conclusion: This study demonstrates BCAT1 increases EV release in U937 cells, moreover, the number of CD63 positive vesicles was increased despite a concurrent decrease in syntenin positive vesicles. Mutation of the CXXC motif increased the syntenin positive vesicle count but decreased vesicle release raising the possibility of a novel BCAT1‐CXXC dependent regulatory mechanism for EV generation in AML cells. Funding: Engineering and Physical Sciences Research Council (EPSRC) PF09.19. Cholesterol binding regulates extracellular vesicle formation and membrane bending by Prominin‐1 Tristan A. Bell ^1; Bridget E. Luce^2; Hiba Dardari^2; Virly Y. Ananda^2; Pusparanee Hakim^2; Tran H. Nguyen^2; Luke H. Chao^2 ^1Massachusetts General Hospital, Department of Molecular Biology, Boston, USA; ^2Massachusetts General Hospital, Department of Molecular Biology, USA Introduction: Prominin‐1 (Prom1, CD133) is a pentaspan transmembrane (TM) protein that regulates plasma membrane curvature and induces secretion of extracellular vesicles (EVs) during stem cell and cancer stem cell differentiation. Prom1 EVs form at the tips of microvilli and cilia as well as through bulk blebbing from cholesterol‐rich domains of the plasma membrane. Patient mutations in Prom1 cause retinal degeneration due to failure of membrane homeostasis. Despite its importance in EV biogenesis during differentiation, the molecular mechanisms underlying EV formation by Prom1 are unclear. Methods: We mutate several sites in the TM of Prom1 to reduce stable cholesterol binding. We reconstitute Prom1 EV formation by transient transfection in Expi293 cells. EVs are purified using ultracentrifugation (30 min at 10,000 x g, 1 hr at 200,000 x g), gradient separation (5‐30% sucrose, 5 hr at 80,000 x g), and size‐exclusion chromatography (qEV2‐35nm columns). We characterize EV morphology and composition using electron microscopy and mass spectrometry. We use immunopurification to quantify cholesterol binding and bifunctional crosslinkers and limited proteolysis to study Prom1 orientation and conformation in purified EVs. Results: Wild‐type (WT) Prom1 and mutants all promote EV formation, but mutant EVs have more deformed membranes. All Prom1 EV membranes are less deformed than those produced by a homolog (Ttyh1) that lacks all cholesterol binding. Structure‐function studies indicate that WT and mutant Prom1 in EVs assume different oligomeric states and conformations. Summary/Conclusion: Cholesterol binding modulates the oligomerization and conformation of Prom1, and is thermodynamically linked to membrane bending and release of Prom1 EVs. We suggest a model where stable interaction of Prom1 with cholesterol inhibits membrane bending and EV release. This mechanism may regulate Prom1 EV release during stem cell differentiation. Funding: This work is supported by an HHWF fellowship to TAB. keywords: Prom1, CD133, Membrane bending PF09.20. Photometric method for dual targeting of surface and surface‐associated proteins on extracellular vesicles in multiparametric test Lee‐Ann Clegg ^1; Jenni Kathrine Sloth^2; Rikke Baek^3; Malene Møller Jørgensen^4 ^1Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark; ^2Hospital Pharmacy Central Denmark Region, Aarhus, Denmark, Aalborg, Denmark; ^3Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark, Aalborg, USA; ^4Department of Clinical Immunology and Medicine, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark Introduction: Extracellular Vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers, however, recent developments in the study of EVs have increased a need for simpler, but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several surface or surface‐associated proteins on EVs, using a multiparametric microarray‐based analysis termed Extracellular Vesicle Array (EV Array), which is developed to catch and phenotypically characterize small EVs (sEVs). Methods: Plasma from six healthy subjects were analyzed with EV Array. Three antibodies against human CD9, CD81, and CD63 were printed onto slides and used to immobilize sEVs. Slides with captured sEVs, were added either biotinylated anti‐human ‐CD9, ‐CD63, ‐CD81, or ‐Flotillin‐1 and ‐HSP90 detection antibodies. Streptavidin‐Cy5 and Cy3‐labelled antibodies were added in various combinations to detect the EV surface or surface‐associated proteins: CD9, CD63, CD81, Flotillin‐1 and HSP90 simultaneously. Fluorescent detection was performed with a microarray scanner. Plasma EVs were isolated with ultracentrifugation and the presence of surface or surface‐associated proteins were validated with western blot. Results: It was observed that it is possible to measure the EV surface or surface‐associated proteins at both 532nm (Cy3) and 635nm (Cy5) simultaneously without a significant change in signal from the detection molecules. This allows us to measure multiple EV marker proteins in a single analysis; thereby, more quickly find complex biomarker patterns in a sample. Summary/Conclusion: This study represents the first time where two signals, measured at different wavelengths, have been applied in the EV Array method. We can conclude that it was possible to measure different EV surface or surface‐associated proteins simultaneously. Overall, this study provides an important analytical advantage within medical diagnostics, since it allows for simultaneously measurement of both general and specific biomarkers in a sample. In the future, the detection of further biomarkers and their compatibility to the EV Array platform should be explored. Funding: Supported by the Novo Nordisk Foundation Tandem program. keywords: EV Array, sEV, biomarker, diagnostics PF09.20. Why do EVs from some cells display “luminal” protein epitopes? Zach A. Troyer ^1; Olesia Gololobova^2; Jane Nguyen^3; Zhaohao Liao^1; Kenneth W. Witwer^4 ^1Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA; ^2Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; ^3Department of Molecular and Comparative Pathobiology, Johns Hopkins University, USA; ^4Johns Hopkins University, Baltimore, USA Introduction: The lipid bilayer defines the topology of EV contents: luminal, transmembrane, and external (including the corona). The membrane is generally impermeable to larger luminal contents, including most proteins. To show that an EV is not simply a membrane fragment, MISEV recommends detection of at least one internal protein, requiring lipid bilayer disruption for antibody‐mediated detection. We observed a case in which EVs from a particular cell type appeared to be positive for the luminal marker Syntenin‐1 even without membrane permeabilization and even when gentle EV separation techniques were used. Methods: EVs were separated from the conditioned media of PM1 and Expi293F cells by methods with different degrees of “gentleness,” including differential ultracentrifugation, ultrafiltration, and size exclusion chromatography. Expi293F cells and their EVs were exposed to various damaging conditions. Single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence was performed with tetraspanin capture chips, EVs, and fluorescently labeled antibodies to proteins including Syntenin‐1 and with and without protease treatment and membrane permeabilization steps. Results: A population of apparently Syntenin‐1+ PM1 EVs was detected even in the absence of membrane permeabilization, and even when EVs were separated with gentle techniques. In contrast, Expi293F EVs were Syntenin‐1‐negative in the absence of membrane permeabilization. We evaluate the relationship of membrane damage and luminal epitope exposure. Summary/Conclusion: EVs from PM1 cells allowed immunolabelling of a canonical luminal EV protein, Syntenin‐1, despite not being permeabilized. This suggests that their membranes are uniquely permeable to antibodies or that some Syntenin‐1 is located outside of the PM1 EV membrane. The phenomenon of “flipped” topology has extensive implications for EV biology and methodology. Funding: This work was funded by the ION‐ARPA initiative (sponsored research agreement with Johns Hopkins University). keywords: EV topology, luminal, syntenin‐1, permeabilization, membrane, SP‐IRIS, immunolabelling, cargo, display PF09.20. Endolysosomal fusion attenuates exosome secretion Ganesh V. Shelke ^1; Chad Williamson^2; Michal Jarnik^2; Juan S. Bonifacino^1 ^1Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892, USA, Bethesda, USA; ^2Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892, USA, USA Introduction: Exosomes are subset of extracellular vesicles secreted from cells that facilitate intercellular communication and clearance of undigested content. Exosomes are formed by the exocytic fusion of multivesicular endosomes (MVE) with the plasma membrane (PM). Alternatively, MVE can fuse with lysosomes, resulting in degradation of intraluminal vesicles. The factors that influence MVE fusion with the PM or lysosomes remain unknown. We hypothesized that proteins like the BLOC‐one‐related complex (BORC) and the small GTPase ARL8, which mediate endolysosome coupling to microtubule motors and tethering/fusion with other organelles, play an important role in modulating exosome secretion. Methods: Exosomes were isolated from cells (HeLa, iPSCs‐derived neurons, HEK 293T/, THP‐1) using differential ultracentrifugation, filtration and iodixanol density gradient centrifugation. Exosome characterization was performed using immunoblotting and transmission electron microscopy (TEM). We employed various CRISPR KO cell lines and siRNA‐mediated knockdown to investigate the role of different proteins that regulate endolysosomal dynamics. Total internal reflection fluorescence microscopy and TEM were performed to visualized MVE‐PM fusion and ILV accumulation. Multiple live‐cell based assays were used to determine the fusion defect in cells. Results: We found that the BORC and ARL8 negatively influence exosome production through recruitment of the ARL8‐effector HOPS complex, which promotes tethering and fusion of MVEs with lysosomes. By silencing BORC, ARL8 or HOPS, intraluminal vesicles are prevented from being degraded in lysosomes, resulting in increased secretion of exosomes. Summary/Conclusion: We demonstrate that the BORC‐ARL8‐HOPS axis reduces exosome yield through increased endolysosomal fusion. Silencing of this axis could be used to increase exosome yield for biotechnology applications. Funding: This work was supported by the Intramural Program of NICHD, NIH, to J. S. Bonifacino. (ZIA HD001607), the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie grant agreement No 754412 and a fellowship from the Swedish Society for Medical Research to G. V. Shelke. keywords: extracellular vesicles, exosomes, endosomes, lysosomes, MVE, MVB, BORC, ARL8, kinesin, HOPS, fusion PF12: EV Labels and Imaging Chairs: Charles Lai, Allaura Cone Location: Hall 4A 16:00 ‐ 18:00 PF12.08. Immunoassay‐type biosensor based on fluorescence strategy for extracellular vesicles surface protein determination Saqer M. Al Abdullah ^1; Kristen Dellinger^2 ^1Department of Nanoengineering, Joint School of Nanoscience and Nanoengineering, North Carolina A&T State University, 2907 East Gate City Boulevard, Greensboro, North Carolina 27401, United States, Greensboro, USA; ^2Department of Nanoengineering, Joint School of Nanoscience and Nanoengineering, North Carolina A&T State University, 2907 East Gate City Boulevard, Greensboro, North Carolina 27401, United States, USA Introduction: Extracellular vesicles (EVs) are receiving mounting attention nowadays due to their ability to diagnose and monitor several diseases, such as cancer and Alzheimer's, in a non‐invasive and cost‐effective manner. Utilizing exosomes as a source of biomarkers is due to the fact that they act as a carrier of several pathogenic proteins on their outer surface. Also, Exosomes can be easily accessed and collected since they are available in almost all biofluids, including blood, urine, and saliva. Methods: We developed a sandwich immunoassay biochip that selectively captures exosomes and detects the presence of multiple proteins on the outer surface of exosomes by using various antibodies. Specifically, capturing EVs is achieved by using a gold surface conjugated with anti‐CD63, which allows the capturing of numerous kinds of EVs since CD63 is a generic protein expressed on the outer surface of the vesicles. Subsequently, a second antibody was added to the captured exosomes to detect the presence of a protein of interest on the outer the surface of each subpopulation of EVs by using fluorescence. Results: The immuno‐biochip is able to selectively capture exosomes based on their outer surface proteins, CD63, and also is able to detect the presence of another protein, namely L1CAM. Summary/Conclusion: This method can be used as a promising tool to design a multiplex biosensor for the early diagnosis of several multifactorial diseases by detecting and quantifying the expression of multiple pathogenic proteins on the outer surface of EVs, such as amyloid beta and tau proteins in AD. PF13: EV Heterogeneity 1 Chairs: Michael Harding, Andre Gorgens Location: Hall 4A 16:00 ‐ 18:00 PF13.08. Comparisons of Tetraspanin profile of Extracellular Vesicles‐derived from Sarcoma Cell Lines Using Exoview 100TM Instrument Thitikan Jirakittisonthon ^1; Orman L. Snyder^2; Hong He^3; Mark Weiss^1 ^1Kansas State University, Manhattan, USA; ^2Kansas State University, Topeka, USA; ^3Kansas State University, USA Introduction: Based on Minimum Information for Studies of Extracellular Vesicles 2018 (MISEV 2018), characterization of EVs using transmembrane protein markers is one of the criteria for characterization. New technology, such as the Exoview 100 TM, has been developed that allows us to “fingerprint” EVs by analysis of multiple protein expression on individual EVs by tetraspanin antigen‐capture chips and then use fluorophore‐conjugated antibodies to directly quantify specific EV membrane tetraspanin expression. Here, using Exoview 100 TM, we characterized tetraspanin (CD9, CD63, CD81) distribution on different cancer cell lines and compared the initial results to those found 2‐weeks later on identical samples to determine whether there was consistency in protein profiles based on manufacturer‐claimed chip shelf life. Methods: EVs from 5 cancer cell lines and one mesenchymal stromal cell line were isolated using ultrafiltration and size exclusion chromatography. EVs were then characterized using nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), TEM, and Exoview 100 TM. Identical EV concentrations were used for each cell line for Exoview 100 TM analysis at day one and day 14. All samples were processed concurrently using a CW‐100 plate washer to minimize chip processing variation. Results: Colocalization analysis of each capture probe on individual sample microchips showed that CD9, CD63, and CD 81 surface markers were specific to each cell type on day one analysis. Two weeks later, three of the original cell lines were re‐analyzed on the Exoview 100TM using identical preparation steps. Results showed distinctly different tetraspanin colocalization profiles when compared to the prior results on identical samples. Summary/Conclusion: Our data provides evidence of distinct EV tetraspanin colocalization profiles in different cancer cell lines and MSCs. Our data further indicates a change in the tetraspanin colocalization profiles on stored chips were not stable or reproducible over a two‐week timeframe. Funding: Terry Johnson Center for Cancer Research, Kansas State University, Midwest