Abstract

Background

   The G protein-coupled receptor 55 (GPR55) is part of an expanded
   endocannabinoid system (ECS), and plays a pro-tumorigenic role in
   different cancer models, including pancreatic cancer. Next to cancer
   cells, various cells of the immune tumor microenvironment (TME) express
   receptors of the ECS that critically determine tumor growth. The role
   of GPR55 in cancer cells has been widely described, but its role in the
   immune TME is not well understood.

Methods

   We intended to uncover the role of GPR55 in tumor immunity in a model
   of pancreatic ductal adenocarcinoma (PDAC). To this end, a KPCY tumor
   cell line or a GPR55-overexpressing KPCY cell line (KPCY55) from murine
   PDAC were subcutaneously injected into wildtype (WT) and GPR55 knockout
   (KO) mice, and immune cell populations were evaluated by flow
   cytometry.

Results

   Deficiency of GPR55 in the TME led to reduced tumor weight and volume,
   and altered the immune cell composition of tumors, favoring an
   anti-tumorigenic environment by increasing the number of CD3^+ T cells,
   particularly CD8^+ T cells, and the expression of PDL1 on macrophages.
   RNA-seq pathway analysis revealed higher T cell activity in KPCY55
   tumors of GPR55 KO vs. WT mice. In addition, tumors from GPR55 KO mice
   displayed increased levels of T cell chemokines Cxcl9 and Cxcl10.
   Migration of T cells from GPR55 KO mice towards CXCL9 was increased in
   comparison to T cells from WT mice, suggesting that a CXCR3/CXCL9 axis
   was involved in T cell influx into tumors of GPR55 KO mice. Notably,
   anti-PD-1 immunotherapy increased tumor burden in WT mice, while this
   effect was absent in the GPR55 KO mice.

Conclusion

   Our study indicates that GPR55 in TME cells may drive tumor growth by
   suppressing T cell functions, such as migration, in a model of PDAC,
   making it an interesting target for immunotherapies.

   Keywords: KPCY model, pancreatic cancer, GPR55, checkpoint inhibitors,
   anti-PD-1 antibody, tumor microenvironment, CXCR3/CXCL9 axis

1. Introduction

   The tumor microenvironment (TME) is comprised of a variety of cell
   types, such as immune, stromal, and endothelial cells, which play a
   crucial role in cancer progression ([46]1). For instance, patients with
   abundant presence of cytotoxic T and natural killer (NK) cells in the
   TME have a better prognosis across multiple cancer types ([47]2,
   [48]3). However, this favorable response may be hampered by other,
   namely immunosuppressive cell types present in the TME, like regulatory
   T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) ([49]4).
   Pancreatic ductal adenocarcinoma (PDAC) has a highly immune-suppressive
   TME, in part because the majority of infiltrating immune cells are
   myeloid in origin ([50]5). Moreover, only a fraction of pancreatic
   cancer patients has infiltrating cytotoxic T cells, which are known to
   be correlated with improved survival ([51]6). Lately, the
   endocannabinoid system (ECS) has attracted attention regarding its
   effects on the behavior of immune cells during inflammation and cancer
   ([52]7, [53]8). Main members of the ECS are the cannabinoid receptors 1
   and 2 (CB[1] and CB[2]), their endogenous ligands (endocannabinoids),
   and enzymes for endocannabinoid metabolism, like monoacylglycerol
   lipase (MGL), the 2-arachidonoylglycerol (2-AG)-degrading enzyme
   ([54]7). Other receptors show responsiveness to endocannabinoids, such
   as the G protein-coupled receptor 55 (GPR55), which is considered part
   of an ‘expanded’ ECS, or the ‘endocannabinoidome’ ([55]9–[56]11).
   Various immune cells in the TME possess cannabinoid receptors ([57]7),
   which can be influenced by components of the ECS ([58]9). We previously
   showed prominent expression of CB[2] and MGL in TME immune cells of
   non-small cell lung cancer (NSCLC) models and human NSCLC tissue
   ([59]12, [60]13). Like CB[2] ([61]14), GPR55 is present in immune
   cells, e.g., B cells ([62]15), T cells ([63]16) and neutrophils
   ([64]17), and also in cancer cells of various origin, for instance, in
   colon and pancreatic cancer cells ([65]18–[66]20). In many types of
   cancer that have been studied, GPR55 primarily plays a pro-tumorigenic
   role ([67]18, [68]20–[69]25). As to pancreatic cancer, genetic ablation
   of GPR55 in a PDAC model clearly improved disease outcome ([70]20).

   Since involvement of GPR55 in cancerogenesis was mostly researched in
   the context of its role in cancer cells ([71]18, [72]19, [73]26), we
   focused on the immune TME in this study using GPR55 KO mice, and
   explored whether the knockout of GPR55 could have an influence on the
   immune cell landscape and the tumor progression in a PDAC mouse model
   ([74]27). To this end, we used KPCY tumor cells (from mouse PDAC), and
   - since GPR55 is highly expressed in human pancreatic cancer cells
   ([75]20) and higher stage pancreatic intraepithelial neoplasia ([76]28)
   - KPCY tumor cells that overexpressed GPR55 (termed KPCY55). KPCY and
   KPCY55 cell lines were subcutaneously (s.c.) injected into the flanks
   of immunocompetent wildtype (WT) or GPR55 knockout (KO) mice. By using
   GPR55 KO mice in this model, we created a situation where GPR55 was
   present in cancer cells, but not in cells of the TME. We can report
   that mice lacking GPR55 had smaller tumors and higher lymphoid cell
   infiltration than WT mice. The results may have importance for
   developing new immunotherapies against PDAC.

