Abstract Cisplatin‐induced ototoxicity remains a clinical dilemma with limited mechanistic understanding and no food and drug administration (FDA)‐approved therapies. Despite emerging roles of small extracellular vesicles (sEV) in drug ototoxicity, their molecular cargo profiles and causal roles to cisplatin‐induced ototoxicity are unexplored. This study systematically investigates sEV derived from cochlear explants treated with cisplatin (Cis‐sEV) and controls (Ctrl‐sEV) using multi‐omics profiling. Through small RNA sequencing, 83 differentially expressed microRNAs (miRNAs) are identified in Cis‐sEV compared to Ctrl‐sEV. Notably, mmu‐miR‐34a‐5p, mmu‐miR‐140‐5p, mmu‐miR‐15b‐5p, mmu‐miR‐25‐3p, and mmu‐miR‐339‐5p are significantly upregulation in Cis‐sEVs. Predicted target pathways of these differentially expressed miRNAs are enriched in apoptosis, inflammation, and cellular damage, indicating their potential involvement in cisplatin‐induced cochlear damage. LC‐MS/MS analysis reveals 90 upregulated and 150 downregulated proteins in Cis‐sEV, with many involved in damage response. Specifically, CLTC, CCT2, ANXA6, and HSPA8 are uniquely upregulated proteins in Cis‐sEV, and CLTC and ANXA6 are exclusively co‐localized in hair cells (HCs) post‐cisplatin exposure, suggesting that Cis‐sEV originate primarily from damaged HCs. Moreover, CLTC in sEV may serve as a potential biomarker for cisplatin‐induced ototoxicity as verified in both in vitro and in vivo models. This study provides novel insights into the molecular mechanisms of cisplatin‐induced ototoxicity and identifies potential biomarker and therapeutic targets. Keywords: biomarker, cisplatin‐induced ototoxicity, CLTC, miRNAs, small extracellular vesicles __________________________________________________________________ Cisplatin causes reactive oxygen species accumulation, leading to apoptosis and inflammation in cochlear hair cells. Small extracellular vesicles primarily derived from the damaged hair cells likely contribute to cisplatin‐induced ototoxicity, carrying a variety of microRNAs and proteins. These differentially expressed components found in these cisplatin‐derived small extracellular vesicles may serve as biomarkers and potential targets for mitigating cochlear damage. graphic file with name ADVS-12-e02627-g001.jpg 1. Introduction Hearing is crucial for social activities but is particularly vulnerable to factors like genetics, ototoxic drugs, noise exposure, and aging, leading to irreversible sensorineural hearing loss (SNHL).^[ [52]^1 ^] Cisplatin, approved by the food and drug administration (FDA) in 1978, is a potent chemotherapeutic drug for malignant solid tumors but often causes severe adverse effects, including bilateral, progressive, and irreversible ototoxicity in 40% to 60% of patients.^[ [53]^2 ^] Cisplatin‐induced ototoxicity predominantly results in injury to hair cells (HCs), stria vascularis (SVs), and spiral ganglion neurons (SGNs) within the inner ear. Moreover, the basal turn of the cochlea, which is particularly sensitive to high‐frequency signals, is the initial damage site of cisplatin ototoxicity and suffers the most severe damage.^[ [54]^3 ^] High‐dose accumulation of cisplatin can also compromise auditory cells' apical and middle regions. Previous investigations have shown that apoptosis and inflammation elicited by reactive oxygen species (ROS), necrosis, ferroptosis, and the accumulation of lipid peroxides are involved in the progression of cisplatin ototoxicity.^[ [55]^4 ^] Nevertheless, the cellular interactions and molecular mechanisms underlying cisplatin‐induced ototoxicity have not yet been completely clarified and still need to be studied. Small extracellular vesicles (sEV) are lipid bilayer vesicles (<200 nm in diameter) secreted by almost all cell types, and they carry multifarious bioactive molecules that reflect the characteristics of their parent cells.^[ [56]^5 ^] sEV‐mediated intercellular communication is a highly conserved process involving the transfer of functional molecules to recipient cells that are nearby or distant (via biofluids and circulation).