Institute for Comparative Stem Cell Biotechnology keywords: Tetraspanin profile, Sarcoma Cell Lines PF15: EV Separation/Technology 2 Chairs: Rossela Crescitelli, Heather Pua Location: Hall 4A 16:00 ‐ 18:00 PF15.11. Enrichment of NCAM‐positive extracellular vesicles from the SH‐SY5Y neuronal cell line using anti‐NCAM immuno‐capture beads Mary E W Collier; Flaviano Giorgini University of Leicester, Leicester, United Kingdom Introduction: The enrichment and analysis of neuronally‐derived extracellular vesicles (nEVs) from blood allows a “window into the brain” since the proteins and nucleic acids in these EVs reflects that of the parent cell from which they were released. However, finding neuron‐specific antigens that can be used to enrich for nEVs has been challenging. The aim of this study was to examine the use of the neuronal adhesion molecule NCAM as a neuronal marker to enrich for NCAM‐positive EVs released from the SH‐SY5Y neuronal cell line. Methods: EVs were isolated from serum‐free conditioned media of SH‐SY5Y cell cultures using an EV precipitation reagent. EVs were characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and immunoblotting analysis. Isolated EVs were incubated with magnetic beads conjugated to antibodies against the neuronal markers NCAM or L1CAM. IgG beads, anti‐calnexin beads or beads alone were used as negative controls. Immunocapture of NCAM positive SH‐SY5Y‐EVs (SH‐EVs) was examined using immunoblot analysis and flow cytometry. Results: SH‐EVs had a mode diameter of 225 nm as determined by NTA. TEM analysis revealed a typical cup‐shaped morphology of the EVs and immunoblot analysis showed the expression of the EV markers CD81 and Tsg101. Immunoblot analysis of EVs captured by anti‐NCAM beads revealed the presence of full length NCAM and Tsg101 but not calnexin. In contrast, L1CAM was not detected by immunoblot analysis of SH‐EVs incubated with anti‐L1CAM beads. SH‐EVs captured by anti‐NCAM beads and then labelled with either an anti‐NCAM‐PE or anti‐CD81‐PE antibody and examined by flow cytometry showed increased median fluorescence intensity compared to negative control beads, confirming the immuno‐capture of NCAM positive SH‐EVs by anti‐NCAM beads. Summary/Conclusion: NCAM may be a suitable neuronal marker to enrich for nEVs. The next stage of this study is to examine the immunocapture of NCAM‐positive nEVs from human plasma samples. Funding: National Institutes of Mental Health PF16: Cancer Microenvironment 2 Chairs: Sara Veiga Location: Hall 4A 16:00 ‐ 18:00 PF16.12. Exosomes produced by adipocytes induce EMT, and tumor metastasis, in both in vivo and in vitro models of TNBC Yuhan Qiu ^1; Rebecca Yu^2; Andrew Chen^2; Pablo Llevenes^2; Manohar Kolla^2; Naser Jafari^2; Isabella Pompa^2; Christina Ennis^2; Kiana Mahdaviani^3; Naomi Ko^3; Stefano Monti^2; Gerald Denis^2 ^1Boston University School of Medicine, Medford, USA; ^2Boston University School of Medicine, USA; ^3Boston Medical Center, USA Introduction: Type 2 Diabetes (T2D) is a chronic disease characterized by inflamed adipose tissue. Patients with triple negative breast cancer (TNBC) and comorbid T2D have higher risk of metastasis and shorter survival. However, mechanisms that couple T2D to TNBC outcomes are unknown. Here we hypothesize that exosomes, small vesicles secreted by tumor microenvironment (TME) breast adipocytes, drive epithelial‐to‐mesenchymal transition (EMT), metastasis in TNBC Methods: Exosomes were purified from conditioned media of 3T3‐L1 mature adipocytes, either insulin‐sensitive (IS) or insulin‐resistant (IR), characterized and quantified by NanoSight. Murine 4T1 cells, a TNBC model, were treated with exosomes in vitro (3 days). For in vivo models, mammary fat pads of BALB/c mice were injected with 4T1 cells. Histology and immunohistochemistry detected exosome‐driven TME differences (EMT, proliferation and angiogenesis). Distant metastases were quantified by clonogenic assay, and mRNA was extracted for RNA‐seq analysis. Exosomal RNAs were profiled by miRNA array to identify potential candidates responsible for driving metastasis. Results: Tumor‐bearing mice exhibited more metastasis in exosome‐treated groups. In primary tumors, vimentin (EMT), ki67 (proliferation) and CD31 (angiogenesis) were elevated in IR exosome group vs. IS exosome and control groups. Clonogenic assay of brain metastases showed more mesenchymal morphology and RNA‐seq revealed enriched EMT pathway genes in IR exosome treated group compared to other groups. miR‐let‐7b is highly differentially expressed between IS and IR, and potentially regulates metastasis. Summary/Conclusion: Thus, IR adipocyte exosomes modify TME, increase EMT and promote TNBC metastasis to distant organs, likely through miRNA pathways. We report similar results in human models. We suggest metabolic diseases (e.g. T2D) reshape TME, promoting metastasis and decreasing survival. Therefore, TNBC patients with T2D should be closely monitored for metastasis, with metabolic medications considered. Funding: R01CA222170‐05 keywords: Triple Negative Breast Cancer, Insulin Resistance, Metastasis, Adipocytes PF18: Plant EVs Chairs: Lida Halilovic Location: Hall 4A 16:00 ‐ 18:00 PF18.11. EVs facilitate cross‐kingdom RNA trafficking between plants and fungal pathogens Baoye He ^1; Shumei Wang^2; Angela Chen^3; Huaitong Wu^2; Qiang cai^4; Hailing jin^2 ^1University of California, Riverside, Riverside, USA; ^2University of California, Riverside, USA; ^3UCR, USA; ^4Wuhan University, China (People's Republic) Introduction: Cross‐kingdom RNA trafficking between plant hosts and pathogens is a crucial process that regulates gene expression in both organisms during infection. Despite its significance, the mechanism of RNA transport between plants and fungal pathogens has remained elusive. Here, we demonstrate that extracellular vesicles (EVs) serve as a means for both plants and fungal pathogens to deliver RNA to their respective interacting partners. Methods: Arabidopsis EVs were isolated from the apoplastic washing fluid of Botrytis cinerea‐infected plants by sequential ultracentrifugation at 100,000xg followed by sucrose gradient fractionations or immunoisolation. Fungal EVs were isolated from liquid cultures by sequential ultracentrifugation at 100,000xg, followed by sucrose gradient fractionations. Both EVs were characterized by western blot, nanoparticle tracking analysis, and transmission electron microscope. Results: We performed RNA seq analysis and revealed that both small RNAs (sRNA) and message RNAs (mRNA) were found in plant EVs. These sRNAs can be transferred to the interacting fungal pathogen B. cinerea, where they can silence fungal virulence‐related gene expression. Meanwhile, the mRNAs can also be transported to fungal cells, where they can be translated into proteins to modulate fungal mitochondrial morphology and functions. Plant tetraspanin proteins, including TET8, are enriched in plant EVs and can be used as a plant EV biomarker. The fungal pathogen B. cinerea can also secrete EVs to deliver fungal sRNAs into plant cells that suppress plant immune‐related gene expression and facilitate infection. B. cinerea EVs can be internalized by plants via clathrin‐mediated endocytosis. B. cinerea tetraspanin protein BcPLS1 serves as a biomarker for B. cinerea EVs and plays an essential role in fungal pathogenicity. Summary/Conclusion: EVs have been identified as a mechanism used by both plants and fungal pathogens to deliver RNAs into one another to modulate the plant‐pathogen interaction. These findings could have significant implications for the development of novel plant protection strategies. Funding: NIH (R35GM136379), NSF (IOS 2020731), USDA (2021‐67013‐34258), USDA (2019‐70016‐29067), and the CIFAR ‘Fungal Kingdom’ fellowship. keywords: cross‐kingdom RNAi PF18.12. Plant defense metabolites localize in extracellular vesicles Lida Halilovic ^1; Rafael Fonseca Benevenuto^2; Hailing jin^3 ^1University of California Riverside, Riverside, USA; ^2University of California Riverside, USA; ^3University of California, Riverside, USA Introduction: As part of the plant defense response to fungal infection, plants synthesize and secrete extracellular vesicles (EVs) that traffic bioactive materials, such as sRNAs, into interacting fungal cells. Further than sRNAs and RNA‐binding proteins, the contents of Arabidopsis thaliana EVs in the context of the plant defense response remains elusive. Our research investigates the trafficking of plant defense metabolites within EVs as part of the response of the model plant Arabidopsis thaliana against fungal pathogen Botrytis cinerea, the causal agent of gray mold disease and the second most economically devastating fungal plant pathogen in agriculture. Methods: Arabidopsis EVs were isolated from the apoplastic washing fluid of plants by differential centrifugation followed by ultracentrifugation at 100,000 x g to pellet EVs. EVs were further characterized by nanoparticle tracking analysis. EVs were extracted from Botrytis cinerea‐infected Arabidopsis plants and uninfected healthy Arabidopsis for control. Ultra‐high performance liquid chromatography‐tandem mass spectrometry was used for the untargeted detection of plant metabolites within EVs. Results: Metabolite detections were validated through peak matching to metabolite databases such as METLIN, Mass Bank of North America, and an in‐house metabolomics database. Enrichment of lipids were detected in both EVs isolated from infected and healthy plants. Notably, several key Arabidopsis defense metabolites were identified in EVs extracted from infected plants and absent in EVs from healthy plants. Summary/Conclusion: Preliminary data indicates that plant defense metabolites localize within plant EVs during pathogen infection. EVs may facilitate the transport of insoluble, nonpolar and semipolar metabolites through the plant apoplast and into invading fungal pathogen cells. Funding: National Institute of Health (R35GM136379) PF18.13. Plant‐derived Extracellular Vesicles enhances drug delivery with radiation mitigation/sensitivity effects zhu zhao UNIV OF ARIZONA, Phoenix, USA Introduction: Extracellular vesicles (EV) have shown great promise working as drugs and drug carriers in drug delivery system. However, their complex and costly production limit their development for clinical use. Recent studies shows that plants can also produce EVs (pEVs), whose chemical and structural properties are close to animal EVs. Plants are a source of many therapeutic compounds, available in large quantity at low cost, and very likely to carry drug candidates into their pEVs. Hence, we propose here to compare pEVs isolation methods for different plants and their ability to modulate radiation response by carrying and delivering natural or synthetic radiomodulator compounds. Methods: Olives and grapes have been blended, and debris removed by serial low‐speed centrifugations. Total pEVs have been then pelleted by ultracentrifugation and purified through sucrose gradient (UC‐SUC) and/or size‐exclusion chromatography (SEC). Nano‐tracking analysis (NTA) and electron microscopy are used for characterization. pEVs intake by human cells have been assessed by lipid membrane labelling and immunofluorescence (IF). Finally, viability IF assay was performed to assess biological activity and effect on radiation response. Results: Olives‐derived and grapes‐derived EVs (ODEVs and GDEVs) have been successfully isolated and have a clean profile with average size 103nm and 183nm respectively. NTA profiles are more monodisperse for ODEVs using UC‐SUC and for GDEVs using SEC. By performing SEC after UC‐SUC, both pEVs showed highest purity. IF showed that PKH67‐labeled pEVs are present in F‐actin‐labeled cells, suggesting cytoplasmic internalization. Interestingly, both pEVs did not show any cytotoxic effect on human cells, but ODEVs had a radiosensitizing effect on cancer cells while no effect on normal cells. Summary/Conclusion: Our results showed that pEVs can be easily isolated, undergo endocytosis by human cells, are not cytotoxic but can affect the biological response. Funding: grant #U19AI067773 from NIAID PF19: EVs and the Central Nervous System Chairs: Huaqi Su, Gagan Deep Location: Hall 4A 16:00 ‐ 18:00 PF19.12. Bioengineering exosomes to enhance brain targeting in a mouse traumatic brain injury model Min Chen^1; Smriti Krishna^2; David N. Haylock ^3; David Reutens^1 ^1The University of Queensland, USA; ^2VivaZome Therapeutics, USA; ^3VivaZome Therapeutics, MELBOURNE, Australia Introduction: This study generated a stable cell line for producing exosomes expressing Rabies Virus Glycoprotein (RVG), attached to a membrane protein, Lamp2b, to enhance brain delivery capacity. Studies were conducted to compare the uptake of native and bioengineered exosomes (RVG‐exosomes) by human neuroblastoma cells and their brain distribution in a mouse traumatic brain injury (TBI) model. Methods: HEK293T cells were transfected with pcDNA GNSTM‐3‐RVG‐10‐Lamp2b‐HA to generate a stable cell line. Native and RVG‐exosomes were concentrated using a Tangential Flow Filtration system. DiO‐stained SH‐SY5Y cells (0.5×105 cells/well) were incubated with DiR‐labelled native or RVG‐exosomes for 2 hours before they were fixed and imaged using a confocal microscope. Adult CD1 mice were subjected to a severe unilateral cortical contusion by compressing the cortex to a depth of 2.0 mm at a velocity of 5 m/s and 100 millisecond duration. Within 15 min post‐TBI, mice were given native or RVG‐exosomes (9 × 1010 particles/mouse, i.v.) labelled with CellMask™ Deep Red Plasma Membrane Stain. Sham‐operated mice received equal volumes of vehicle (PBS). Bio‐distribution and brain delivery were examined using in vivo optical fluorescent imaging 2 hours after injection. Results: Confocal images showed cell uptake of RVG‐exosomes (red) by SH‐SY5Y cells (green) (A) and the presence of RVG‐exosomes along the membrane of the cell body (a) and neurites (b). Optical images demonstrated high uptake of both native exosomes and RVG‐exosomes in the liver, spleen, GI tract, and lungs. The RVG‐exosome treatment increased signal in the brain, with an average radiant efficiency 41% higher than for native exosome treatment (B). Summary/Conclusion: The expression of Lamp2b‐RVG increases the uptake of exosomes by neuroblastoma cells and the accumulation of exosomes in the brain after systemic administration. keywords: Brain injury, mouse model, bioengineering, exosomes, rabies virus glycoprotein nfant formula products: Moving towards next‐generation infant nutrition Natalie P. Turner ^1; Pawel Sadowski^2; Murray Mitchell^1 ^1Queensland University of Technology, South Brisbane, Australia; ^2Central Analytical Research Facility, Queensland University of Technology, Brisbane City, Australia Introduction: The first thousand days of life are critical to establishing optimal health in the human infant. Up to 75% of infants receive infant formula (IF) by 6 months of age, despite human milk (HM) imparting significant health advantages related to improved immune function. Most IF has a protein base derived from cow's milk (CM), however the difference to HM is not well‐understood. Small extracellular vesicles (sEVs; 50 – 200 nm) are present in milk and have been successfully recovered by our group from IF products (unpublished data). Here, we compared the proteomes of sEVs recovered from HM, CM and IF. Methods: sEVs were isolated from 200 mL of HM, CM and IF by sequential differential centrifugation coupled with size‐exclusion chromatography (Vaswani et al., 2017). sEV enriched fractions were pooled and characterised by western blot (WB; FLOT‐1, CD9, CD81, SYN‐1, TSG101, ALB, GAPDH, CALNX), NTA and TEM. Proteomic analysis was performed on a SCIEX5600+ TripleTOF mass spectrometer using sequential window acquisition of all theoretical mass spectra (SWATH‐MS). Raw data were processed in DIA‐NN (False discovery rate = 0.01), and output files were processed in R Studio using homologous peptides for cross‐species comparison. Gene ontology was performed using the PANTHER online tool. Results: Mean particles sizes for HM, CM, and IF were 129.9 nm, 124.3 nm and 101.5 nm, respectively. CD81, TSG101, and FLOT‐1 were detected by WB and mass spectrometry in all samples. 