2. Materials and methods

2.1. Cancer cell lines

   The KPCY cell lines were generated from late-stage primary pancreatic
   tumors from C57BL/6 KPC mice expressing a YFP lineage tag (KPCY)
   (Trp53^L/+) ([77]27). One T-cell high clone (2838c3) was purchased from
   Kerafast (Boston, MA, USA). The KPCY cells were maintained in DMEM with
   10% FBS (Life Technologies) and 1% penicillin/streptomycin (P/S, PAA
   Laboratories) at 37°C and 5% CO[2] in a humidified atmosphere.

   A GPR55-overexpressing KPCY cell line (KPCY55) was generated in our lab
   from the original KPCY parental cell line using a lentivirus for
   transduction that carried a GPR55 Puro cassette (or a control cassette)
   for stable overexpression of GPR55 (VectorBuilder). Puromycin (0.5
   µg/ml, Thermo Fisher, A1113803) was used for positive selection of
   clones, and overexpression was confirmed with RT-qPCR.

2.2. Mice used in the study

   C57BL6/J mice were purchased from Charles River (Germany) and bred in
   house. GPR55 KO mice (B6;129S-Gpr55^tm1Lex/Mmnc) were obtained through
   MMRRC (Mutant Mouse Regional Resource Center; USA). The strain was
   backcrossed with C57BL6/J mice for ten generations, and was also bred
   in house ([78]18). Experimental procedures were approved by the
   Austrian Federal Ministry of Science, Research and Economy (protocol #
   2022-0.748.851) and performed in strict accordance with international
   guidelines. All experimental procedures were performed on 7-12-week
   male mice.

2.3. PDAC mouse model

   KPCY and KPCY55 cells were dissociated into single cells, washed with
   PBS twice and counted before s.c. injection. Cells (5×10^5) were
   resuspended in 450 μL Dulbecco’s phosphate buffered saline (PBS, Gibco)
   and injected into the flanks of mice on day 0. Tumors were harvested at
   the experimental endpoint upon reaching the appropriate size, i.e., on
   day 21 for the KPCY tumors, and on day 28 or 29 for the KPCY55 tumors.
   Tumors were then subsequently weighed, measured with a digital caliper
   ex vivo, and submitted to analysis. Tumor volume was calculated based
   on the following formula: V = length x width x height x π/6 ([79]29).

2.4. Anti-PD1 antibody treatment

   KPCY55 tumor-bearing WT or GPR55 KO mice were injected i.p. with 200 µg
   rat monoclonal anti-mouse PD-1 antibody (clone 29F.1A12, BioXCell,
   Lebanon, NH) or rat IgG2a isotype control (clone 2A3, BioXCell,
   Lebanon, NH) 6 times (as shown in the treatment protocol scheme) over a
   period of two weeks.

2.5. Single-cell suspensions

   Single cell suspensions of s.c. tumors were prepared as previously
   described ([80]13). Briefly, tumors were cut into small pieces with
   scissors. The tumor pieces were afterwards digested with collagenase
   for 30 minutes at 37°C (CLS-1; 4.5 U/ml; Worthington) and DNase I (160
   mU/ml; Worthington; [81]LS002006), while rotating at 1000 rpm. After
   digestion, tissue was passed through a 40 μm-strainer, washed with
   staining buffer (SB; PBS + 2% FBS), followed by a wash in PBS only.

2.6. Flow cytometric phenotyping of immune cell populations

   In order to exclude dead cells, single cell suspensions were incubated
   for 20 min in Fixable Viability Dye (FVD) (eFluor™ 780; eBioscience,
   #65-0865-18) in the dark at 4°C. The cells were initially incubated
   with 1 μg TruStain™ FcX (Biolegend, #101320), and afterwards stained in
   the dark at 4°C for 30 min with antibodies listed in [82]Supplementary
   Table S1 . From this point onwards, the staining protocol differed for
   intracellular immunofluorescent staining. For surface staining, samples
   were fixed with eBioscience™ IC Fixation Buffer (ThermoFisher
   Scientific, #00-8222-49) in the dark for 10 min at 4°C, resuspended in
   SB, and acquired within two weeks on a BD LSR Fortessa, running on
   FACSDiva software (BD Biosciences). Samples requiring intracellular
   staining were fixed with Fix/Perm solution from the BD
   Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences,
   #554714) for 20 min in the dark at 4°C. Afterwards, they were washed
   twice with 1x Perm/Wash buffer and then incubated with the antibody in
   Perm/Wash buffer for 30 min at 4°C. The cells were washed twice in 1x
   Perm/Wash buffer and resuspended in SB prior to analysis. FlowJo
   software (v10.20, Treestar) was used for analysis and compensation. See
   [83]Supplementary Figures S1 and [84]S2 for gating strategies.

2.7. RNA extraction and RT-qPCR

   RNA was extracted from tissue using Trizol (Life Technologies,
   #15596026). Samples were then treated either with a DNA-free™ DNA
   Removal Kit (Invitrogen, #10729525) or RNase-Free DNase set (Qiagen,
   #79254). Quality and concentration of RNA were determined using a
   NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Reverse
   transcription of purified RNA (1 μg) was performed by High-Capacity
   cDNA Reverse Transcription Kit (Applied Biosystems, #4368814). Gene
   expression was assessed by reverse transcription-quantitative
   polymerase chain reaction (RT-qPCR) using SsoAdvanced Universal SYBR
   Green Supermix (Bio-Rad, #1725271). Primers were acquired from Eurofins
   ([85] Supplementary Table S2 ). Relative gene expression was calculated
   using 2^-ΔCT methods ([86]30). Hprt was used as a housekeeping gene.

2.8. Bulk RNA-seq: sample preparation and analysis

   RNA was extracted from tissue using Trizol (Life Technologies,
   #15596026). Samples were afterwards treated with the DNA-free™ DNA
   Removal Kit (Invitrogen, #10729525), and the concentration and quality
   of RNA was measured using Agilent Bioanalyzer 2100. After poly-A
   enrichment and library prep, RNA-seq was performed on Illumina NovaSeq
   (6000 and the X Plus), with 20 million reads per sample (GENEWIZ,
   Azenta Life Sciences). The raw data were aligned to GRCm39 using STAR
   2.7.10b, after running FastQC 0.11.9. Differential gene expression was
   assessed by RStudio 4.4.1 using edgeR package (version 4.2.0) ([87]31).
   For pathway enrichment analysis, pathfindR was used (version 2.4.1)
   ([88]32). Heatmaps were generated after normalization to transcripts
   per million and subsequent z-scaling. All RNA-seq data are deposited at
   GEO database, accession number [89]GSE280636.