^[ [57]^5 ^] Increasing evidence indicates that sEVs play a significant role in physiological and pathological processes, including the development and progression of cancer, neurodegenerative disease progression, immune regulation, cell survival, and apoptosis.^[ [58]^6 ^] In addition, sEV exhibit heterogeneity, primarily characterized by variations in their cargoes of proteins and microRNAs (miRNAs).^[ [59]^7 ^] Under various stress conditions and pathological states, this diversity becomes particularly pronounced, prompting sEV to potentially serve as disease biomarkers for monitoring specific disease development or assisting in diagnosis.^[ [60]^8 ^] sEVs are critical mediators of intercellular crosstalk in the inner ear,^[ [61]^9 ^] yet their molecular specificity in cochlear pathologies remains enigmatic. While a recent study revealed sEV‐mediated protection of vestibular HCs against aminoglycoside toxicity,^[ [62]^10 ^] the cochlea – an anatomically complex microenvironment – exhibits fundamentally distinct sEV dynamics under cisplatin‐induced ototoxicity. Crucially, the cargo composition (miRNAs/proteins) of sEV secreted in response to cisplatin‐induced ototoxicity remains unclear,^[ [63]^9a ^] and whether these cisplatin‐modified sEV actively drive sensory cell death and reflecting damage, which are the main focuses of this study. A deeper understanding of the role of sEV in cisplatin‐induced ototoxicity is imperative to developing effective strategies to prevent or mitigate cisplatin's ototoxic effects and therapy optimization for chemotherapy patients. This study analyzed the miRNAs and proteins from organotypic cochlea‐secreted sEV in cisplatin‐induced ototoxicity and their potential roles in cisplatin‐induced damage. Our findings indicate that the differentially expressed miRNAs in the Cis‐sEV group probably mediate several important cellular and molecular regulation pathways – such as apoptosis, inflammation, and cell survival – which provides a novel perspective for elucidating the downstream effects that contribute to the cochlea's unique vulnerability to cisplatin‐induced injury. Furthermore, several sEV miRNAs are being verified and might be potential therapeutic targets for mitigating cisplatin ototoxicity. Additionally, through LC‐MS/MS analysis and validation, we identified four proteins – clathrin heavy chain 1 (CLTC), T‐complex protein 1 subunit beta (CCT2), annexin A6 (ANXA6), and heat shock cognate 71 kDa protein (HSPA8) – that exhibited a specifical increase expression in Cis‐sEV in the cochlea explant model. Moreover, the damaged HCs are primarily the parent cells of Cis‐sEV, as indicated by the exclusively co‐localized of CLTC and ANXA6. By using the cisplatin‐induced House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cell model and in vivo mice model, CLTC was also enriched expression in Cis‐sEV and could serve as a potential biomarker for cisplatin‐induced ototoxicity. The detailed mechanisms by which the miRNAs and proteins in Cis‐sEV respond to disease progression require further investigation in future studies. 2. Results 2.1. Establishment of an Ex Vivo Model of Cisplatin‐Induced Ototoxicity The cochlear explants from postnatal day 3 (P3) mice were treated with 50 µM cisplatin for 48 h to establish an ex vivo model of cisplatin‐induced ototoxicity (Figure [64]1A). Compared to the control group, the group exposed to 50 µM cisplatin exhibited significant damage to HCs along the cochlear basilar membrane by 24 h, with the severity of damage increasing from the apex to the base, leaving ≈70% of HCs intact (Figure [65]1B,C,D). As the duration of drug exposure extended to 36 h and 48 h, the resulting damage progressively worsened, leading to a time‐dependent decrease in HC survival rates across all turns of the cochlea (Figure [66]1B,C,D), which aligns with previous studies.