216 proteins were common to HM and CM; 126 were enriched and 17 depleted in CM compared to HM. Among the enriched were immunomodulatory proteins lactadherin, xanthine oxidase and butyrophilin, G proteins (24%) and metabolite interconversion enzymes (22%). CM and IF had 208 proteins in common, of which enzymes and binding proteins represented 37% and 41%, respectively. Summary/Conclusion: The workflow captured species‐specific differences, thus demonstrating potential for ongoing characterisation of milk sEVs relating to infant health. Lyophilised IF contains sEVs whose molecular contents are preserved regardless of processing, which could be leveraged to optimise IF products in the future. Further investigation into the functional capacity of IF EVs should focus on changes to the EV protein corona and surfaceome, as this may influence cellular uptake of lyophilised sEVs. Funding: This project was funded by Reckitt Benckiser/Mead Johnson. keywords: Extracellular vesicles; milk; infant formula; nutrition; proteomics; mass spectrometry; SWATH Saturday 20 May 2023 Late Breaking Poster PS01: Therapy Adipose and MSC‐Derived EVs Chairs: Sujata Mohanty, Yves DeClerck Location: Hall 4A 16:50 ‐ 18:50 PS01.10. Mesenchymal Stem Cell‐derived Extracellular Vesicles: a new potential tool for restoring altered GABA switch in vitro Silvia Coco ^1; Genni Desiato^2; Chiara Adriana Elia^2; Desireè Ficarra^3; Irene Corradini^4; Marco Grisetti^1; Ramona Meanti^1; Maria Luisa Malosio^5; Antonio Biagio Torsello^1; Michela Matteoli^6; Davide Pozzi^7 ^1University of Milano‐Bicocca, School of Medicine and Surgery, Monza (MB), Italy; ^2Humanitas Clinical and Research Center ‐ IRCCS, Rozzano, Milan, Italy; ^3School of Medicine and Surgery, and PhD program in Neuroscience,University of Milano‐Bicocca, Monza (MB), Italy; ^4Humanitas Clinical and Research Center ‐ IRCCS and CNR Institute of Neuroscience, Milano, Italy, Rozzano, Milan, Italy; ^5CNR Institute of Neuroscience, Milano, Italy and Humanitas Clinical and Research Center ‐ IRCCS, Rozzano, Milan, Italy; ^6CNR Institute of Neuroscience, Milano and Humanitas Clinical and Research Center ‐ IRCCS, Rozzano, Milan, Italy; ^7Humanitas Clinical and Research Center ‐ IRCCS and CNR Institute of Neuroscience, Milano, Rozzano, Milan, Italy Introduction: Neurodevelopmental diseases (NDD), as epilepsy and autism spectrum disorders (ASD) are associated with an altered GABA switch. Such a switch oversees the transition of GABA excitatory depolarizing (embryonic age), to inhibitory hyperpolarizing actions (adult). The GABA switch must occur within a precise time window to achieve proper brain development. If not, any alterations may end in long‐lasting detrimental outcomes. Effective therapeutic strategies to restore the correct timing of the GABA switch are still lacking. Recently, stem cell therapy is increasingly considered a useful chance. Among these, Mesenchymal Stem Cells (MSC) and their derived EVs possess a plethora of abilities making them the most promising clinical candidates. Methods: Bone marrow mouse MSCs are maintained in a serum free‐neuronal medium for 24 hours. The EV‐containing medium is collected and purified according to Thiery's protocol (2006). Both MVs and Exosomes (Exo) are characterized by NTA (Nanosight) and WB. MVs and Exo are added to cultured neurons (3ug protein/die), both transiently (1‐4 DIV) and chronically (1‐12 DIV) and the GABA switch is assessed through calcium and chloride imaging approaches. Results: We found that MSC‐EVs affect the regulation of GABA transmission, through a significant reduction of the [Cl‐]i levels in cultured hippocampal neurons, thus influencing the GABA developmental switch. Further, MSC‐EVs modulated mRNA expression levels of Cl‐ transporters, with MVs able to upregulate KCC2 and Exo to downregulate NKCC1. Moreover, MVs increased BDNF expression levels and two GABA‐related genes (GAD67 and the vGAT). Summary/Conclusion: Our data indicate that MSC‐EVs can accelerate the GABA developmental switch through mechanisms involving the production of BDNF and modulation of chloride homeostasis. Given the need to develop new tools to restore a proper GABA developmental switch in several NDDs, MSC‐EVs may be exploited as a therapeutic strategy in these human disorders. keywords: Mesenchymal stem cells, extracellular vesicles, GABA switch, hippocampal neurons PS01.11. Characterization and manufacturing of clinical‐grade small extracellular vesicles derived from mesenchymal stromal cells for free or confined applications in joints and periodontal defects Natasha Blanco^1; Eliseo Parra^2; Aliosha I. Figueroa‐Valdés^1; Yessia Hidalgo^2; Celeny Figuera^2; Maria Ignacia Cádiz^2; Francisca Alcayaga‐Miranda^1; Maroun Khoury^2; Jimena Cuenca ^1 ^1IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Centro de Investigación e Innovación Biomédica (CiiB), Universidad de los Andes, Santiago, Chile, Santiago, Chile; ^2IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Centro de Investigación e Innovación Biomédica (CiiB), Universidad de los Andes, Santiago, Chile., Santiago, Chile Introduction: Introduction: High expectations are being held for Small extracellular vesicles from mesenchymal stromal cells (MSC‐sEV) to reach their projected clinical milestone and, hopefully, satisfy the current unmet medical needs. Optimizing the up‐/downstream process development, quality control, and potency test, is essential for their translational pathway. SEV variability of their content depends largely on their production and isolation processes. MSC‐sEV have been considered promising agents for treating various degenerative/inflammatory diseases including osteoarthritis and periodontal defects. Nonetheless a challenge in MSC‐sEV is developing a scalable and consistent clinical‐grade product supported by robust release criteria and validated potency test. Developing compatible vehiculized sEV products is also needed for local applications, including delivery in joints and dental cavities. Methods: Methods: Umbilical cord (UC)‐MSC were obtained under GMP conditions. The process development required transferring all the manufacturing steps into a GMP‐compliant process that included concentration by ultrafiltration, purification with size exclusion chromatography and finally, another concentration step using ultrafiltration. sEV were characterized following the MISEV2018 guidelines by NTA, flow cytometry, western blot and TEM. A potency test was developed to measure Thrombospondin 2, followed by a functional validation based on macrophage‐polarization assays. For the delivery vehicle of sEV, a clinically validated, xenofree plasma‐based scaffold. Results: Results: Isolated MSCs‐sEV showed a modal size of 120–145 nm and characteristic cup‐shape morphology; their identity was confirmed by the presence of CD63, CD9, CD81, Flotillin‐1, and Syntenin‐1, and their purity, was verified by the absence of Calnexin/TOMM20. Functional test showed sEV polarize macrophages towards an anti‐inflammatory M2 phenotype. The results ensured sEV sterility and showed stability at ‐80°C for up to 5 months without affecting their modal size, number, and identity. sEV shelf life at 2–8°C display a 24‐hour window without significantly modifying their characteristics. Summary/Conclusion: Summary/Conclusion: The analytical needs in the EV field are enormous at all stages (mechanism of action, product and process development, GMP manufacturing, release criteria). The findings demonstrated that the established MSC‐sEV standard operating procedures support EV production conditions, enabling chromatography‐based scalable processes under aseptic conditions useful for cell‐free therapeutic applications. Using immunofunctional potency test reinforces their standardization in compliance with regulatory guidance for assay validation. Funding: Funding: FONDECYT #1211376, Fondef ID23I10325, IMPACT #[48]FB210024, ANID, Chile; Cells for Cells S.A., Chile; Consorcio Regenero S.A., Chile; EVast Bio, USA PS02: Neurodegenerative Diseases and EVs 2 Chairs: Yiyao Huang, Ursula Sandau Location: Hall 4A 16:50 ‐ 18:50 PS02.09. Whole blood isolated astrocyte‐derived extracellular vesicles have increased levels of TDP‐43 in amyotrophic lateral sclerosis patients Tanya H. Butt ^1; Lina Marcinczyk^2; Cherine Jaaffar^2; Madeleine Strait^3; Mohammad Alayyoub^2; Roheeni Saxena^2; Diane Re^2 ^1Columbia University, New York, USA; ^2Columbia University, USA; ^3University of Washington, USA Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, typically fatal within 1–5 years of symptom onset. ALS diagnosis and treatment are currently hampered by the lack of reliable disease biomarkers. Extracellular vesicles (EVs) produced by astrocytes may be promising biomarkers in ALS due to the significant role of glial cells in motor neuron degeneration. Pathologically, ALS is characterized by cellular aggregates of TAR DNA‐binding protein‐43 (TDP‐43), including its phosphorylated form (pTDP‐43). Pathological conditions that promote TDP‐43 aggregation increase the excretion of TDP‐43 via EVs. We hypothesized that astrocyte‐derived EV TDP‐43 and pTDP‐43 will be different in ALS patients compared to healthy controls. Methods: We isolated astrocyte‐EVs starting with 0.5mL of whole blood using a direct immunoprecipitation protocol, adapted from previous methods ([49]EV170062). We incubated samples with an antibody against an extracellular domain of EAAT1, a glutamate transporter specific to astrocytes. We used nanotracking particle analysis and transmission electron microscopy to characterize the EVs. Astrocyte‐specific and neuron‐specific protein markers, as well as CD81, CD63, TDP‐43, and pTDP‐43, were detected by enzyme‐linked immunoassays (ELISA) to determine EV isolation efficiency and enrichment. Data was analyzed using a Student's t‐test and R studio. Results: Astrocyte‐derived EVs were highly enriched in astrocyte markers (GFAP, GLAST, GLUL) and less enriched for neuron‐specific markers (NEFL, SYP). CD81‐normalized TDP‐43 and pTDP‐43 were significantly elevated amongst ALS patient astrocyte‐EVs (n = 40) compared to age‐ and sex‐matched healthy controls (n = 40) (p<.005). Summary/Conclusion: EV TDP‐43 from ALS patient plasma was previously suggested to increase with disease progression in a small sample sized study. In this larger study, our results indicate that astrocyte‐EV TDP‐43 and pTDP‐43 may be used as biomarkers for ALS disease status, although its disease‐specificity warrants further investigation. Funding: Funding for this research was provided by the NIH and NIEHS grants: R01ES029971, R01TS000285, TL1TR001875 PS02.09. CNS‐Originating Extracellular Vesicle‐Associated α‐Synuclein as a Promising Biomarker for Parkinsonian Disorders: Systematic Review, Meta‐Analysis and Meta‐regression Hash Brown Taha Department of Integrative Biology & Physiology, University of California Los Angeles, Boulder, USA Introduction: Parkinsonian disorders, such as Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), and corticobasal syndrome (CBS), are neurodegenerative diseases with shared early motor symptoms but distinct pathology. Accurate premortem diagnosis is challenging due to overlapping clinical features, highlighting the need for reliable biomarkers. Extracellular vesicles (EVs) carry cell‐specific biomolecules and cross the blood‐brain barrier into the blood, offering insights into brain processes. They serve as a promising tool for minimally invasive diagnostics in parkinsonian disorders. This study assesses the potential of blood‐derived neuronal and oligodendroglial EVs (nEVs and oEVs) with α‐syn proteoforms as biomarkers for distinguishing these disorders. Methods: I conducted comprehensive meta‐analyses using Cohen's d standardized mean differences (SMD) using 12 eligible studies measuring nEVs or oEVs α‐syn in patients with PD and at least one other group: MSA, DLB, PSP, CBS, or healthy controls (HCs), and at least two methods for quantifying or characterizing isolated EVs. I systematically evaluated α‐syn levels using holistic and subgroup analyses based on the medium, antibody clone for immunoprecipitation of CNS‐EVs, analytical method, different centers/cohorts, sub‐diagnoses, and various combinations. I also performed meta‐regressions using age, gender, disease stage, motor impairment severity, and cognition as covariate predictors. In total, the meta‐analysis includes 1,472 patients with PD, 206 with MSA, 21 with DLB, 172 with PSP, 152 with CBS, and 882 HCs. Results: The results from all meta‐analyses indicate that nEVs α‐syn concentrations are lower in parkinsonian disorders compared to HCs (k = 19, SMD = ‐0.34; 95% CI ‐0.38, 0.00; p = 0.047). However, they are not useful for distinguishing specific parkinsonian disorders from one another. Furthermore, α‐syn levels in oEVs showed no significant differences among patients with PD, MSA, or HCs, even when combined with nEVs α‐syn, which contradicts previous literature. Meta‐regressions revealed that demographic and clinical variables were not significant predictors of α‐syn concentrations in nEVs or oEVs for any of the patients or HCs across all analyses. Summary/Conclusion: These findings highlight the need for adopting more rigorous, standardized procedures and independent validations across all laboratories studying biomarkers in CNS‐originating EVs. Additionally, there is a pressing need for improved biomarkers to distinguish among parkinsonian disorders more effectively. keywords: Parkinson's disease, L1CAM, synucleinopathies, α‐synuclein, diagnosis, parkinsonism. PS04: Cardiovascular EVs 2 Chairs: Robert Myette, Dylan Burger Location: Hall 4A 16:50 ‐ 18:50 PS04.11. A minimally invasive method to detect complement deposition on endothelial cells using endothelial‐derived extracellular vesicles from patient plasma Elena Tarabra ^1; Ellen E. Millman^2; Tobin J Cammett^1 ^1Alexion RDU, New Haven, USA; ^2Alexion RDU of Astrazeneca, New Haven, USA Introduction: Thrombotic microangiopathy (TMA) is a syndrome of rare and devastating disorders involving vascular endothelial cell (EC) injury. Pathogenic causes of TMA include dysregulation of the innate immune complement pathway. Specifically, dysregulation of complement leads to surface assembly of membrane attack complex (MAC), including vascular ECs and cytolytic damage. Clinically, no soluble antigen tests exist to detect complement involvement and tissue biopsy is often inconclusive or not readily available. Here, we present preliminary design and data for a relatively high‐throughput, minimally invasive alternative to traditional tissue biopsy, for the detection of complement deposition on EC‐derived extracellular vesicles (ECEVs). Methods: ECEVs from normal human donors, in matched plasma matrices, were characterized by NTA and surface phenotyped for canonical EV tetraspanins, EC‐specific