2.9. Isolation of splenic pan-T cells and migration assays

   Pan-T cells were isolated from WT and GPR55 KO mouse spleens using
   EasySep™ Mouse T Cell Isolation Kit (Stemcell, #19851), according to
   the manufacturer’s protocol. T cell migration assay was performed using
   a 96-well MultiScreen plate (Millipore, MAMIC5S10) with a 5-μm
   pore-size polycarbonate filter. A series of dilutions of the chemokine
   (C-X-C motif) ligand 9 (CXCL9 Biolegend, #578204) in assay buffer
   containing Ca^2+ and Mg^2+ was pipetted into the bottom well, after
   which T cells were added to the top well in the same buffer (1x10^5 per
   75 μl). The cells were left to migrate in the plate for 90 min at 37°C.
   Migrated cells in the bottom wells were counted on a BD Accuri™ C6 Plus
   flow cytometer. Chemotactic index was defined as the number of cells
   migrating towards CXCL9 divided by the number of cells migrating
   towards negative control ([90]17).

2.10. In situ hybridization and immunofluorescence

   Tumors were fixed in acid-free phosphate-buffered 10% formaldehyde
   solution (Roti^®-Histofix 10%, pH 7, Roth, P087.1) for 24-48 hrs at
   room temperature, and further processed for paraffin embedding,
   according to standard procedures. Tissue was cut in 5 μm-sections,
   baked at 60°C for 1 hr, dewaxed, and rehydrated. ISH was performed
   according to the manufacturer’s protocol, and as recently published by
   us ([91]12). In brief, three ZZ probes for GPR55 (targeting bases 2-907
   of [92]NM_001033290.2) (RNAScope ™ RED kit; Advanced Cell Diagnostics
   [ACD], #322360) were used to detect the corresponding mRNAs in tumors.
   Sections with tumor tissue were treated with H[2]O[2] for 10 min, which
   was followed by target retrieval, using the Brown FS3000 food steamer
   for 15 min. Each step was followed by washes in distilled water. The
   sections were digested with Protease IV at 40°C for 20 min, washed,
   incubated at 40°C for 2 hrs, and stained with FastRed. GPR55 KO and WT
   mice samples were put on one slide for comparison. GPR55 KO mice lacked
   expression of GPR55 outside of KPCY tumor cells ([93] Supplementary
   Figure S3 ).

   An antibody against GFP (1:500; Abcam #ab290) was used to label tumor
   cells (YFP-tagged). Antibodies against CD8^+ (1:100; Abcam # ab203035),
   CD11b^+ (1:100; Novus #NB11089474), F4/80^+ (1:500, Cell Signaling
   #70076), and CD4^+ (1:400, Abcam #ab183685) were used to stain cell
   types of the immune TME co-localizing with GPR55 mRNA. After ISH,
   tissue sections were first blocked with 5% goat serum (Sigma-Aldrich)
   in 0.1 M PBS containing 0.3% Triton X-100. Afterwards, primary
   antibodies were applied in 0.1 M PBS containing 0.3% Triton X-100 and
   1% goat serum over night at 4°C. As second antibodies, Alexa Fluor^®
   488 goat anti-rabbit (1:500; Jackson Immuno Research; #111-5491144) or
   Cy5-labeled anti-rabbit IgGs (1:500; Jackson Immuno Research;
   #711-175-152) were used. Sections were then mounted with Vector^®
   TrueVIEW^® Autofluorescence Quenching Kit containing DAPI (Vector
   Laboratories, #SP-8500-15), and images were taken by an Olympus IX73
   fluorescence microscope (Olympus) connected with a Hamamatsu ORCA-ER
   digital camera (Hamamatsu Photonics K.K.). Images were processed with
   Olympus CellSense^® 1.17 imaging software (Olympus). Only contrast,
   brightness, and color balance of images were adjusted. ImageJ software
   and QuPath (version 0.4.3) were applied to quantify expression and
   colocalization with the ISH probe ([94]29).

2.11. Statistical analysis

   GraphPad Prism 10.0.3 (GraphPad^® Software) was used to perform
   statistical analyses. Data are presented as means ± standard deviation
   (SD), or as medians with 25^th and 75^th percentiles, and min-max
   values. Statistically significant differences between two experimental
   groups were determined by unpaired Student’s t-test, two-way ANOVA with
   Šídák’s multiple comparisons or Tukey’s post-hoc test, and by ordinary
   one-way ANOVA with Dunnett’s multiple comparisons test. P values <0.05
   were considered significant and denoted with 1, 2, 3 or 4 asterisks
   when lower than 0.05, 0.01, 0.001, or 0.0001, respectively. For RNA-seq
   data analysis, edgeR (version 2.4.0) was used to obtain differential
   gene expression according to the respective vignette.

3. Results

3.1. GPR55 deficiency reduces KPCY-induced tumor weight and volume

   Tumors were induced by s.c. injection of KPCY cells and harvested after
   21 days ([95] Figure 1A ). Ex vivo measurements revealed that the
   tumors of GPR55 KO mice were smaller in weight and volume than those of
   WT mice ([96] Figure 1B ). Although we measured more live cells in
   tumors from KO mice, percentages of CD45^+ cells did not differ
   significantly between the two groups ([97] Figure 1C ). Percentages of
   CD3^+ T cells, CD8^+ T cells, and pan-dendritic cells were higher in
   tumors of GPR55 KO vs. WT mice ([98] Figures 1D, E ), while the number
   of neutrophils was lower ([99] Figure 1E ). We observed no changes in
   CD8^+ and CD4^+ T cell subtypes ([100] Figure 1F ). Expression of PD-1
   was higher on CD8^+ T cells in tumors of GPR55 KO vs. WT mice ([101]
   Figure 1G , left), whereas PD-L1 expression was higher on CD45^- cells
   ([102] Figure 1H ). GPR55 mRNA expression (which should only derive
   from GPR55-expressing tumor cells) was significantly lower in tumor
   samples of GPR55 KO vs. WT mice ([103] Figure 1I ).