^[ [67]^3 , [68]^4 ^] ≈80% of the HCs in each cochlear turn were lost after 48 h of cisplatin treatment (Figure [69]1D). Additionally, at 48 h, the protein expression of TOMM20 and p‐SAPK/JNK showed a noticeable increase in the cisplatin group, indicating the accumulation of ROS and an inflammatory response following cisplatin administration (Figure [70]1E). The accumulation of ROS in the mitochondria of HCs exposed to cisplatin was further confirmed by Mito‐Sox fluorescence staining (Figure [71]1H). Moreover, the protein level of apoptosis markers, including cleaved‐Caspase9 and cleaved‐Caspase3, was increased in response to cisplatin‐induced injury, as shown by western blotting and immunofluorescence results (Figure [72]1E,G). This indicates that the damaged HCs underwent apoptosis following cisplatin stress. Furthermore, cisplatin treatment led to a significant increase in the expression of mRNA for pro‐apoptosis genes such as Bax, Caspase3, Caspase8, and Caspase9, as well as the ROS‐related gene Tomm20 (Figure [73]1F). In summary, we successfully established an ex vivo model of cisplatin‐induced ototoxicity, characterized by ROS accumulation, apoptosis, and an inflammatory response. Figure 1. Figure 1 [74]Open in a new tab ROS accumulation, apoptosis, and inflammatory responses of auditory HCs in the ex vivo cisplatin‐induced ototoxicity model. A) The flow chart of ex vivo cochlear explant culture with 50 µM cisplatin. B,C) Representative confocal images of cochlear explants treated with various durations of 50 µM cisplatin (including 24 h, 36 h, and 48 h) are presented, including the complete cochlear explants (B) and local segments of the apical, middle, and basal turns of the cochlear explants (C). The control group of cochlear explants was cultured for 48 h without drug treatment. Myosin7a (cyan) marked HCs. Scale bars are 200 µm (B) and 50 µm (C). D) A quantitative analysis of HCs in each cochlear turn at different cisplatin treatment periods is shown, expressed as the number of HCs per 100 µm of the basilar membrane. Data are shown as mean ± SD, with “*” indicating p < 0.05, “**” indicating p < 0.01, and “***” indicating p < 0.001. “n” represents the number of cochlear explants counted. E) The levels of TOMM20 protein, phosphorylated SAPK/JNK, cleaved‐Caspase3, and cleaved‐Caspase9 in the cochlear explants were all significantly increased after cisplatin‐induced injury at 48 h. β‐Actin served as the reference protein. F) The mRNA levels of Bax, Caspase3, Caspase8, Caspase9, and Tomm20 were significantly increased when the cochlea was exposed to cisplatin for 48 h; n = 3. All results are presented as the mean ± SD, with statistical significance indicated as follows: “*” for p < 0.05, “**” for p < 0.01, and “***” for p < 0.001. G) Immunostaining with cleaved‐Caspase3 (red fluorescence) and anti‐Myosin7a (green fluorescence) showed that HCs in the cisplatin group exhibited a high level of the pro‐apoptosis factor. Scale bar, 20 µm. H) Mito‐Sox fluorescence staining labeled mitochondrial ROS levels in living HCs, showing significant ROS accumulation and oxidative stress in HCs after cisplatin administration. Scale bar, 20 µm. 2.2. Isolation and Characterization of Tissue‐Derived sEV from Cisplatin‐Treatment Cochlear Explants Tissue‐derived sEV are found in the intercellular space of tissues and play significant roles in facilitating intercellular communication within the tissue microenvironment. Therefore, they closely reflect the characteristics of the parental cells and the surrounding tissue environment.^[ [75]^11 ^] To investigate changes in sEV secretion patterns associated with the progression of cisplatin‐induced toxicity, we isolated the sEV from the supernatant of the cochlear explant culture medium over a continuous timeframe from 0 to 48 h. This isolation process involved a series of differential centrifugation, ultrafiltration, and ultracentrifugation steps, as illustrated in the flow diagram presented in Figure [76]2A. The sEVs collected from the cisplatin‐treated group are designated as Cis‐sEV, while those derived from the control group are designated as Ctrl‐sEV. TEM analysis revealed typical oval and cup‐shaped structures in both types of sEVs, with sizes of ≈200 nm (Figure [77]2B,C). Additionally, the NTA results showed that both Ctrl‐sEV and Cis‐sEV were within the size range of 200 nm (Figure [78]2D,E). Statistical analysis revealed that the median size of the Ctrl‐sEV group was 149.6 ± 6.461 nm, while the median size for the Cis‐sEV group was 152.0 ± 5.606 nm, showing no significant difference in median size between the two sEV groups (Figure [79]S1, Supporting Information). Additionally, the mean quantity of derived