markers and regulators of complement pathway using the Luminex xMAP technology. Optimized method conditions were then used to detect deposition of complement proteins on ECEVs enriched from patients diagnosed with TMA. Results: Initial evaluation of normal donor K2EDTA and NaCitrate plasma by immuno‐bead capture for CD9, CD63, CD81 and detection of EC‐specific markers (endoglin and VE‐cadherin) confirms acceptable performance of matrix, antibodies and analytical platform for enrichment of ECEVs. ECEVs were enriched using anti‐Endoglin and anti‐VE‐cadherin coated capture beads and then surface phenotyped for complement membrane regulatory proteins (CD46, CD55, CD59) resulting in positive signals for CD46, CD55 in both K2EDTA and NaCitrate. Using these optimized conditions, we then evaluated ECEVs enriched from TMA patient plasmas for deposition of complement effectors proteins. Preliminary results showed significantly higher signal for deposition of complement effectors proteins, including MAC, compared to normal donor plasma. Summary/Conclusion: Taken together these results confirm the clinical potential for this platform as a minimally invasive method utilizing plasma to monitor complement mediated vascular endothelial damage associated with TMA and possibly other systemic diseases associated with complement dysregulation. keywords: Complement, Endothelial cells injury, Plasma EVs PS04.12. Extracellular vesicles derived from human fibroblast and loaded with pro‐vascular transcription factors induce endothelial cells differentiation and mature vascularization María A. Rincon‐Benavides ^1; Tatiana Z. Cuellar Gaviria^2; Natalia Mendonca^3; Lilibeth Ortega‐Pineda^4; Ana I. Salazar‐Puerta^5; Britani Blackstone^4; Heather Powell^6; Daniel Gallego‐Perez^7; Natalia Higuita‐Castro^8 ^1Biophysics program and Biomedical Engineering Department, The Ohio State University, USA; ^2Biomedical Engineering, The Ohio State University, Columbus, USA; ^3The Ohio State University, Columbus, USA; ^4The ohio state university, USA; ^5Biomedical Engineering, The Ohio State University, USA; ^6The ohio state, USA; ^7Biomedical Engineering and General Surgery, The Ohio State University, USA; ^8Department of Biomedical Engineering and Department of Surgery, The Ohio State University, USA Introduction: Ischemic disorders and tissue injury can affect several organs such as the brain, skin, and heart. Efficient vascularization has an important role in functional tissue regeneration and healing. Although endothelial cell (ECs) transplantation has shown the potential to increase vascularization, sources for donor cells are scarce. To overcome this limitation, direct cell reprogramming can be used to generate induced endothelial cells (iECs) from non‐vascular cells. Direct cell reprogramming can be achieved through the delivery of pro‐vascular transcription factors. However, finding effective vehicles to deliver transcription factors remains a significant challenge. Engineer EVs (eEVs) hold a potential promise for targeted drug delivery. EVs can cross biological barriers without rejection while exhibiting low toxicity, and immunogenicity compared to other standard delivery methods. We propose the implementation of eEVs loaded with pro‐vascular transcription factors to direct the reprogramming of patient‐human dermal fibroblasts (PHDFs) into iECs. Methods: PHDFs were transfected using electroporation with plasmids encoding pro‐vascular transcription factors and derived PHDFs EVs loaded with the pro‐vascular cargo were isolated from the culture media using a total exosome reagent. Packing of the genetic material inside the eEVs was confirmed using RT‐PCR. eEV size distribution and concentration were analyzed via Nanosight and cryo‐TEM. eEVs were used to reprogram PHDFs into iECs in vitro. Additionally, mouse models were used to evaluate the angiogenic potential of the eEVS, where iECs suspended in a Matrigel plug were subcutaneously implanted in nude mice to characterize Vasculogenesis. Results: Our results show that eEVs can effectively reprogram fibroblast into iECs, with positive expression of the endothelial markers 7 days after treatment. Our study indicates that the iECs reprogrammed via eEVs induced vasculature in Matrigel plugs transplanted. Summary/Conclusion: These findings demonstrate that eEVs can induce direct reprogramming of human fibroblasts into iECs and have significant potential as a novel therapy of regenerative medicine. Funding: This work was partially funded by NIH Award No. R01AR079485 to N.H.C., and NIH Awards Nos. DP1DK126199 and DP2EB028110 to D.G.P. keywords: Tissue engineering, vasculogenesis, and engineering extracellular vesicles PS04.13. Genetically engineered human induced pluripotent stem cells for the production of brain‐targeting extracellular vesicles Hans B. Liu ^1; Wenshen Wang^2; Chengqian Zhou^1; Irfaan Karim^3; Mingyao Ying^4; Guanshu Liu^5; Pan P. Li^1 ^1Department of Psychiatry and Behavioral Sciences, Division of Neurobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, USA; ^2F.M Kirby Center, Kennedy Krieger Institute; Department of Radiology, Johns Hopkins University,, USA; ^3Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, Maryland, USA, USA; ^4Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, Maryland, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, USA; ^5F.M Kirby Center, Kennedy Krieger Institute; Department of Radiology, Johns Hopkins University, Baltimore, USA Introduction: Extracellular vehicles (EVs) are are cell‐secreted membrane vehicles that have become a promising, natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture, Human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However, hiPSC‐EVs only have a moderate ability for brain delivery. Herein, we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG‐Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor. Methods: An RVG‐Lamp2b‐HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9‐assisted homologous recombination. Karyotyping was performed to detect the potential chromosomal abnormality. Immunocytochemistry was used to detect the expression of pluripotent markers in the RVG‐edited hiPSCs. The differentiation potential of the RVG‐edited hiPSCs was validated by differentiation into three germ layers. Western blot was used to detect the expression of RVG‐Lamp2b‐HA in RVG‐edited hiPSCs as well as EVs derived from RVG‐edited hiPSCs. Biodistribution and brain delivery of intravenously injected unmodified and RVG‐edited EVs in wild‐type nude mice was examined using in vivo fluorescent imaging. Results: An RVG‐edited hiPSC line was successfully generated using a CRISPR/Cas9 approach. The RVG‐edited iPSCs have normal karyotype, express pluripotency markers, and have differentiation potential. Expression of RVG‐Lamp2b‐HA was detected in total cell extracts as well as EVs derived from RVG‐edited (vs. control) hiPSCs. The preliminary biodistribution study confirmed that EVs derived from RVG‐edited hiPSCs possesses higher brain delivery efficiency. Summary/Conclusion: We have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain‐targeted drug delivery. Funding: This work was supported by the Maryland Stem Cell Research Fund, and NIH (NS125350). PS04.14. Identification of exosome‐associated miRNAs expression signature in patients with Aortic Valve Stenosis (AVS) Neda Mohammadi ^1; Staffan Hildebrand^1; Philip Roger Goody^2; Denise Nehl^2; Felix Jansen^2; Mohammed Rabiul Hosen^2; Alexander Pfeifer^3 ^1Institute of Pharmacology and Toxicology, University Hospital Bonn, Bonn, Germany, Germany; ^2Department of Internal Medicine‐II, Heart Center Bonn, University Hospital Bonn, Bonn, Germany, Bonn, Germany; ^3Institute of Pharmacology and Toxicology, University Hospital Bonn, Bonn, Germany, Bonn, Germany Introduction: Exosomes have recently emerged as potential contributors to the pathophysiology of cardiovascular diseases. However, their role in Aortic Valve Stenosis (AVS), a prevalent valvular disease in the elderly, remains poorly understood. In this study, we aimed to identify the profile of exosome‐derived miRNAs, their potential target genes, and signaling pathways in AVS. Methods: Exosomes were isolated from aortic valve tissues obtained from AVS patients who underwent surgical aortic valve replacement (SAVR) [control, aortic insufficiency (n = 5) vs. AVS (n = 10)]. Exosome characterization was performed by Scan Electron Microscopy (SEM), Zeta view, and Flow Cytometry. A small RNA library was constructed, and sequencing was carried out using Illumina. Significant dysregulation of miRNAs was validated by qPCR, and gene ontology analysis was performed using DAVID and DIANA online tools. Results: Our results confirmed the successful isolation of exosomes with size (99.2nm), presence of exosome markers (CD63, CD81, and CD9), and cup‐shaped vesicles in SEM. We identified nine significantly dysregulated miRNAs in AVS patients compared to controls. Gene ontology analysis revealed the enrichment of cellular component terms, such as cytosol and exosome, and molecular function terms, including TGF‐β binding and SMAD binding. Using qPCR, we validated the upregulation of miR‐486‐5p and the downregulation of Let‐7c‐5p and miR‐30‐5p in AVS patients. Further in silico analysis revealed that these miRNAs targeted several genes involved in the PI3K, TGF‐β, and Wnt signaling pathways, which are known to play a significant role in the pathophysiology of AVS. In conclusion, our study identified dysregulated exosome‐derived miRNAs and their potential target genes and signaling pathways in AVS. Summary/Conclusion: Our findings may provide new insights into the underlying mechanisms of AVS, leading to the development of novel therapeutic approaches. Funding: This work was part of the TRR259 program, funded by the German Research Foundation (DFG). keywords: Exosomes, miRNAs, and Aortic Valve Stenosis. PS05: EV Cargo Characterization II Chairs: Martin van Rooijen Location: Hall 4A 16:50 ‐ 18:50 PS05.17. Cellular origin of extracellular vesicle influences the targeting – intercellular communication between blood cells Petra Ilvonen^1; Reetta Pusa^1; Kai Härkönen^1; Ulla Impola^1; Saara Laitinen ^2 ^1Finnish Red Cross Blood Service, Helsinki, USA; ^2Finnish Red Cross Blood Service, Helsinki, Finland Introduction: Blood‐derived extracellular vesicles (EVs) hold great therapeutic potential. As the whole blood and plasma contain mixed EV populations, it is challenging to study EVs originating from different cells separately. Blood cell concentrates manufactured in blood banks offer a good non‐invasive source of blood cell‐specific EV populations making it possible to study separately platelet or red blood cell EVs and their functions. Methods: In this study, EVs derived from platelet (TREVs) and red blood cell (EryEV) concentrate were used to investigate the EV interaction, targeting, and potential uptake into different populations of blood mononuclear cells. EVs were characterized for their purity, size, and shape as well as surface markers, using nanoparticle tracking analysis, electron microscopy and western blot analysis. Using imaging flow cytometry, we were able to evaluate the interaction and uptake of EVs into different mononuclear cells. To further understand their functional effect, we also studied the T cell activation capacity of these two EV populations using in vitro activation assay. Results: Our findings are in correlation with the previous results by Danesh A. et al. 2014. Blood. and Weiss R. et al. 2018 Sci Rep. results further showing that TREVs and EryEVs interact with CD3‐/CD11c+ mononuclear cells but TREVs also interact with CD3‐/CD19+ B‐lymphocytes. TREVs are interacting significantly more with CD11c+ cells compared to EryEVs. The interaction is also clearly different with the CD3‐/CD11c+ mononuclear cells as seen using imaging flow cytometry. We were able analyze the cellular localization of the EVs strengthening our previously reported result using label‐free targeting of the blood cell derived EVs to PC‐3 cells. These two EV populations have very different targeting properties and the cellular origin of the EVs effects uptake of the target cell. Summary/Conclusion: We can verify that blood cell derived EVs from healthy donors when cell specific can be studied using imaging flow cytometry with the accuracy that reveals differences in targeting and uptake. We also show that blood cell derived EVs used here are naturally non‐immunogenic increasing their potency either as therapeutic components in tissue repair or as drug delivery vehicles although further studies are needed. We believe that our results help to find previously ignored, new applications for donated blood components. Funding: This study was funded by Finnish Red Cross Blood Service and Business Finland Grant EVE Consortium 2346/31/2019 keywords: red blood cell; erythrosome; mononuclear cell; microvesicle; exosome; targeting PS05.18. Interindividual variance of plasma EVs in healthy adults and the influence of clinical, demographic, and lifestyle parameters Marija Holcar ^1; Ivica Marić^2; Katja Goričar^1; Tobias Tertel^3; Nina Mavec^4; Ana Plemenitaš^1; Urška Čegovnik Primožič^5; Darko Černe^5; Bernd Giebel^6; Metka Lenassi^1 ^1Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia; ^2Department of Immunohaematology, Blood transfusion centre of Slovenia, Ljubljana, Slovenia, Slovenia; ^3Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany, Essen, Germany; ^4Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, Slovenia; ^5Clinical Institute for Clinical Chemistry and Biochemistry, University Medical Centre Ljubljana, Ljubljana, Slovenia, Slovenia; ^6Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany, Essen, USA Introduction: Blood is commonly used for EV biomarker research of disease, yet little is known about plasma EVs in healthy population, limiting interpretation of the biomarker data. Here, we first systematically analyzed plasma EVs in healthy adults and the influence of clinical, demographic, and lifestyle parameters on interindividual variability. Methods: Blood and clinical (height, weight, blood pressure, menopause), demographic (sex, age), and lifestyle (exercise, smoking) data were collected from 208 blood donors (ethically approved, informed consent). Analysis on blood: complete blood count (CBC); on serum: hsCRP, insulin, ApoA1, ApoB and on plasma: CBC, PF4, hemoglobin. 