Figure 1.

   [104]Figure 1
   [105]Open in a new tab

   Deficiency of GPR55 reduces tumor weight and volume in KPCY-induced
   tumors. (A) GPR55 KO and WT mice were subcutaneously (s.c.) injected
   with 5x10^5 KPCY cells on day 0. On day 21, tumors were measured ex
   vivo and processed for analysis. (B) Tumor mass and volume are
   expressed by medians, 25^th 75^th percentiles, and min-max values from
   three pooled independent experiments. n= 25-27. (C–H) Flow cytometric
   analysis of lymphoid and myeloid populations in single cell suspensions
   from KPCY tumors. Data indicate medians, 25^th and 75^th percentiles,
   and min-max values from three pooled independent experiments. n=14-25.
   (I) Relative gene expression of GPR55 mRNA in KPCY tumors. n=12-14.
   Statistical differences were evaluated by using unpaired Student’s
   t-test (B, C, H, I) or two-way ANOVA with Šídák’s multiple comparisons
   test (D, E, G). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. WT,
   wildtype; KO, knockout; CD8^+ T mem, CD8^+ T memory cells; panDCs,
   pan-dendritic cells; cDCs1, dendritic cell type 1; mDCs,
   monocyte-derived dendritic cells; NK cells, natural killer cells; ns,
   not significant; γδ T cells; TME, tumor microenvironment.

3.2. GPR55 deficiency also reduces KPCY55-induced tumor weight and volume

   Knowing that GPR55 is highly expressed in pancreatic cancer ([106]20,
   [107]28), we generated a KPCY cell line (KPCY55) that stably
   overexpressed GPR55 ([108] Figure 2A ). KPCY55 cells were injected s.c.
   into the flanks of mice, and tumors were harvested after 29 days ([109]
   Figure 2B ). Similar to KPCY cell-induced tumors, KPCY55 tumors from
   GPR55 KO mice were smaller in both mass and volume than those from WT
   mice ([110] Figure 2C ). Percentages of CD45^+ cells remained unchanged
   between the two groups ([111] Figure 2D ). The number of lymphoid cells
   was higher in tumors of GPR55 KO mice, with increases in CD3^+, CD8^+ T
   and NK cells ([112] Figure 2E ). There was no difference between the
   presence of myeloid cells between the two groups ([113] Figure 2F ).
   Among lymphoid subtypes, we measured increases in CD4^+ T effector and
   CD8^+ T memory cells ([114] Figure 2G ). PD-1 expression on CD8^+ T
   cells remained unchanged, but it was lower in CD4^+ T cells from tumors
   of GPR55 KO vs. WT mice ([115] Figure 2H ). On the other hand,
   expression of PD-L1 was higher on macrophages [known to be the main
   source of PD-L1 in the TME ([116]33)] in KPCY55 tumors of GPR55 KO mice
   ([117] Figure 2H , right). Total mRNA expression in KPCY55 tumor
   samples differs widely between GPR55 KO and WT mice, indicating the
   lack of GPR55 in cells of the TME, and the reduction of tumor cells in
   KO mice ([118] Figure 2I ). In [119]Figure 2J , representative ISH
   images and colocalization of GPR55 mRNA with tumor cells are shown in
   KPCY55 tumors from GPR55 KO and WT mice, demonstrating similar
   colocalization ([120] Figure 2J , right).

Figure 2.

   [121]Figure 2
   [122]Open in a new tab

   GPR55 deficiency reduces tumor weight and volume in tumors with a
   GPR55-overexpressing clone (KPCY55). (A) Relative gene expression of
   GPR55 in KPCY and KPCY55 cell lines. n=3. (B) GPR55 KO and WT mice were
   subcutaneously (s.c.) injected on day 0 with 5x10^5 KPCY55 cells. On
   day 29, tumors were measured ex vivo and used for analysis. (C) Tumor
   mass and tumor volume from ex vivo measurements of KPCY55 tumors. Data
   indicate medians, 25^th and 75^th percentiles, and min-max values from
   three experiments. n=21-24. (D–H) Flow cytometric analysis of single
   cell suspensions from KPCY55 tumors showing changes in the lymphoid and
   myeloid cell populations. (I) RT-qPCR of KPCY55 whole tumor samples
   from WT and GPR55 KO mice. n=4. (J, left) GPR55 ISH signals (red;
   representatively indicated by arrows) in tumor cells (YFP tagged) of
   KPCY55 tumors labeled with anti-GFP antibody (green). Nuclei are
   stained with DAPI (blue). Calibration bars: 20µm. (J, right) Percentage
   (%) colocalization of GPR55 mRNA ISH signals with KPCY55 tumor cells.
   Sections from 3 animals were evaluated. Data indicate medians, 25[th]
   and 75[th] percentiles, and min-max values from one (G), two (H), or
   three experiments (D–F). n=4-24. Statistical differences were assessed
   by using two-way ANOVA with Šídák’s multiple comparisons test (E–G) or
   unpaired Student’s-t test (A, C, D, I, J right). *p<0.05; **p<0.01,
   ***p<0.001,****p<0.0001; **p<0.01; ****p<0.0001. WT, wildtype; KO,
   knockout; CD8+ T mem, CD8+ T memory cells; panDCs, pan-dendritic cells;
   cDCs1, dendritic cell type 1; mDCs, monocyte-derived dendritic cells;
   NK cells, natural killer cells; ns, not significant γδ T cells, gamma
   delta T cells; TME, tumor microenvironment.