sEV particles per cochlear explant in the control group was measured at 3.27 × 10^7 ± 1.52 × 10^7, compared to 5.38 × 10^7 ± 3.80 × 10^7 sEV particles per cochlear explant in the cisplatin‐treated group, also demonstrating no statistical difference between the two groups (Figure [80]2G). Typical protein markers of sEV (CD63, TSG101, and Flotillin‐1) were confirmed by western blotting in both the isolated sEV and tissue lysate, while Calnexin, an sEV‐negative marker, was detected only in the tissue lysate and not in either the Cis‐sEV or Ctrl‐sEV groups (Figure [81]2F). Taken together, these results demonstrated that sEVs were effectively isolated and purified from the conditioned culture medium of cochlear explants, which could be used for further research. Figure 2. Figure 2 [82]Open in a new tab Isolation and characterization of cochlear tissue‐derived sEV form cochlear explants. A) The flow diagram for isolating sEV from the conditioned culture medium of the cochlear explants involves a 48‐h treatment. B,C) TEM analysis of the morphology of Cis‐sEV and Ctrl‐sEV. Scale bars, 50 nm (B) and 100 nm (C). D,E) Particle size distribution of Cis‐sEV and Ctrl‐sEV was determined by NTA. F) Western blotting analysis of isolated sEV and cochlear explants tissue lysate with typical positive markers for sEV, such as CD63, TSG101, and Flotllin‐1, and the negative marker Calnexin, normalized according to BCA protein quantification results. G) Statistical analysis of the number of sEV secreted per cochlear explant in the control group and cisplatin‐treated group. Ctrl group: n = 5 samples; Cis group: n = 5 samples. The “n.s” represent the no signification. 2.3. Differential miRNA Profiles in Cis‐sEV Compared to Ctrl‐sEV Small RNAs, particularly miRNAs, are crucial components of sEV and play a direct role in intercellular communication by regulating gene expression at the post‐transcriptional level.^[ [83]^12 ^] To detect the differentiation of miRNA molecular profiles between the Cis‐sEV and Ctrl‐sEV groups, a small RNA‐seq analysis was performed with three biological replicates for each group. First, PCA analysis revealed a clear discrepancy between the two groups (Figure [84]S2A, Supporting Information). Additionally, correlation analysis indicated a strong correlation among replicates within each group (Figure [85]3A). Notably, the miRNA composition in the sEV RNA categories was found to be more abundant in the Cis‐sEV group, when compared to the Ctrl‐sEV group (Figure [86]3B). 810 miRNAs were identified across all samples. Furthermore, the analysis revealed 799 miRNAs in the Cis‐sEV group and 400 miRNAs in the Ctrl‐sEV group, with 389 miRNAs being common in both groups (Figure [87]3C). The level of miRNAs in the Cis‐sEV group was markedly higher than that in the Ctrl‐sEV group, potentially offering valuable insights into the pathological changes associated with cochlear damage induced by cisplatin exposure. The expression levels of the top 40 miRNAs in both the Cis‐sEV and Ctrl‐sEV groups were presented in Figures [88]S2B,C, Supporting Information, respectively. Furthermore, differential expression analysis revealed that 83 miRNAs were significantly altered between the two groups, of which 74 miRNAs were upregulated (including 7 novel miRNAs) and 9 miRNAs (including 3 novel miRNAs) were downregulated in the Cis‐sEV group compared to the Ctrl‐sEV group (Figure [89]3D,E). The seven novel miRNAs that exhibited differentially enriched expression in the Cis‐sEV group were cataloged in Table [90]S1, Supporting Information. Additionally, the expression levels of numerous upregulated miRNAs were markedly increased in the Cis‐sEV group (Figure [91]3E). This includes several miRNAs known to regulate ROS reactions, inflammatory responses, and apoptosis, such as mmu‐miR‐100‐3p,^[ [92]^13 ^] mmu‐miR‐124‐3p,^[ [93]^14 ^] mmu‐miR‐129‐2‐3p,^[ [94]^15 ^] mmu‐miR‐140‐5p,^[ [95]^16 ^] mmu‐miR‐153‐3p,^[ [96]^17 ^] mmu‐miR‐15b‐5p,^[ [97]^18 ^] mmu‐miR‐17‐5p,^[ [98]^19 ^] mmu‐miR‐25‐3p,^[ [99]^20 ^] mmu‐miR‐339‐5p,^[ [100]^21 ^] mmu‐miR‐34a‐5p,^[ [101]^22 ^] and mmu‐miR‐370‐3p.^[ [102]^23 ^] Eight of these miRNAs, along with their potential functions and relevant references were summarized in Table