25 surface markers were analyzed on EVs of 60 plasma samples (imaging flow cytometry). Alternatively, EVs were enriched from 208 plasma samples (UC on a 20% sucrose cushion, sUC) and analyzed for concentration (NTA), 37 surface markers (MACSPlex), and ApoA1, ApoB100 (ELISA). The variance in EV characteristics was determined and correlation with individuals’ parameters statistically evaluated. Results: Study subjects were equally distributed between sex (M/F), age groups (18‐29/30‐44/ 45–54/55‐65 years) and were healthy (normal ranges of CBC, insulin, CRP, lipoproteins). Plasma was not hemolyzed and had low residual/activated platelets. The average concentration of EV subtypes in plasma was 2.2*10^7/mL, but interindividual variance within the same EV type was from 13‐fold (CD9) to 5249‐fold (CD206). There was up to a 468‐fold difference between medians of different EVs (CD90+: 14.5*10^4/mL vs. CD227+: 67.8*10^6/mL) and up to a 121‐fold difference between the median of EVs, originating from the same cell type (e.g. endothelial: CD90+ vs. CD71+: 17.5*10^6/mL). After enrichment of EVs with sUC (median 5.65*10^9 particles/mL plasma), mostly blood‐cell and endothelial markers were detected on CD9+/CD81+/CD63+ EVs, with the highest signal for platelet‐derived EVs. The concentration of EVs (in plasma or enriched) did not correlate with CBC, clinical, demographic or lifestyle parameters, however, smoking correlated with the increase in enriched EVs of platelet, endothelial and leukocyte origin. Summary/Conclusion: There is a high interindividual variance in concentrations of plasma EVs in healthy adults, which is, except for smoking, not correlated to analyzed clinical, demographic and lifestyle variables, including sex and age. PS07: EV Heterogeneity 2 Chairs: Sarah Catherine Baker, Dolores DiVizio Location: Hall 4A 16:50 ‐ 18:50 PS07.12. EXO‐NET enriched salivary small extracellular vesicles in periodontitis Pingping Han^1; Chun Liu^1; Carlos Palma^2; Saso Ivanovski^1; Ramin Khanabdali ^3; Gregory E. Rice^4 ^1School of Dentistry, Epigenetics nanodiagnostic and therapeutic group, Center for Oral‐facial Regeneration, Rehabilitation and Reconstruction (COR3), Australia; ^2Inoviq Limited, Notting Hill, Victoria, Australia., Australia; ^3Inoviq Limited, Notting Hill, Australia., USA; ^4Inoviq Limited, Notting Hill, Victoria, Australia., Notting Hill, Australia Introduction: Salivary small extracellular vesicles (sEVs) have emerged as promising candidates for the diagnosis of periodontitis1, a common chronic inflammatory oral disease that is associated with microbial dysbiosis and host immune response2. Information on the isolation and characterization methods of saliva sEVs, as well as the use of sEV cytokines cargo as biomarkers for periodontitis, however, remains limited. The aims of this study were: (i) to compare the enrichment of salivary sEVs from 12 periodontitis‐free patients using size exclusion chromatography (SEC) and bead‐based immunoaffinity capture (EXO‐NET®); and (ii) assess the differential expression of EXO‐NET® enriched EVs assocaited inflammatory cytokines between individuals with (n = 20) and without periodontitis (n = 12). Methods: The study was approved by the Human Research Ethics Committee of The University of Queensland (HREC No. 2018001225) and by Metro North Hospital and Health Service (approval No. 54584). Whole unstimulated saliva (0.3 ml) was collected from 12 periodontitis‐free (4 females, 8 males) and 20 patients with periodontitis (7 females and 13 males). EVs were isolated according to manufacturer's instructions. Nanoparticle concetration of SEC‐EVs was determined by nanoparticle tracking analysis (NanoSight, Malvern Panalytical). EV‐associated protein was determined using BCA and infalmmatory cytokines (interleukin (IL) ‐ 6, IL‐1β, IL‐8 and IL‐10) were quantified by ELISA. Bacterial pathogens were determined by genomic DNA qPCR for 5 periodontal pathogenes (Tannerella forsythia, Eikenella corrodens, Porphyromonas gingivalis, Peptostreptococcus anaerobius, and Treponema denticola). Results: EVs isolated by immunoaffinity capture contained more protein content than ‐sEVs iolated by SEC without altering the CD9 sEV subpopulation. Furthermore, immunoaffinity‐enriched salivary sEVs contained less periodontal pathogens than SEC‐sEVs, that contained abundant bacteria‐derived DNA. Additionally, immunoaffinity‐enriched sEVs from periodontitis patients contained higher amounts of inflammatory cytokines than those from periodontitis‐free patients. Summary/Conclusion: The results indicate that immunoaffinty capture is a reliable method for enriching salivary sEVs without contamination from bacteria‐derived EVs or DNA. Moreover, the study suggests that immunoaffinty capture ‐enriched sEVs can serve as biomarkers for periodontitis, with increased expression of pro‐inflammatory cytokines observed in sEVs from patients with periodontitis. keywords: Saliva derived EVs, EVs in periodontitis, EXO‐NET captured EVs, PS09: Preeclampsia, Reproduction and Development 2 Chairs: Metka Lenassi, Rienk Nieuwland Location: Hall 4A 16:50 ‐ 18:50 PS09.08. Novel oligonucleotide barcode tagging of semen‐derived extracellular vesicles (SEV) Phuong Nguyen ^1; Shahrokh Paktinat^2; Elizabeth Nance^3; Lucia Vojtech^4 ^1Molecular Engineering, University of Washington, Seattle, USA; ^2Obstetrics & Gynecology, University of Washington, USA; ^3Chemical Engineering, University of Washington, USA; ^4Obstetrics & Gynecology, University of Washington, Seattle, USA Introduction: Immune tolerance to the fetus is essential for a successful pregnancy. Semen contains extracellular vesicles (SEV) that may interact with antigen presenting cells (APCs) to confer fetal tolerance. Investigating the mechanisms through which SEVs confer tolerance through APCs could improve health outcomes for mothers and inform future diagnostics and therapeutics for pregnancy complications. We optimized and deployed novel barcode oligonucleotide (oligo) labeling to quantify SEV association with APCs in vaginal tissue. Methods: SEV were purified from diluted semen by ultracentrifugation at 100,000xg over 25% sucrose cushions for 12h, then washed with PBS and concentrated in 100 kDa spin filters. SEV were characterized by nanoparticle tracking, electron microscopy, and western blotting. As previously published, SEVs are then click‐conjugated to an oligo barcode (oligo‐SEV) and directly exposed to human vaginal cells for 6 and 24h. Unconjugated oligo and cells alone are used as negative controls. APCs are separated with magnetic bead isolation against CD11c, and both CD11c+ cells and non‐APC remaining cells were analyzed. Direct PCR was used to prepare cell lysate for amplification, followed by stem loop RT‐qPCR to detect target oligo expression. Results: In vitro oligo‐SEV exposure experiments on human vaginal cells yielded 3 key results: 1) Oligo‐SEV conjugates remained detectable in cell culture at 6h and up to 24h of direct exposure, 2) cells preferentially uptake oligo‐SEVs compared to unconjugated oligo by a million‐fold, and 3) oligo‐SEVs were significantly localized with APCs compared to other cell types by 50‐fold. Summary/Conclusion: These results demonstrate the stability of oligo‐SEV conjugations in physiological conditions and suggest early and preferential colocalization of SEVs to APCs following acute exposure. Our novel oligo‐SEV tagging is a promising platform to quantify EV trafficking to different cell populations. In addition, our results support the hypothesis that SEVs, in tandem with APCs, may play an important role in the conferral of immune tolerance during pregnancy. Funding: R01AI153342 (PI Vojtech) keywords: Semen EVs, oligonucleotide barcoding, EV tagging PS09.09. Isolation, characterization, and determination of the immunomodulatory potential of small extracellular vesicles derived from menstrual fluid from nulliparous and multiparous women Paz Cerda‐Castro^1; Hugo Tobar^2; Stephanie Acuña‐Gallardo^3; Javiera De Solminihac^4; Karina Pino‐Lagos^4; Patricia Valdebenito^5; Vicente Peragallo‐Papic^6; Jimena Cuenca^2; Sebastián Illanes^5; Francisca Alcayaga‐Miranda ^2 ^1IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Centro de Investigación e Innovación Biomédica (CiiB), Universidad de los Andes, Santiago, Chile, Chile; ^2IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Centro de Investigación e Innovación Biomédica (CiiB), Universidad de los Andes, Santiago, Chile, Santiago, Chile; ^3Laboratory of Reproductive Biology, CiiB, Universidad de los Andes, Santiago, Chile, Santiago, Chile; ^4Facultad de Medicina, CiiB, Universidad de los Andes, Santiago, Chile, Santiago, Chile; ^5Laboratory of Reproductive Biology, CiiB, Universidad de los Andes, Santiago, Chile., Santiago, Chile; ^6Laboratory of Reproductive Biology, CiiB, Universidad de los Andes, Chile., Santiago, Chile Introduction: Introduction: Data from our laboratory demonstrate that mononuclear cells in menstrual fluid (MF) from nulliparous and multiparous women show a different immune signature. Since extracellular vesicles (EVs) have an established immunomodulatory role, this research seeks to determine if MF‐derived EVs of nulliparous and multiparous (N/M) donors possess different immunomodulatory potentials on lymphocytes establishing a protocol for the isolation and characterization of MF‐derived EVs and defining their immunomodulatory potential. Methods: Methods: EVs were isolated from MF of N/M donors through a protocol based on multiple filtrations and centrifugations. EVs were characterized by concentration, size, morphology, and protein markers. The immunomodulatory potential was assessed through a lymphocyte immunosuppression assay. Results: Results: An efficient protocol for isolation of MF‐derived EVs was established. MF‐derived EVs from N/M donors possess a similar concentration of nanoparticles that share morphological characteristics. However, MF‐derived EVs from M showed lower expression of exosomal markers with respect to N donors. Interestingly, MF‐derived EVs from N donors showed more immunomodulation capacity than those obtained from M women. Summary/Conclusion: Summary/Conclusion:: This study characterized the EVs obtained from MF of healthy (N/M) donors, providing baseline data and information on EVs of this unique source that are not currently recorded. Funding: ANID—Basal funding for Scientific and Technological Center of Excellence, IMPACT, #[50]FB210024 & ANID‐FONDECYT#1190411 PS10: Lung EVs Chairs: Brian Dobosh, Augusto Zani Location: Hall 4A 16:50 ‐ 18:50 PS10.13. Artificial Intelligence to Accelerate New Drug Discovery: Data‐driven patient stratification and proposal of novel drug targets using exosome proteomics and medical information of idiopathic pulmonary fibrosis (IPF) patients Yoshito Takeda ^1; Mari Nogami‐Itoh^2; Jun Adachi^3; Yayoi Natsume‐Kitatani^4; Atsushi Kumanogoh^1 ^1Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Japan; ^2Artificial Intelligence Center for Health and Biomedical Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Japan; ^3Laboratory of Proteome Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Japan; ^4Artificial Intelligence Center for Health and Biomedical Research, National Institutes of Biomedical Innovation, Health and Nutrition, Japan Introduction: If the data‐driven drug target discovery using human data is possible with technologies like machine‐learning, the benefit will be particularly enormous for intractable diseases for which the mechanisms of disease development are not fully understood at the molecular level. We constructed a database of serum exosome proteome data that is linked with their medical information on interstitial pneumonia including IPF and developed a novel machine learning algorithm for patient stratification called subset binding. Methods: We collected clinical data through electronic medical record entries (medical record and initial medical questionnaire, CT imaging interpretation report, blood test data) to input data generation. Total 602 patients were enrolled in Osaka University Hospital. In order to isolate exosome, phosphatidylserine‐positive extracellular vesicles were purified from 200 μl of serum using MagCapture isolation kit (Fujifilm Wako). Pretreated samples were subjected to LC‐MS/MS analysis using the Data independent acquisition (DIA) method. Results: We obtaining 6,506 (6,282 attributes from CT image interpretation reports, 171 attributes from blood test results and 53 attributes from medical records) ×602 cases (with overlap from 403 patients and 39 controls) of medical information and 2,445 protein ID ×602 cases matrices. In this study,, subset binding (SB), a newly developed algorithm was used to detect patient stratification rules using structured medical information and proteome data. Thus, we found 20 proteins that were co‐occurring with items of medical information related to IPF. We further identified the upstream regulators of these proteins by network analysis and found several novel drug target candidates. We have experimentally validated their relevance using lung tissue from IPF patients, metabolites in blood and urine, and an epithelial‐mesenchymal transition test in human airway epithelial cells. Summary/Conclusion: Our approach demonstrated that data‐driven linkage by subset‐binding between phenotype‐level data and biomolecule‐level data of diseases can be used for drug target discovery through patient stratification. Funding: Cabinet Office of Japan Government for the Public/Private R&D Investment Strategic Expansion PrograM (PRISM) keywords: exosomes, AI, New Drug Discovery, proteomics,: heterogeneous data integration, biomarker, patient stratification, idiopathic pulmonary fibrosis (IPF) PS10.14. Cancer stem cell derived extracellular vesicle secretion mediated by RAB27B drives stem like phenotype in NSCLC Prita Pandya ^1; Kayleah Meneses^1; Jennifer Lindemann^2; Dania Al‐Qasrawi^3; Ryan Argo^3; Celeste Weems^4; Danielle Beetler^1; Irene Yan^5; Tushar Patel^5; Joy Wolfram^6; Verline Justilien^7 ^1Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA, USA; ^2University of North Florida, Jackonsville, FL, USA, USA; ^3Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA, USA; ^4University of North Florida, Jacksonville, FL, USA, USA; ^5Department of Transplantation, Mayo Clinic, Jacksonville, FL, United States, Jacksonville, USA; ^6School of Chemical Engineering/Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia, USA; ^7Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, United States, USA Introduction: The cancer stem cell (CSC) population within NSCLC tumors have been shown to be involved in metastasis, relapse, and therapeutic resistance. Therefore, understanding the mechanisms by which CSCs mediate the aggressiveness may provide insights for developing more effective NSCLC therapeutic interventions. Based on the RNA sequencing data, we identified RAB27B, which functions as a small GTPase, significantly upregulated gene in CSCs, compared to bulk cancer cells (BCC). The objective of our current study is to determine the mechanism by which RAB27B promotes an aggressive stem‐cell like phenotype. Since RAB27B plays a role in extracellular vesicle (EV) secretion; we hypothesize that RAB27B mediates the aggressive phenotype of NSCLC through EV‐directed pathways. Methods: CSC and BCC‐derived EVs were isolated by ultracentrifugation and characterized by transmission electron microscopy, western blotting and nanoparticle tracking analysis based on MISEV 2018 guidelines. For phenotypic assay, BCCs and CSCs were treated with increasing dilutions of BCC‐derived and CSC‐derived EVs and monitored for expansion and migration. For EV uptake experiment, BCCs were incubated with equivalent DiI‐labelled CSC‐ or BCC‐derived EVs in growth medium depleted of exogenous EVs. Results: We found that RAB27B is required for the maintenance of metastatic stem‐like cell population in NSCLC. Moreover, CSCs secrete significantly more EVs compared to BCCs and knockdown of RAB27B leads to a decrease in the levels of EVs secreted. Also, CSC‐derived EV cargo molecules are internalized more compared to BCC‐derived EVs. Furthermore, delivery of CSC‐derived to BCCs induces spheroid growth, clonal expansion, and invasion in BCCs. Summary/Conclusion: To summarize, we show that CSCs which show significant upregulation of RAB27B, is associated with increase in EV secretion. CSC‐derived EVs are preferentially internalized by BCCs, and which leads to increased clonal efficiency, spheroid growth and invasion. Funding: This work is supported by a grant from the V Foundation. Prita Pandya is supported by the Mayo Clinic Graduate