3.3. GPR55 is present in immune and cancer cells of tumors

   We performed ISH combined with immunofluorescence and localized GPR55
   mRNA expression in sections of KPCY and KPCY55 tumors from WT mice
   ([123] Figure 3 ). GPR55 largely colocalized with CD11b^+ cells (66.8%
   ± 7.9%), CD8^+ T cells (31.6% ± 5.7%), CD4^+ T cells (43.5% ± 4.9%),
   but only little with F4/80^+ macrophages (12.6% ± 4.3%) in KPCY tumors.
   GPR55 mRNA colocalized with KPCY55 tumor cells by 43.4% ± 13.4%, and
   with KPCY tumor cells by 31.9% ± 11% (all data are means ± SD).

Figure 3.

   [124]Figure 3
   [125]Open in a new tab

   Presence of GPR55 in KPCY and KPCY55 tumors. ISH/immunofluorescence for
   immune and tumor cells in sections of KPCY and KPCY55 tumors from WT
   mice. Representative arrows point at cells expressing GPR55 mRNA
   signals (red). Antibodies against immune cell markers CD8^+, CD4^+,
   CD11b^+, and F4/80^+ were used (all in green). Tumor cells (YFP^+) of
   KPCY and KPCY55 tumors were labeled with anti-GFP antibody (green).
   Nuclei were stained with DAPI (blue). Calibration bars=20 µm. 30-900
   cells were counted per section; sections from 3 animals used. ISH, In
   situ hybridization; WT, wildtype; TME, tumor microenvironment.

3.4. GPR55 deficiency leads to upregulation of genes involved in immune cell
functions in KPCY55-induced tumors

   Since changes in the immune TME of GPR55 KO mice were more pronounced
   in KPCY55 than KPCY tumors, and high expression of GPR55 was reported
   in human pancreatic cancer ([126]28), we used KPCY55 tumors for RNA-seq
   evaluation and for further in vivo experiments.

   Gene expression analysis revealed 768 differentially expressed genes
   (DEGs) in KPCY55 tumors (see volcano plot in [127]Figure 4A ) of GPR55
   KO vs. WT mice, of which 569 were up- and 199 downregulated (|log FC|≥1
   and FDR<0.05) (see also [128]Supplementary Table S3 ). PCA analysis of
   the top 500 genes with greatest significant differences between KPCY55
   tumors of GPR55 KO and WT mice revealed clear separation of gene
   expression profiles ([129] Figure 4B ). In [130]Figure 4C (and
   [131]Supplementary Figure S4 ), pathway enrichment analysis of RNA-seq
   data showed that the DEGs particularly correspond, among others, to
   pathways related to immune cell functions, such as antigen
   processing/presentation, signaling and differentiation of T cells, NK
   cell cytotoxicity, and checkpoint protein expression, indicating strong
   anti-tumor immune responses in KPCY55-induced tumors of GPR55 KO vs. WT
   mice. Normalized read counts of the top fifty differentially expressed
   genes from individual mice of each group further highlight the
   differences in gene expression between the two groups (wt1-wt6 vs.
   ko1-ko6; [132]Figure 4D ).

Figure 4.

   [133]Figure 4
   [134]Open in a new tab

   RNA-seq reveals that GPR55 deficiency leads to upregulation of genes
   involved in enhanced immune cell functions in KPCY55-induced tumors.
   (A) Volcano plot showing the log of fold-change (logFC) on the x axis
   and the −log[10] false discovery rate (−log[10]FDR) on the y axis of
   RNA-seq data. The cut-off values were 1 for fold-change, and 0.05 for
   FDR, respectively. (B) PCA plot of log gene expression data showing the
   first and second principal components. (C) Pathway analysis of RNA-seq
   data, showing enrichment in processes linked to immunity. The number of
   genes enriched in a pathway is represented by the size of the dot. The
   color of the dot represents the −log[10] (lowest-p) value of the
   pathway. (D) Heatmap of the top 50 differentially expressed genes in
   KPCY55 tumors from WT and GPR55 KO mice. The resulting Z-score scaling
   indicates that genes exhibiting high expression relative to the mean
   have positive Z-scores (dark red), while low-expression genes exhibit
   negative Z-scores (white). Ko (ko), knockout; WT (wt), wildtype; TME,
   tumor microenvironment.

3.5. Cell cycle genes and Ki-67 are changed in KPCY55-induced tumors of GPR55
KO vs. WT mice

   Our RNA-seq dataset additionally revealed decreased expression of
   stratifin (Sfn; a protein involved in cell cycle control and used as a
   biomarker of poor prognosis in PDAC) ([135]34), and cyclin D2 (Ccnd2;
   involved in cell cycle regulation and malignancy ([136]35)) in KPCY55
   tumors of GPR55 KO vs. WT mice ([137] Supplementary Figure S5A ).
   Interestingly, there were no changes in the expression of proliferation
   marker Ki-67 in our bulk RNA-seq data from whole tumor samples ([138]
   Supplementary Figure S5A ). However, by measuring Ki-67 mRNA in KPCY55
   tumor cells of ISH sections, we observed lower expression in GPR55 KO
   vs. WT mice ([139] Supplementary Figure S5B ). GPR55 overexpression did
   not influence the viability and proliferation of KPCY55 cells in
   culture ([140] Supplementary Figures S6A, B ).