School of Biomedical Sciences. PS10.15 Increased generation of extracellular vesicles (EVs) from primary lung epithelial cells after ethanol and e‐cigarette (e‐Cig) exposure and EVs paracrine effects via purinergic P2×7r signaling Naveen Mekala ^1; Jayshil Trivedi^2; Namdev Shivaji Togre^2; Slava Rom^3; Uma Sriram^2; Yuri Persidsky^2 ^1Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA, Philadephia, USA; ^2Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA, Philadelphia, USA; ^3Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA, Phiadelphia, USA Introduction: Exposure to alcohol and e‐cigarette (e‐cig) smoke stimulates inflammation. ATP and its signaling via the P2×7r promote proinflammatory response in many cells. Previously, we reported elevated P2×7r expression, Ca2+ accumulation, and ATP release after ethanol (ETH), acetaldehyde (ALD), or e‐cig exposure in human brain microvascular endothelial cells (BMVEC), which were normalized by a P2×7r antagonist, A804598. In the current study, we examined extracellular vesicle (EV) generation and P2×7r distribution in EVs from primary human pulmonary alveolar epithelial cells (PAECs) after ETH, ALD or e‐cig exposure. Methods: PAEC (ABC‐TC3770, AcceGen) pretreated with A804598 (4473, Tocris BioSci) were exposed to ETH (100mM), ALD (100μM), and 1.75μg e‐cig‐conditioned media (1.8% nicotine) overnight. EVs were isolated from media using Exoquick‐TC (EXOTC50A‐1, SBI). EV size and concentration were analyzed by Nanosight NS300. ATP levels in isolated EVs were measured using an ATP detection kit (ab113849, Abcam). P2×7r presence on EVs was assessed by flow cytometry using the Tetraspanin Exo‐Flow Capture kit (EXOFLOW150A‐1, SBI). Paracrine signals were evaluated in BMVECs, treated with EVs from PAEC, and intracellular Ca2+ levels were assessed by Ca2+ assay (ab102505, Abcam). Results: Exposure to ETH, ALD, and e‐cig increased the size (by 20–30%) and number of EVs (7‐fold by ETH, 1.4‐fold by ALD, and 2.3‐fold by e‐cig). The A804598 pretreatment reduced ATP levels to control levels in EVs. Flow cytometric analysis of EVs revealed increased P2×7r expression (median fluorescence intensity, MFI) after ETH (1.6‐fold), ALD (1.5‐fold) and 1.8% e‐cig exposure (1.43‐fold), which was reduced to control levels by A804598. BMVECs, treated with EVs from PAEC, increased intracellular Ca2+ levels (2‐fold by ETH and 4‐fold by ALD or e‐cig), while EVs, derived from A804598‐pretreated PAEC, had no effect on Ca2+ accumulation in BMVEC. Summary/Conclusion: PAEC exposure to ETH, ALD and 1.8% e‐cig‐conditioned media increased EVs and their ATP/P2×7r content. These lung secreted EVs, by transporting secondary messengers, affect BMVEC, and thus changing Blood Brain Barrier function. keywords: P2×7r; ethanol, electronic cigarette; A804598; lung epithelial cells PS10.16. Untargeted metabolomic analysis of an ARDS murine model treated with engineered extracellular vesicles loaded with anti‐inflammatory cytokines Tatiana Z. Cuellar Gaviria ^1; Ana I. Salazar‐Puerta^2; María A. Rincon‐Benavides^3; Julian Aldana^4; Daniel Gallego‐Perez^5; Natalia Higuita‐Castro^6 ^1Biomedical Engineering, The Ohio State University, Columbus, USA; ^2Biomedical Engineering, The Ohio State University, USA; ^3Biophysics program and Biomedical Engineering Department, The Ohio State University, USA; ^4Biochemistry Program, The Ohio State University, USA; ^5Biomedical Engineering and General Surgery, The Ohio State University, USA; ^6Department of Biomedical Engineering and Department of Surgery, The Ohio State University, USA Introduction: Acute respiratory distress syndrome (ARDS) causes the retention and accumulation of fluid in the lungs, preventing oxygen exchange. Currently, there is no cure for ARDS. In our lab, we developed a drug‐delivery system based on engineered EVs (eEVs) loaded with anti‐inflammatory cytokines. Treatment with this eEVs have shown to reduce inflammation in an ARDS mouse model, causing a reduction in the secretion of pro‐inflammatory cytokines, neutrophil infiltration, as well as tissue damage. In this work, we performed an untargeted metabolomics approach to elucidate the global changes in the metabolites of the lung induced upon treatment with eEVs. Methods: eEVs were obtained from adult mouse dermal fibroblasts transfected with the corresponding molecular cargo. eEVs isolation was performed using a combination of centrifugation, centrifugal concentration using vivaSpin 500 with a MWCO of 100 kDa, and EV content was subsequently isolated using Total exosome isolation reagent. eEVs contents were characterized using qRT‐PCR, western blotting, and ELISA. Size distribution was determined via NanoSight and CryoTEM. BALB/c mice challenged with LPS were used as model of ARDS. Treatment with eEVs was done 6 h post LPS treatmet and bronchoalveolar lavage fluid (BALF) was collected 6 h after treatment with eEVs. Metabolomic profiles of LPS‐challenged mice, healthy mice, LPS‐challenged mice post‐treated with eEVs, or control eEVs (i.e., Sham) were compared in this study. Data acquisition was carried out in a 6545 Q‐TOF LC/MS coupled to a HPLC system (Agilent 1290 Infinity LC), using a SB‐C18 column. Samples were processed in positive and negative ionization modes. Feature detection was carried out in MZmine v.2.53 and statistical analyses were done using Metaboanalyst 5.0. Results: Metabolomic profiles in BALF samples of LPS‐challenged mice was clearly impacted by treatment with eEVs loaded with anti‐inflammatory cargo, but also significantly different than the treatment with Sham eEVs. We identified the presence of several metabolites with anti‐inflammatory and antioxidant properties in the BALF of eEV treated animals. Summary/Conclusion: Different metabolomic responses were elicited depending on the molecular payload of the eEVs and higher concentrations of anti‐inflammatory and anti‐oxidant metabolites were observed in the eEVs. To our knowledge, this is the first report to show that eEVs have the potential to shift the metabolomic profiles in animals with acute lung inflammation. Funding: This work was partially funded by The Ohio State University Office of Research and the NIH grants 1R01AR079485‐01A1 (awarded to NHC), DP1DK126199 and DP2EB028110 (awarded to DGP), and to the College of Engineering LEGACY postdoctoral program (awarded to TCG) keywords: Engineered extracellular vesicles, drug delivery, metabolomics PS12: EVs in Cancer Progression Chairs: Alissa Weaver Location: Hall 4A 16:50 ‐ 18:50 PS12.01. Circulating extracellular vesicles derived from tumor endothelial cells blunt the immune response via broadly increasing myeloid‐derived suppressor cells Malvina Koni^1; Tatiana Lopatina^1; Cristina Grange^2; Alessandro Sarcinella^1; Stefania Bruno^3; Massimo Cedrino^4; Fabrizio Buffolo^1; Giovanni Camussi^5; Maria Felice MF Brizzi ^1 ^1University Of Turin, Torino, Italy; ^2Department of Medical Sciences, University of Turin, Turin, Italy; ^3Department of Medical Sciences, University of Turin, Turin, Italy, Turin, Italy; ^43. Molecular Biotechnology Center, Fondazione per le Biotecnologie, University of Turin, Torino, USA; ^5Department of Medical Sciences, University of Turin, Torino, Italy Introduction: Circulating tumor‐derived extracellular vesicles were supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumor‐endothelial cells (TEVs) in the expansion of the metastatic bulk. Particular attention has been dedicated to the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model. Methods: Mice were intravenously injected with 4T1 cells and treated with TEVs starting from day 7 till sacrifice on day 18. Lung, spleen, and bone marrow were recovered for the analyses. Results: We demonstrated that TEV‐treated mice display an increased number of lung metastasis. A significant swelling of the spleen and an immune reaction were detected in the animals treated with TEVs. FACS and immunohistochemically analyses revealed that CD11b+/F4‐80+/Ly6G+ cells (immunosuppressive macrophages) as well as CD11b+/Ly6G+/F4‐80‐ (myeloid‐derived suppressor cells: MDSCs) were significantly enriched in the lung tissue of TEVs‐injected animals. Matching populations were also increased in the spleen and bone marrow suggesting a systemic and local immune suppression associated with the presence of circulating TEVs. The increased expression of PD‐L1 and PD‐1 positive cells in the tumour mass also supports TEV‐immune‐suppressive action. Consistently, in vitro TEVs were able to upregulate the expression of LAG3, CTLA4 and PD‐1 in T‐cells and PD‐L1 in myeloid cells and cancer cells. ROS and NOS release and an impaired T‐cell‐mediated cytotoxic effect confirmed the suppression of immune response in vitro. Consistent with in vivo results, showing an increased level of granulocyte‐colony stimulating factor (G‐CSF) in the presence of circulating TEVs, in vitro experiments demonstrated that G‐CSF is crucial for TEVs‐mediated immunosuppression. Summary/Conclusion: In conclusion, we provide evidence that circulating TEVs boost the metastatic bulk and that this effect was associated with TEV‐mediated immune‐cell reprogramming resulting in an impaired anti‐tumor‐immune response. Funding: University of Turin,ex 60% keywords: tumor‐endothelial cell‐derived EVs, TNBC, Immune cells PS12.19. Aberrant EV Secretion by Phosphoserine Aminotransferase 1 Promotes Cancer Metastasis Tomofumi Yamamoto ^1; Jun Nakayama^2; Kagenori Ito^3; Yusuke Yamamoto^3; Takahiro Ochiya^4 ^1Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjuku‐ku, Japan; ^2Laboratory of Integrative Oncology, National Cancer Center Research Institute, Tokyo, Japan; ^3Laboratory of Integrative Oncology, National Cancer Center Research Institute, Chuo‐Ku, Japan; ^4Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan Introduction: Extracellular vesicles (EVs) play an important role in the formation of the cancer microenvironment. In general, serine synthesis is promoted in cancer cells to facilitate glycolysis. We reported that phosphoserine aminotransferase 1 (PSAT1) promoted serine synthesis, was expressed in many types of cancer cells, and increased EV secretion. Interestingly, the protein level of PSAT1 in metastatic cancer cell lines was higher than that of their parental cell lines; however, the aberrant secretion of EVs by PSAT1 is not known to contribute to metastasis. Methods: Co‐culture system was used for the functional assay of EVs. Raw 264.7 cells were seeded in lower side, and bone metastatic cancer cells were seeded in the upper chamber (pore size: 0.4 μm). After the co‐culture, we measured the activation of osteoclasts by tartrate‐resistant acid phosphatase (TRAP) staining. To understand the functions the EVs from PSAT1‐overexpressing (OE) cells, EVs were isolated from PSAT OE MCF‐7 cells and treated with parental MCF‐7 for 2 weeks. In animal studies, cancer cells (MCF‐7 and MDA‐MB‐231) were injected through caudal artery and caudal vein, and bone metastasis and lung metastasis were monitored with in vivo imaging system. PSAT1 knockout (PSAT1 KO) cells were established by CRISPR‐cas9 system. Results: Firstly, we established PSAT1‐overexpressing cell lines (MCF‐7 PSAT1 OE and MDA‐MB‐231 PSAT1 OE). These cell lines secreted 2–5 times more EVs than the parental cell lines. According to the co‐culture assay, PSAT1‐overexpressing cell lines promoted the differentiation of osteoclasts. Two weeks of EV education from PSAT1 overexpressing cell lines also enhanced osteoclasts differentiation. These results indicated that EVs infuluenced not only osteoclasts but also the cancer cells themselves. In animal models of bone metastasis, our data clearly showed that PSAT1‐overexpressing cells promoted bone metastasis. The same trend was also found in the lung metastasis model. In contrast, PSAT1 KO cell lines reduced the EV secretion. This suppression of EV secretion was rescued by PSAT1 overexpression. Summary/Conclusion: These data suggested that aberrant EV secretion by PSAT1 enhanced bone and lung metastasis in breast cancer cells. keywords: Serine Metabolism, metastasis PS12.20. Plasma exosomes from type 2 diabetic patients drive expression of EMT‐related genes in prostate cancer models Michael D. Seen ^1; Yuhan Qiu^2; Pablo Llevenes^3; Rebecca Yu^3; Naser Jafari^4; Andrew Chen^3; Isabella Pompa^4; Christina Ennis^3; Joakin Mori^5; Kiana Mahdaviani^6; Meredith Halpin^5; Gretchen Gignac^5; Christopher Heaphy^5; Stefano Monti^3; Gerald Denis^3; Heejoo Kang^3; Manohar Kolla^3 ^1Boston University, Boston, USA; ^2Boston University School of Medicine, Medford, USA; ^3Boston University School of Medicine, USA; ^4Boston University‐Boston Medical Center Cancer Center, USA; ^5Section of Hematology and Medical Oncology, Department of Medicine, Boston University School of Medicine and Boston Medical Center, USA; ^6Boston Medical Center, USA Introduction: Uncontrolled diabetes is associated with poorer outcomes among prostate cancer patients. We hypothesize that differences in the miRNA payload of plasma exosomes among individuals with Type 2 diabetes (T2D) may contribute to poorer prostate cancer outcomes by promoting tumor aggressiveness. Methods: Exosomes were isolated from platelet‐free plasma using size‐exclusion chromatography. NanoSight was used to measure exosome sizes and concentrations. Exosomal miRNA profiles were obtained using a commercially available PCR array. DU145 cells were treated with exosomes for 2 days. Total RNA was extracted from the exosome‐treated cells. Relative expression of genes related to epithelial‐to‐mesenchymal transition (EMT) was measured using commercially available PCR array. RNA sequencing and Principal Component Analysis was used to measure changes in global transcription. Results: The plasma exosome miRNA profiles of the T2D group differ significantly from that of the ND group. Treatment of DU145 cells with T2D plasma exosomes resulted in increased expression of EMT‐related genes when compared with DU145 cells treated with ND plasma exosomes. Individual recombinant miRNAs represented in the profile are able to drive EMT transcription. Cells treated with ND exosomes displayed global transcription patterns similar to each other as well as to cells treated with media‐only control exosomes. By contrast, DU145 cells treated with T2D plasma exosomes displayed wide variance in global transcription pattern. Summary/Conclusion: The miRNA payload of plasma exosomes appears to be altered in T2D and has functional impact on tumor cells, thereby contributing to a poorer prostate cancer outcome among patients with co‐morbid T2D. PS12.21. Priming anti‐tumor response with poxvirus‐induced extracellular vesicles Lucas WALTHER ^1; Caroline TOSCH^2; Jules DEFORGES^2; Christine Carapito^3; Magali ROMPAIS^4; Marie‐Christine CLAUDEPIERRE^2; Christelle REMY^2; Nathalie SILVESTRE^2; Kuang‐Jing HUANG^5; Kaidre BENDJAMA^2; Eric QUEMENEUR^2; Jacky G. Goetz^6; Vincent HYENNE^6; Karola RITTNER^2 ^1TRANSGENE / CRBS Strasbourg, Illkirch‐Graffenstaden, France; ^2TRANSGENE, France; ^3Laboratoire de Spectrométrie de Masse Bio‐Organique, Institut Pluridisciplinaire Hubert Curien, UMR 7178, CNRS‐Université de Strasbourg, Strasbourg, France., Strasbourg, France; ^4Laboratoire de Spectrométrie de Masse Bio‐Organique, Institut Pluridisciplinaire Hubert Curien, UMR 7178, CNRS‐Université de Strasbourg, Strasbourg, France., France; ^5Tumor Biomechanics Lab, INSERM UMR_S1109, Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Equipe Labellisée Ligue contre le Cancer, Strasbourg, France., France; ^6Tumor Biomechanics Lab, INSERM