3.6. Migration of T cells from GPR55 KO mice towards CXCL9 is enhanced

   Since we observed increased T cell activity in RNA-seq pathway
   analysis, as well as increased numbers of lymphoid cells in KPCY55
   tumors of GPR55 KO mice, we set out to investigate a possible mechanism
   behind the higher T cell counts, focusing on cytokines/chemokines
   involved in T cell migration. In our RNA-seq data, significant
   increases in normalized RNA counts for the T cell-attracting and
   interferon (IFN)-γ-inducible ligands Cxcl9 and Cxcl10, and for their
   receptor Cxcr3 ([141]36) were detected (although levels for Cxcr3 and
   INF-γ were quite low) ([142] Figure 5A ). We subsequently validated
   these targets using RT-qPCR ([143] Figure 5B ) and investigated a
   potential migratory effect of CXCL9 on T cells involving GPR55. To this
   end, we assessed the migration of splenic pan-T cells from GPR55 KO and
   WT mice in response to CXCL9. We counted more T cells from KO than from
   WT mice migrating towards increasing concentrations of CXCL9 ([144]
   Figure 5C ).

Figure 5.

   [145]Figure 5
   [146]Open in a new tab

   Role of the CXCR3/CXCL9 axis in the migration of T cells. (A)
   Differential expression of Cxcl9, Cxcl10, Cxcr3, and Ifnγ in the TME of
   KPCY55 tumors from WT and GPR55 KO mice, presented by normalized counts
   (transcripts per million) with p-adjusted values (p.adj) from bulk
   RNA-seq. n=6. (B) Relative gene expression (2^^-dCT) of Cxcl9, Cxcl10,
   and Ifnγ normalized to Hprt. Statistical differences were evaluated by
   unpaired Student’s t-test, **p<0.01, *p<0.05. n=6. (C) Chemotaxis assay
   of pan-T cells isolated from healthy spleens of GPR55 KO and WT mice,
   migrating towards different concentrations of CXCL9 in the bottom
   wells. Statistical differences were evaluated using two-way ANOVA and
   Tukey’s post hoc test. *p<0.05. n=4-5 (D) Geometric means of CXCR3
   expression on lymphocytes in KPCY55 tumors from WT and GPR55 KO mice.
   Statistical differences were evaluated using two-way ANOVA with Šídák’s
   multiple comparisons test. ***p<0.001, ****p<0.0001. n=4-5. In all
   datasets, data indicate medians, 25^th and 75^th percentiles, and
   min-max values. (E) Schematic showing the comparison between the GPR55
   WT and KO TME. p.adj, adjusted p-value; ns, not significant; WT,
   wildtype; KO, knockout; TME, tumor microenvironment. Created in
   BioRender. Ristic, (D) (2024) [147]https://BioRender.com/j25e259.

   Presence of CXCR3 on T cells in KPCY55 tumors was then evaluated using
   flow cytometry ([148] Figure 5D ). There was a decrease of CXCR3 on
   CD3^+ and CD8^+ T cells in KPCY55 tumors of GPR55 KO vs. WT mice.
   Expression of CXCR3 was not different between splenic T cells of
   healthy WT and GPR55 KO mice ([149] Supplementary Figure S7 ). A
   schematic summarizing the migration assays is shown in [150]Figure 5E .

3.7. Deficiency of GPR55 influences anti-PD-1 antibody treatment in mice with
KPCY55-induced tumors

   In [151]Figure 2H , we showed an increase of PD-L1 on macrophages by
   flow cytometry in KPCY55 tumors of GPR55 KO vs. WT mice. By use of
   RNA-seq and RT-qPCR, we also detected higher mRNA expression of immune
   checkpoint and co-stimulatory molecules, e.g., CD27, CD86, ICOS and
   CD40 ([152] Figures 6A, B ). Since PD-L1 is one of the most reliable
   biomarkers for responding to PD-1 blockade ([153]37), we injected
   KPCY55 cells s.c. into GPR55 KO and WT mice, and after 14 days, treated
   the mice with 6 rounds of anti-PD-1 antibodies (or isotype control)
   ([154] Figure 6C ). We confirmed the results on tumor weight and volume
   from [155]Figure 2C in this set of experiments (see [156]Supplementary
   Figures S8A, B ). Notably, when comparing isotype control and anti-PD-1
   antibody treatment, we observed that tumor volume and mass increased in
   WT ([157] Figure 6D ), but not in GPR55 KO mice ([158] Figure 6E ). The
   treatment, however, did not further reduce tumor burden in GPR55 KO
   mice ([159] Figure 6E ). To elucidate whether PD-1 inhibition was more
   effective in GPR55 KO than WT mice, we evaluated the % differences (Δ)
   in tumor weights/volumes between isotype control and anti-PD-1 antibody
   treatment in both groups. As a result, we noticed differences in
   changes of tumor weight/volume that were greater between
   anti-PD-1-antibody-treated than isotype control-treated mice (p=0.07),
   suggesting that anti-PD-1 antibodies are more effective in KPCY55
   tumors of GPR55 KO than WT mice ([160] Supplementary Figure S8C ).

Figure 6.

   [161]Figure 6
   [162]Open in a new tab

   GPR55 deficiency in the TME of KPCY55 tumors influences anti-PD-1
   treatment. (A) RNA-seq-derived normalized counts (transcripts per
   million with p-adjusted values (p.adj)) and (B) RT-qPCR data
   (normalized to Hprt) for a select number of immune checkpoint and
   costimulatory molecules. n=6. (C) GPR55 KO and WT mice were
   subcutaneously (s.c.) injected on day 0 with 5x10^5 KPCY55 cells.
   Starting on day 14, six rounds of anti-PD1 antibodies or isotype
   control were injected i.p. into mice. On day 28, tumors were measured
   ex vivo and used for analysis. (D, E) Ex vivo measurement of tumor mass
   and volume in isotype control (Isotype) and anti-PD1 antibody (PD-1)
   treated mice. n=8-10 per group. Statistical differences were evaluated
   using unpaired Student’s t-test. (F) Flow cytometric analysis of
   monocytes and macrophages positive for CXCL9 expression in KPCY55
   tumors from WT or GPR55KO mice. Statistical differences were evaluated
   using two-way ANOVA with Šídák’s multiple comparisons test. In all
   datasets, data indicate medians, 25^th and 75^th percentiles, and
   min-max values. *p<0.05, **p<0.01, ***p<0.001. n=7-10. WT, wildtype;
   KO, knockout; p.adj, adjusted p-value; PD-1, anti-PD-1 antibody;
   Isotype, isotype control; TME, tumor microenvironment.

   With regard to a potential effect of PD-1 blockade on the CXCR3/CXCL9
   axis, we found that macrophages from tumors of GPR55 KO mice expressed
   significantly more CXCL9 than those from tumors of their WT
   counterparts, while noticing a strong trend in the same direction for
   monocytes ([163] Figure 6F ). However, CXCL9 expression did not differ
   between isotype control and anti-PD-1 antibody treatment ([164]
   Figure 6F ) (nor was there a difference in CXCR3 expression on CD3^+
   and CD8^+ T cells between the groups; see [165]Supplementary Figure S8D
   ), indicating that the CXCR3/CXCL9 axis was unaffected by anti-PD1
   antibody treatment.

4. Discussion

   Even though pancreatic cancer is the eleventh most common cancer in the
   world, it is the seventh leading cause of cancer-related deaths
   ([166]38). Since involvement of the immune system is low in this type
   of cancer, new therapeutic approaches are hard to implement, and often
   do not prolong long-term survival of the patient ([167]39). Recent
   research suggests that stratification of patients according to immune
   infiltration could be of benefit ([168]40). As such, a higher number of
   infiltrating CD8^+ T cells can predict a better response to
   immunotherapy ([169]40).

4.1. GPR55 in cancer cells

   Studies have shown that GPR55 plays a pro-tumorigenic role in many
   types of cancer ([170]20, [171]21, [172]25, [173]41–[174]43). As to
   pancreatic cancer, inhibition of GPR55 revealed antitumor effects in a
   human pancreatic cancer cell line ([175]44). In addition, genetic
   ablation of GPR55 in a PDAC mouse model significantly prolonged the
   survival of mice ([176]20). In our experiments, we also saw less Ki-67
   expression in tumor cells of GPR55 KO mice, suggesting reduced
   proliferation through deficiency of GPR55. Its influence on the
   metabolism of pancreatic cancer cells has been also demonstrated
   through the actions of (R, R’)-4’-methoxy-1-naphthylfenoterol, which,
   by modulating GPR55 signaling, altered L-lactose metabolism in PANC-1
   cells and mice xenografts ([177]45).

   We did not see differences in proliferation and viability between KPCY
   and GPR55-overexpressing KPCY cells in culture. However, our ISH
   findings indicated that KPCY tumor cells, although hardly expressing
   GPR55 mRNA in culture ([178] Figure 2A ), upregulated GPR55 mRNA in
   situ ([179] Figure 3 ). Importantly, we observed that GPR55 was present
   in several types of immune cells of the TME, suggesting that GPR55 may
   influence tumor growth via actions of these cells. In line with this
   hypothesis, we previously reported smaller tumors and higher
   infiltration of CD3^+, CD4^+, and CD8^+ T cells, but lower infiltration
   of myeloid suppressor cells into tumors of GPR55 KO vs. WT mice with
   colorectal cancer, indicating a possible T cell- suppressive role of
   GPR55 ([180]18). A pronounced immune response could also be the reason
   why KPCY55-induced tumors grew slower (appropriate tumor size for
   harvesting after 29 days) than KPCY-induced tumors in GPR55 KO mice
   (harvested after 21 days).

4.2. GPR55-deficiency favors an anti-tumorigenic immune cell profile

   Next to KPCY and KPCY55 tumor cells, we identified GPR55 in numerous
   immune cells of the TME (CD4^+ and CD8^+ T cells, macrophages, and
   CD11b^+ myeloid cells) indicating a role for GPR55 in immune cell
   functions, such as migration and cytokine release ([181]16, [182]46).
   Although similar changes in tumor weight and volume were measured for
   KPCY and KPCY55 tumor-bearing GPR55 KO mice, we observed differences in
   the composition of their immune TME. Lack of GPR55 in the TME of KPCY55
   tumors from KO mice caused a shift in the immune cell profile towards
   increased numbers of CD8^+ T and NK cells, which are known for their
   cytotoxicity against cancer cells ([183]3, [184]47). Among T cell
   subtypes, CD4^+ effector T cells and CD8^+ memory T cells were
   increased, indicating enhanced adaptive immune response ([185]48). In
   KPCY tumors of GPR55 KO mice, we also detected a decrease of
   neutrophils vs. WT tumors, which fits with the mostly pro-tumorigenic
   role of neutrophils in pancreatic cancer ([186]49). In addition, KPCY
   tumors from GPR55 KO mice had a higher number of pan-dendritic cells
   than tumors from WT mice. Dendritic cells are known to cross-present
   antigens to CD8^+ T cells ([187]50), and to enhance T cell priming
   ([188]51). Spleen from healthy GPR55 KO mice do not show increased
   levels of CD3^+ T cells as compared to healthy WT mice ([189]
   Supplementary Figure S9 ) indicating that the regulation of the immune
   cell profiles were tumor specific.

4.3. RNA-seq data indicate enhanced immune cell functions in KPCY55 tumors of
GPR55 KO mice

   By employing pathway analysis of differentially-expressed genes, we
   noticed strong shifts, in particular, towards pathways related to
   immune cell functions, such as antigen processing/presentation and T
   cell signaling in KPCY55 tumors of GPR55 KO mice. We further observed
   upregulation of the immunoproteasome genes Psmb8, 9 and 10, which are
   known to be involved in enhanced immunogenicity in PDAC ([190]52).
   These findings may be of importance, as restoring immunity, such as the
   expression of MHC I on the pancreatic tumor cell surface ([191]53), is
   of prime interest for new immunotherapies against PDAC. It therefore
   seems that GPR55 deficiency in the TME favors an anti-tumorigenic
   environment by overriding T cell suppression.