UMR_S1109, Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Equipe Labellisée Ligue contre le Cancer, Strasbourg, France., Strasbourg, France Introduction: Viruses‐based therapies show promise in inducing systemic anti‐tumor immunity. Over the past years, modified poxviruses were developed to induce a sustained antitumoral immune response. We previously reported MVA induced T‐cell response against HPV oncoviral antigens E6 and E7 in HPV anogenital cancers ([51]NCT03260023). In this work, we inquired whether extracellular vesicles (EVs), known as mediators of intercellular communication, play a role in these therapeutic vaccination processes. Methods: Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors, or DC2.4 mouse dendritic cell line were cultivated in serum EV‐depleted medium and infected by three strains of GFP‐expressing poxviruses at a multiplicity of infection (MOI) of 1 for 20h or 10h respectively. Small EVs were isolated from cell culture supernatant by differential centrifugation, filtered on 0.1μm and ultracentrifuged at 100,000 g for 2h (2 cycles), before being resuspended in PBS. Results: Our data showed that 0.1μm filtration allowed the complete separation of EVs from infectious viral particles. We demonstrate that infection of PBMCs with Poxviruses led to a 5‐fold increase in the amount of secreted EVs. Next, we investigated the molecular content of EVs fractions isolated from MVA‐infected PBMCs by mass spectrometry. We observed differences in the abundance of human proteins and several viral proteins, including virus‐encoded GFP were detected in EVs from infected cells. Finally, virus‐free EVs were isolated from DC2.4 mouse dendritic cell line infected with therapeutic MVA vaccine candidate encoding for GFP fused to OVA and additional cargoes that cannot be disclosed due to confidentiality reasons. Systemic injections of these EVs in C57BL/6 murine E.G7‐OVA tumor model induce a reduction of tumor size. Summary/Conclusion: We successfully separated small EVs from poxviruses using 0.1μm filtration. Poxviral infection increases EVs secretion from infected cells (PBMCs & DC2.4 cell line) and modifies their protein content. Previously described EVs contain GFP‐OVA fusion protein and are effective to reduce tumor growth in EG.7‐OVA tumor‐bearing mice upon iv injection (n = 1). Further analyses are required to fully characterize the molecular content and immune function to highlight the underlying mechanisms of these EVs. Funding: This work was supported by Transgene S.A. PS13: EV Production for Therapeutics Chairs: Anthony Yan‐Tang Wu, Sander Koojimans Location: Hall 4A 16:50 ‐ 18:50 PS13.20. Comparison of active drug loading methods and effect on EV properties Neona M. Lowe ^1; Rachel R. Mizenko^2; Alyssa Panitch^3; Randy P. Carney^2 ^1University of California, Davis, USA; ^2University of California, Davis, Davis, USA; ^3Georgia Institute of Technology and Emory University, Davis, USA Introduction: Since engineered EVs have emerged as a promising drug delivery system, diverse methods have been used to load EVs with active pharmaceutical ingredients (APIs) in preclinical and clinical studies. However, there is rarely optimization of methods or comparison across different methods for specific API. Furthermore, the heterogeneity of loading across EV subpopulations is largely unexplored. There is therefore a need for direct comparison of optimized methods with single particle analysis across APIs and EV types. Methods: EVs were collected from HEK2943T cells via ultracentrifugation and labeled with carboxyl fluorescein succinimidyl ester (CFSE). Rhodamine B, the surrogate cargo, was incubated with CFSE labeled EVs, and active drug loading methods of sonication, electroporation, and freeze‐thaw were performed. EVs were isolated from free dye via size exclusion chromatography and analyzed with ExoView chips to determine the number of CD9/CD63/CD81 captured particles with CFSE and rhodamine fluorescence. Results: Sonication was found to significantly increase the number of rhodamine particles (surrogate cargo) compared to incubation. However, the number of CFSE particles (protein label for EV tracking) was significantly greater in sonicated samples compared to incubated samples, which suggests that sonication is increasing the number of EVs. Data also suggests that sonication leads to aggregation of EVs. For electroporation, increasing voltage was found to decrease rhodamine and CFSE particle numbers. This is potentially due to leakage of CFSE and rhodamine from within the EVs. The number of freeze‐thaw cycles was not found to significantly increase the number of rhodamine particles. Summary/Conclusion: Here we demonstrate that active drug loading methods of sonication and electroporation affect EV properties and characteristics with single particle analysis. These changes could have functional implications such as cellular uptake. Funding: The research was supported by T32 [52]HL007013 (NML). keywords: drug loading PS14: EV Therapy Chairs: Tom Driedonks, Minh Le, Sai Kiang Lim Location: Hall 4A 16:50 ‐ 18:50 PS14.15. Extracellular vesicles (EVs) with loaded microRNA to treat HER2‐positive breast cancer Hanine A. El Itawi ^1; Shakhlo Aminova^1; Jeannie Lam^2; Yuki Harada^1; Masako Harada^1 ^1Michigan State University, East Lansing, USA; ^2Michigan Stae University, USA Introduction: Overexpression of human epidermal growth factor receptor 2 (HER‐2) is associated with an aggressive type of breast cancer with poor prognosis. Extracellular vesicles (EVs) naturally transport microRNAs, thereby, ideal delivery vehicles for therapeutic miRNAs such as miR‐203. Engineering the EV surface with cell‐type specific molecules enables targeted delivery. In this study, HER2‐specific EV‐mediated miRNA delivery and miR function on tumor cells are evaluated. Methods: EVs loaded with microRNAs were generated by co‐transfecting synthetic miRNAs to Human Embryonic Kidney (HEK‐293T) cells. After 24‐h, the cells were washed, and the culture media was replaced with DMEM supplemented with serum‐free additives and cultured for another 24‐h for EV generation. EVs were isolated by a series of differential centrifugations followed by ultracentrifugation. After isolation, engineered EVs (eEVs) were characterized for size, morphology, and surface markers respectively with nanoparticle tracking analysis, super‐resolution microscopy, and western blotting. EV binding to BT474 and SKBR3 (HER2‐positive cells) and MCF‐7 (HER2‐negative cells) was assessed by bioluminescence imaging using gaussia luciferase (gLuc) labeled EVs and by fluorescent microscopy using mCherry labeled EVs. The functional effect was tested by examining cell growth and target gene downregulation. Results: eEVs packaging of exogenous miR‐203 was confirmed. The binding assay showed that HER2‐targeting‐eEVs specifically bound to HER2‐positive cells in culture and transferred encapsulated miR‐203 to inhibit target gene expression and reduced cell proliferation. Summary/Conclusion: These findings suggest that miRNA replacement therapy might improve HER2 cancer treatment by harnessing targeted delivery via engineered EVs. keywords: Keywords: Extracellular vesicles, miRNA‐based delivery, HER2‐overexpressing breast cancer PS14.18. The abstract is about “exRP250: An Extracellular Vesicle‐based Natural Microbial Product for Anti‐inflammatory Therapy with Sugar Moiety Conjugated GlycoRNA as an Immunomodulator and Anti‐Biofilm Agent” Vinay Kumar Tiwary Exrna Therapeutics, Bodhagaya, Gaya, India Introduction: Psoriasis is a chronic, autoimmune skin disease characterized by red, scaly patches that can be itchy and painful. Current treatments include topical and systemic therapies, but they often have limitations and side effects. exRNA Therapeutics Limited has developed a natural microbial product called exRP250, which is an extracellular vesicle (EV) containing different amino acids, vitamins, lipids, nucleic acids, etc. exRP250 shows excellent anti‐psoriatic activity by RNAi therapy and reducing amino acid starvation. Moreover, exRP250 is conjugated with sugar moieties, i.e., glycoRNA, which enhances its stability and reduces toxicity. This study explores the potential of exRP250 as an immunomodulator and anti‐biofilm agent for the treatment of psoriasis. Methods: exRP250 was tested in both invitro as well as in‐vivo pre‐clinical experiments to demonstrate its anti‐psoriatic and anti‐biofilm activity. The extracellular vesicle‐like structure of exRP250 was characterized, and its conjugation with sugar moieties, i.e., glycoRNA, was evaluated for its stability, toxicity, and ability to deliver the drug to the target site. Further research is ongoing to investigate the mechanisms of action underlying these findings. Results: The results of this abstract include: exRP250, a natural microbial product, shows excellent anti‐psoriatic activity by RNAi therapy and reducing amino acid starvation. exRP250 is an EV‐like structure conjugated with sugar moieties, i.e., glycoRNA, which enhances its stability, reduces toxicity, and helps in delivering the drug to the target site. exRP250 has anti‐biofilm activity by depressing (p)ppGpp levels in live bacterial cells by RNAi mechanism, preventing biofilm formation, and containing different amino acids and RNases that help in the breakdown of biofilm. Further research is ongoing to investigate the possible mechanisms underlying these findings. Summary/Conclusion: This abstract describes exRNA Therapeutics Limited's development of a natural microbial product called exRP250 for the treatment of psoriasis. exRP250 is an extracellular vesicle (EV)‐like structure conjugated with sugar moieties, i.e., glycoRNA, that enhances its stability, reduces toxicity, and helps in delivering the drug to the target site. exRP250 exhibits anti‐psoriatic activity by RNAi therapy and reducing amino acid starvation, as well as anti‐biofilm activity. Further research is ongoing to investigate the mechanisms underlying these findings. PS14.20. Targeting the immunosuppressive tumour microenvironment with virally generated small extracellular vesicles Victoria A. Jennings ^1; Reah Rumbold‐Hall^1; Gemma Migneco^2; Nicola Ingram^1; Carolina Ilkow^3; John bell^4; Alan Melcher^5; Fiona Errington‐Mais^1; Graham Cook^1 ^1University of Leeds, United Kingdom; ^2Drexel Univeristy, USA; ^3Ottawa Hospital Research Hospital, Ottawa, Canada; ^4Ottawa Hospital Research Hospital, Canada; ^5Institute of Cancer Research, United Kingdom Introduction: Tumour‐induced immunosuppressive mechanisms within the tumour microenvironment (TME) are a major reason for the limited success of many cancer immunotherapies. Reversing this immunosuppressive milieu will be critical to generate effective anti‐tumour immunity. Dysregulation of cellular miRNA profiles has been reported for the majority of cells that constitute the TME, including tumour and immune cells. Restoring miRNA profiles within these cell populations, to abrogate their tumour promoting phenotype and restore their anti‐tumour activity provides novel opportunities for cancer interventions. As oncolytic viruses (OVs) preferentially replicate in tumour cells, and virally expressed miRNA get packaged into extracellular vesicles, OVs are ideal vehicles to deliver miRNA mimetics to cells within the TME. Methods: Specific 22nt mature miRNA sequences, known to promote an anti‐tumour phenotype in tumour associated macrophages (TAM), or a control miRNA sequence were constructed into the pre‐miR30 cassette and cloned into oncolytic Rhabdoviruses (ORV‐miRNA). Tumour derived small extracellular vesicles (sEV) were isolated by sequential ultracentrifugation; electron microscopy and immunoblots were performed, and the size and quantity of sEV determined by NanoSight. Results: Mature 22nt miRNA sequences were processed from the pre‐miR30 cassette following infection of malignant cells without impeding replication or cytotoxicity. ORV‐miRNA infection resulted in the increase production of sEV, and virally encoded miRNA sequences detected. Tumour derived sEV were taken up by TAM and the virally expressed miRNA specifically knocked down both RNA and protein expression in the recipient cell. TAMs treated with virally induced sEV, containing specific miRNA, reversed the suppressive phenotype and function of these cells compared to control sEV. Summary/Conclusion: OVs are ideal vehicles to deliver therapeutic miRNA via sEVs to the TME to target not only tumour cells but also tumour associated immune cells. Funding: Ovarian Action, UK keywords: Oncolytic viruses (OVs) Tumour microenvironment (TME) miRNA PS14.21. The effects of Stevia rebaudiana cell suspension‐derived EVs on wound healing Oğuz Kaan Kırbaş ^1; Derya Sağraç^2; Batuhan Turan Bozkurt^1; Özgün Cem Çiftçi^1; Gökçeçiçek Özdemir^1; Cem Derman^1; Bahar Soğutmaz Soğutmaz Özdemir^3; Fikrettin Şahin^4; Pakize N. Taşlı^4 ^1Yeditepe University, USA; ^2Yeditepe University, Atasehir, Turkey; ^3Yeditepe University, Istanbul, USA; ^4Yeditepe University, Istanbul, Turkey Introduction: One of the important biological tools in intercellular communication is EVs. These vesicular structures with a bilayer lipid membrane have many physiological and pathological roles. In addition to mammalian systems, EVs also have several functions in plants. In many studies, therapeutic properties (anti‐oxidant, anti‐inflammatory, wound healing effect, etc.) of plant‐derived EVs have been discovered. However, the use of fruits or plant parts as a source in these studies leads to some difficulties. The use of a conditioned medium obtained from plant suspension as an EV source provides an important advantage. In this study, the wound healing efficiency of EVs obtained from Stevia rebaudiana (SDEVs) suspension culture was investigated. Methods: SDEVs were isolated by using the ATPS isolation method. EVs were subjected to NTA for quantification and size determination. The size and integrity of EVs were also exhibited by SEM Imaging. Uptake analysis was performed with or without of addition of Heparin. EVs and cells were stained with CMTPX and PKH‐67 dyes, respectively. MTS cell toxicity measurement assay was performed to investigate the effect of SDEVs on cell viability. DCF‐DA ROS measurement assay was selected for the determination of the antioxidant activity of SDEVs. Scratch assay was used to represent wound closing activity of SDEVs in HaCat cells. Lastly, tube formation assay was conducted in Huvec cells. Results were analyzed by One‐Way ANOVA with Tukey Post‐Hoc test. Differences between groups were investigated and results with p< 0,05 were accepted as statistically significant. Results: Our results showed that SDEVs were successfully isolated from plant cell suspension medium. NTA, SEM, and uptake analysis were performed to characterize SDEVs. Also, It was shown that SDEVs have the ability to close scratch line in keratinocytes within 20 hours and induce angiogenesis three‐fold. Also, SDEVs have ROS scavenging activity in both HaCat (%56) and Huvec (72%) cells. Summary/Conclusion: It can be said that SDEVs may be used as a healing agent in the near future. Utilizing plant EVs in the future wound healing science will be accelerated owing to EV studies. Funding: This study was supported by Yeditepe University. keywords: Wound healing, plant suspension cells, extracellular vesicles PS14.22. Milk Exosome Augments Antibiotic Efficacy Against Mastitis‐Causing Staphylococcus Aureus JITENDRA KUMAR ^1; PRIYA SHARMA^2; Suneel Kumar Onteru^2; Dheer Singh^2 ^1ICAR‐NDRI, Karnal‐ Haryana ‐India, India; ^2ICAR‐NDRI, USA Introduction: Mastitis poses a major burden on the dairy industry and is becoming increasingly difficult to treat due to the development of antibiotic resistance. And overuse of antibiotics for the treatment of mastitis exacerbates the problems. Antibiotic therapy for mastitis is currently ineffective due to the intracellular persistence of Staphylococcus aureus (S.aureus) and less bioavailability of antibiotics at the site of infection. Therefore, the aim of present study was to develop an effective therapeutics for intracellular delivery of antibiotic into mammary epithelial cells to tackle S.aureus Methods: In this study, milk exosomes (mEs) were explored for their unique potential both as drug delivery vehicles and treatment modulators against S.aureus. The milk exosome (mEs), pre‐characterized by zetasizer and high resolution transmission electron microscopy were used for encapsulation of D‐(‐)‐α‐ aminobenzylpenicillin (AMP) antibiotic. The mEs‐loaded AMP (mEs‐AMP) was contrived and tested against mastitis Results: The mEs‐AMP had a significantly (p < 0.001) higher therapeutic efficacy than unloaded‐AMP at the same dosage and frequency of treatment. The mEs‐AMP had 4.08‐fold lower minimum inhibitory concentration (MIC) than that of AMP, and the mEs‐AMPs killed S.aureus more effectively than AMP (74.35% vs 34.46%). The antimicrobial and therapeutic efficacies of mEs‐AMP were tested in subclinical and clinical mastitis of Sahiwal cows. Furthermore, mEs‐AMP reduced the number of somatic cells (4.53 0.69 to 3.39 0.51 lakh/mL; P 0.001) and bacterial log CFU (2.2 0.15 to 1.7 0.37; p < 0.05) in treated animal milk compared to AMP. Summary/Conclusion: Therefore, the mEs‐AMP could be used as a therapeutic tool for effective control of mastitis in dairy animals. Funding: NA keywords: Mastitis, Antibiotic resistance, Staphylococcus aureus, Milk‐exosomes, Aminobenzylpenicillin, Therapeutic efficacy. PS14.22. Applying regenerative medicine sEV knowledge to the cosmetic aesthetics market Rob Knight Cellese, Inc., Irvine, USA Introduction: Stem cells or their conditioned medium have been used in topical cosmetic products for almost 25 years (starting in 1999). The rationale for the use of stem cell based cosmetic products is that stem cells are capable of stimulating cell growth and repair leading to more youthful and healthier looking skin. With the growing amount of evidence and research in the regenerative function of extracellular vesicles it was only time before exosomes or sEVs started to appear and replace the use of stem cell conditioned media in topical cosmetic products. The use of exosomes or small extracellular vesicles in cosmetic products is a promising approach within the field of cosmetic aesthetics; however, the appropriate reporting and alignment with MISEV2018 is essential for both clarity and safety. The inclusion of any EV product into a cosmetic is an attractive avenue for developing therapeutics due to the reduction in regulatory requirements to get a product on the market. Methods: Cell culture and all liquid handling was conducted in either a class 5 or class 7 ISO certified clean room. sEVs were isolated from both bone marrow and umbilical cord derived MSCs. Cells were cultured in either vertical wheel spinning bioreactions (PBS Bio) or in large 5‐layer cell stacks (Corning). Cells were cultured until approximately 10L of conditioned media was collected for sEV isolation. sEVs were isolated from pre‐clarified conditioned medium in a combination process of tangential flow filtration (TFF) and size exclusion chromatography (SEC). Following purification sEVs were characterized in accordance to MISEV2018. NTA was carried out to determine particle size and concentration (ParticleMetrix, ZetaView), protein expression was carried out by ELISA and ExoView analysis and sEVs were visualized by Cryo‐TEM. sEVs were then lyophilized along with a cryoprotectant (25mM Trehalose) before in vitro functional testing. Anti‐inflammatory function, fibroblast proliferation and migration. Results: Results demonstrated the presence of sEVs and after lyophilization the sEVs retained their characteristic morphology and function in the in vitro testing. Safety testing including endotoxin testing and sterility testing demonstrated a sterile final sEV product that once combined with a diluent could be applied topically to the treatment area. Summary/Conclusion: The application of sEVs in cosmetic aesthetics is a novel and exciting opportunity for the knowledge and research being done within the drug development and regenerative medicine fields. Applying this knowledge and understanding to a cosmetic product allows for improvements and the development of novel anti‐ageing cosmetic products. Funding: All work was funded by Cellese, Inc. keywords: Small Extracellular Vesicles, Exosome, Skin, Cosmetics, Aesthetics PS14.23. Salivary gland epithelial progenitor cell derived exosome ameliorates salivary duct obstruction induced fibrosis via regulating activin signaling Yeo‐Jun Yoon ^1; Sunyoung Park^2; Yongpyo Hong^3; Haeun Yu^4; Jianning Yu^5; Jae‐Min Cho^3; Kyung‐A Hyun^2; Hyo‐Il Jung^6; Jae‐Yol Lim^7 ^1Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Republic of Korea, Seoul, Republic of Korea; ^2School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea, Seoul, Republic of Korea; ^3Yonsei University College of Medicine, Republic of Korea; ^4Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, USA; ^5Department of Biomedical Laboratory Science, College of Software and Digital Healthcare Convergence, Yonsei University, Republic of Korea; ^6Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea; ^7Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Republic of Korea, Republic of Korea Introduction: Since exosomes are considered avatars of cells, salivary gland stem/progenitor cells (SGSPC)‐derived exosomes may heal or regenerate damaged salivary glands. We aimed to identify the effect and mechanisms of SGSPC‐derived exosomes on impaired salivary glands. Methods: We isolated and propagated human parotid gland‐derived SGSPCs using a serum‐free keratinocyte expansion medium with a combination of small molecules. Next, the characteristics of the SPSPC and secreted exosomes were identified. The exosomes were delivered retrogradely to a submandibular gland duct ligation mouse model that mimics obstructive sialoadenitis. In addition, the mechanisms of SGSPC‐derived exosomes were verified using an ex vivo human parotid organoid model. Results: SGSPCs had the potency to passage more than 50 times and showed salivary basal‐like characters. Microscopic examinations revealed that exosome‐treated group showed reduced tissue damage compared to the PBS‐delivered group. More reduced fibrosis was also found in the exosome‐treated group than the PBS‐injected control group. In addition, acini were more in the exosome‐treated group than in the PBS‐injected group, which was confirmed by PAS staining. Exosome treatment also increased saliva volume and improved saliva lag times. In the exosome‐treated group, salivary gland epithelial cell‐related markers, NKCC1 (acinar cell), KRT5 (basal cell), KRT7 (luminal cell), and ACTA2 (myoepithelial), were clearly increased compared to the PBS‐treated group. On the other hand, the expression of KI67, a marker of cycling cells, was higher in the duct ligation model groups than in the sham group, but the exosome‐treated group showed higher expression than the PBS group, indicating active tissue regeneration. These effects were maintained for up to 6 months even when exosomes were frozen and thawed. The Ex vivo organoid model showed that salivary gland acinar genes were increased in the SGSPC‐derived exosome treatment group, and epithelial to mesenchymal transition genes were decreased through inhibition of activin signaling. Summary/Conclusion: SGSPC‐derived exosomes can be a potential therapeutic strategy by inhibiting fibrosis and restoring salivary function due to ductal obstruction‐induced sialoadenitis. Funding: Ministry of Science and ICT 2020M3A9I4039045, NRF 2022R1I1A1A01072997 keywords: salivary gland, epithelial stem cell, conditional reprograming culture, fibrosis, activin PS14.25. Development of an exercise‐based EV therapy to improve skeletal muscle recovery following immobilization Alexander M. Fliflet ^1; Ray A. Spradlin^2; Thomas R. Bludgen^3; Madeleine Meehan^4; Marni D. Boppart^5 ^1University of Illinois ‐ Urbana‐Champaign, Urbana, USA; ^2University of Illinois, Urbana‐Champaign, USA; ^3University of Illinois at Urbana‐Champaign, Naperville, USA; ^4University of Illinois at Urbana‐Champaign, Urbana, USA; ^5University of Ilinois at Urbana‐Champaign, Urbana, USA Introduction: Immobilization is necessary for recovery from illness or bone fractures; however, prolonged immobilization can result in significant loss of muscle mass and function. It is critical to recover muscle mass quickly following immobilization as severe muscle atrophy is correlated with increased likelihood of metabolic disorders, disability, and mortality. Physical rehabilitation can improve outcomes but is not always feasible. The purpose is to determine the extent to which EVs circulating in blood after endurance exercise training (EnVs) act as a safe and effective therapy to recover skeletal muscle mass following disuse. Methods: Donor C57BL/6 mice (n = 8–10, 4‐month‐old) were provided access to horizontal running wheels in a locked (sedentary) or unlocked (exercise) condition. After 4 weeks of voluntary running, EVs (SedVs or EnVs) were isolated from plasma using size exclusion chromatography according to manufactures guidelines, and concentration confirmed via nanoparticle tracking analysis (NanoSight NS3000). Recipient C57BL6/J mice (n = 5–8, 4‐month‐old) were subjected to single limb immobilization for 14 days. Upon remobilization, mice received a single intramuscular injection of PBS, SedVs, EnVs, or endurance plasma proteins, and remobilized for 14 days. Muscle samples were collected after remobilization and assessed for muscle cross‐sectional‐area (CSA) and capillarization. Results: EnVs did not significantly improve global fiber CSA and fiber‐type specific CSA (Type IIa, Type IIx or Type IIb). However, a significant treatment effect was observed for capillary density (P = 0.0445), with increases observed for both SedV (P = 0.034) and EnV (P = 0.0117). Summary/Conclusion: EVs isolated from plasma after exercise can significantly improve skeletal muscle vascularization after immobilization, which may represent an important step toward full recovery of skeletal muscle mass. Funding: Partially support by NIH (NIAMS R01AR072735). keywords: Disuse, exercise, recovery, EV PS14.26. Development of an isolation method for plasma extracellular vesicles excluding lipoproteins Kosuke Otani ^1; Tomoko Kodama^2; Muneyoshi Okada^3; Hideyuki Yamawaki^3 ^1School of Veterinary Medicine, Kitasato University, Towada City, Japan; ^2School of Veterinary Medicine, Kitasato University, Japan; ^3School of Veterinary Medicine, Kitasato University, Towada, Japan Introduction: The high concentration of lipoproteins in plasma, which have similarity in size, density, and contents to extracellular vesicles (EVs), hampers analysis on plasma EVs. To overcome this issue, we aimed to develop an effective isolation method for plasma EVs excluding lipoproteins, by using polyanion and a divalent cation. Methods: Human plasma was mixed with 1) heparin and manganese (II) chloride, 2) polyethylene glycol, or 3) phosphotungstic acid and magnesium chloride. The mixture was separated into a supernatant and pellet, and expression of Apo A‐I (HDL marker), Apo B (LDL and VLDL marker), and CD9 (EV marker) was measured by ELISA. Results: When 1) heparin and manganese (II) chloride or 2) polyethylene glycol at optimal concentration was mixed with plasma, the expression of Apo A‐I and Apo B was high in the supernatant, while the CD9 expression was high in the pellet. When 3) phosphotungstic acid and magnesium chloride at optimal concentration was mixed with plasma, the expression of Apo A‐I and CD9 was high in the supernatant, while the Apo B expression was high in the pellet. Summary/Conclusion: These results may be attributed to the differences in surface charge and structure between EVs and lipoproteins. Further research is necessary to compare this method with conventional EV isolation ones. keywords: Plasma, Biological fluids, Lipoproteins, Extracellular vesicles, Isolation method PS14.27. Engineered small extracellular vesicles displaying ACE2 variants on the surface protect against SARS‐CoV‐2 infection Hark Kyun Kim ^1; Dong‐Gyu Jo^2 ^1Sungkyunkwan University, Suwon, Republic of Korea; ^2School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin‐converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID‐19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. Methods: With excellent biocompatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS‐CoV‐2. Moreover, to enhance the sACE2‐S binding interaction, we employ sACE2 variants. Results: sACE2‐loaded sEVs exhibit typical sEVs characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry of wild‐type (WT), the globally dominant D614G variant, Beta (K417N‐E484K‐ N501Y) variant, and Delta (L452R‐T478K‐D614G) variant SARS‐CoV‐2 pseudovirus, and protect against authentic SARS‐CoV‐2 and Delta variant infection. Of note, sACE2 variants harboring sEVs show superior antiviral efficacy than WT sACE2 loaded sEVs. Therapeutic efficacy of the engineered sEVs against SARS‐CoV‐2 challenge was confirmed using K18‐hACE2 mice. Summary/Conclusion: The current findings provide opportunities for the development of new sEVs‐based antiviral therapeutics. Funding: the National Research Foundation of Korea, Grant/Award Numbers:NRF‐2019R1A2C3011422, NRF‐2019R1A5A2027340, NRF‐2022R1C1C2012084; the Ministry of Oceans and Fisheries’ R&D project, Korea,Grant/Award Number: 1525011845;KoreaBasic Science Institute (National Research Facilities and Equipment Center); the Ministry of Education, Grant/Award Number: 2020R1A6C101A191; Korea Disease Control and Prevention Agency, Grant/Award Number: KCDC2021‐ER1602‐00; the Korea National Institute of Health fund, Grant/AwardNumber: 2021‐NI‐026‐00 keywords: SARS‐CoV‐2, ACE2, sEV, Extracellular vesicles, SPIKE, COVID‐19 __________________________________________________________________ Articles from Journal of Extracellular Vesicles are provided here courtesy of Wiley (BUTTON) Close ACTIONS * [53]View on publisher site * [54]PDF (1.2 MB) * (BUTTON) Cite * (BUTTON) Collections * (BUTTON) Permalink PERMALINK https://pmc.ncbi.nlm (BUTTON) Copy RESOURCES (BUTTON) Similar articles (BUTTON) Cited by other articles (BUTTON) Links to NCBI Databases Cite (BUTTON) * (BUTTON) Copy * [55]Download .nbib .nbib * Format: [NLM] Add to Collections ( ) Create a new collection (*) Add to an existing collection Name your collection * ____________________ Choose a collection Unable to load your collection due to an error [56]Please try again (BUTTON) Add (BUTTON) Cancel Follow NCBI [57]NCBI on X (formerly known as Twitter) [58]NCBI on Facebook [59]NCBI on LinkedIn [60]NCBI on GitHub [61]NCBI RSS feed Connect with NLM [62]NLM on X (formerly known as Twitter) [63]NLM on Facebook [64]NLM on YouTube [65]National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 * [66]Web Policies * [67]FOIA * [68]HHS Vulnerability Disclosure * [69]Help * [70]Accessibility * [71]Careers * [72]NLM * [73]NIH * [74]HHS * [75]USA.gov (BUTTON) Back to Top References