4.4. GPR55 is involved in the migration of T cells towards CXCL9

   Although macrophages, neutrophils, mast cells, and tumor cells treated
   with a GPR55 antagonist decrease their migratory behavior ([192]17,
   [193]19, [194]46, [195]54), we observed increased migration of pan-T
   cells, isolated from healthy GPR55 KO mice. This is well in line with a
   previous study that showed enhanced migration of γδ T cells in GPR55 KO
   mice, indicating that GPR55 can also function as a migration-inhibitory
   receptor ([196]16). Our RNA-seq data revealed upregulation of the T
   cell chemokines Cxcl9/Cxcl10 and their receptor, Cxcr3, in KPCY55
   tumors of GPR55 KO mice. CXCL9, CXCL10 and CXCL11 have been previously
   shown to be effective in supporting an adequate antitumoral response in
   various cancers ([197]55), although results in pancreatic cancer have
   been conflicting ([198]56, [199]57). CXCR3 is almost exclusively
   expressed in immune cells ([200]36), and together with its ligands, it
   has been demonstrated to play an important role in anti-tumor T cell
   development in the spleen ([201]57). Our RNA-seq data showed
   significantly upregulated Cxcr3 transcripts in KPCY55 tumors of GPR55
   KO vs. WT mice, despite the decrease of CXCR3 protein on CD3^+ and
   CD8^+ T cells in the TME. This observation may be explained by a study
   showing that expression of CXCR3 on T lymphocytes decreases as a
   consequence of internalization and degradation in the presence of its
   ligands ([202]58). With regard to the source of CXCL9, we identified
   monocytes and macrophages as main producers ([203]59), which is in line
   with a report demonstrating that depletion of CXCL9-expressing
   tumor-associated macrophages hamper anti-tumor responses ([204]60).

4.5. GPR55 influences anti-PD-1 antibody treatment

   Pancreatic cancer has one of the lowest response rates to
   immunotherapy, recently demonstrated in a clinical trial, where 3.1% of
   patients with metastatic PDAC responded to dual immunotherapy with
   durvalumab (PD-L1 blocking antibody) and tremelimumab (CTLA-4 blocking
   antibody), and 0% to durvalumab monotherapy ([205]61). It is known that
   tumor mutational burden and expression of PD-L1 on tumor cells are
   robust predictive factors for the success of immunotherapy ([206]37).
   Our RNA-seq and RT-qPCR data revealed upregulated PD-L1 and PD-1
   expression in KPCY55 tumors of GPR55 KO mice. In addition, flow
   cytometry revealed higher PD-L1 expression on macrophages in KPCY55
   tumors from GPR55 KO mice, which suggested a favorable response to
   immunotherapy. Interestingly, anti-PD-1 antibody treatment increased
   the tumor burden in WT, but not GPR55 KO mice. The treatment did not
   further decrease tumor growth in GPR55 KO mice, most likely because it
   had already been robustly reduced by GPR55 deficiency alone. Regarding
   the increase in tumor burden in GPR55 WT mice, it should be noted that
   cancer patients can paradoxically respond with increased tumor
   progression to immune checkpoint inhibitor therapy (hyperprogressive
   disease) ([207]62, [208]63), something that has been now also reported
   for advanced pancreatic cancer ([209]64). This phenomenon describes an
   increase in tumor growth post PD-1/PD-L1 blockade, and was observed in
   several cancers with solid tumors (rev. in Champiat et al.) ([210]62).
   Reported frequencies lie between 9-29% and are associated with poor
   outcome ([211]62). Mechanisms for hyperprogressive disease have not
   been clarified yet but several explanations are discussed, such as T
   cell exhaustion, a modulation of pro-tumorigenic T cells, or enhanced
   oncogenic signalling. A case study in advanced pancreatic cancer with
   hyperprogression treated with PD-1 blockade and chemotherapy identified
   amplification of MDM4 (a p53 regulating protein) as a risk factor
   ([212]65). From our experiments, it is not clear what caused
   hyperprogression. We can only speculate that overexpression of GPR55 in
   tumor cells may have taken part in the tumor progression of WT mice
   after PD-1 blockade.

4.6. Limitations of the study

   One of the limitations of our study is that we only performed mouse
   experiments but did not use human samples. GPR55, however, has been
   found to be highly expressed in human PDAC specimens and
   patient-derived xenografts ([213]20), suggesting that the receptor may
   also play an important role in human PDAC. In addition, the abundance
   of CD8^+ T cells, in particular of the PD-1 CD8^+ T cell population, in
   tumors of mouse models injected with KPCY cells, can clearly predict
   the response to immunotherapy with checkpoint blockers ([214]27). This
   is in accordance with the situation in human tumors, where a high CD8^+
   T cell population is crucial for a successful immunotherapy with
   checkpoint inhibitors ([215]47). The findings indicate that our KPCY
   mouse model can recapitulate the situation of human PDAC, therefore
   being of high translational value.

5. Conclusions

   We can demonstrate that deficiency of GPR55 in the TME of murine PDAC
   tumors leads to improved immune cell infiltration and upregulation of
   genes involved in T cell activity and function, indicating that
   TME-derived GPR55 may promote a “cold” tumor. A CXCR3/CXCL9/CXCL10 axis
   could drive T cell infiltration into KPCY55 tumors of GPR55 KO mice,
   suggesting that GPR55 suppresses this pathway in PDAC, thereby
   promoting tumor growth. Tumor cell-derived GPR55 does not seem to play
   a role in the differences seen in tumor burden between GPR55 KO and WT
   mice, but we cannot exclude that stromal TME cells like fibroblasts or
   endothelial cells, which are known to express GPR55, may contribute to
   it. Finally, our data suggest that GPR55 could be an interesting target
   for immunotherapies against PDAC by using GPR55 inhibitors as an immune
   adjuvants to promote T cell trafficking and infiltration, or in an
   adoptive immunotherapy approach.

Acknowledgments