Siglec15 Shapes A Non-Inflamed Tumor Microenvironment And Induces Immunotherapy Resistance In Bladder Cancer

Background and Purpose:Bladder cancer(BLCA)is the second most common urinary cancer with increasing morbidity.Anti-cancer immunotherapy significantly improved the survival of BLCA.However,only a minority of BLCA patients are sensitive to immunotherapy.Therefore,it’s urgent to explore the immunotherapy resistance mechanisms and then develop new immunotherapy targets.Recently,Siglec15 emerged as a broad-spectrum target for normalization cancer immunotherapy,and it was complementary to PD-L1.Siglec15 promotes tumor growth by inhibiting the proliferation of CD8~+T cells,and Siglec15 inhibitors relieve this immunosuppression.Though the results of phase Ⅰ clinical trial in advanced non-small cell lung cancer indicated that Siglec15 inhibitors achieved a promising clinical response(NCT03665285),the phase Ⅱ clinical trial failed to confirm the survival benefit of anti-Siglec15 immunotherapy in more cancer types.In addition,the role of Siglec15 in BLCA remains uncertain.In this study,we aimed to identify the optimal cancer type to receive anti-Siglec15 immunotherapy.Then,we aimed to explore the role of Siglec15 in shaping tumor immune microenvironment of BLCA.Methods:We comprehensively evaluated the expression pattern and immunological role of Siglec15 using pan-cancer analysis based on RNA sequencing data obtained from The Cancer Genome Atlas(TCGA).We then systematically analyzed the relationship between Siglec15 and immunological characteristics in the BLCA tumor microenvironment(TME),such as immunomodulators,cancer immunity cycles,tumor-infiltrating immune cells(TIICs),immune checkpoints,T cell inflamed score(TIS),in several independent BLCA cohorts.Functional enrichment analysis was used to explore the regulatory mechanisms of Siglec15 in shaping the TME immune status of BLCA.We then established Siglec15 overexpressed and down-expressed BLCA cell lines.RNA-Seq,high throughput liquid protein microarray,RT-PCR,WB,and ELISA were used to detect the level of cytokines or chemokines regulated by Siglec15.Chemotaxis assay,flow cytometry,and Tissue FAXS Cytometry panoramic tissue quantification assay were applied to identify the immune cell types mostly regulated by Siglec15.T cell-mediated tumor cell killing assay and co-culture of T cells and tumor cells were utilized to explore the direct impact of Siglec15 on CD8~+T cells.Transwell assay,MTT,and wound healing assay were used to detect the invasiveness,proliferation,and migration ability of BLCA cell line.Finally,the regulatory mechanisms of Siglec15 on TME immune status and immunotherapy resistance in BLCA were validated in C57 mouse models and in a real-world neoadjuvant immunotherapy cohort.Results:Siglec15 was specifically overexpressed in the TME of various cancers including BLCA.The most significant immunosuppressive effect of Siglec15 on TME was observed in BLCA,which suggested that BLCA may be a suitable candidate for anti-Siglec15immunotherapy.We demonstrated that Siglec15 shaped a non-inflamed TME in BLCA based on the evidence that Siglec15 negatively correlated with immunomodulators,TIICs,cancer immunity cycles,immune checkpoints,and TIS.Siglec15 overexpression resulted in immunotherapy resistance.Mechanically,Siglec15 overexpression inhibited the expression of several pro-inflammatory cytokines and chemokines,such as CCL2,CCL3,CCL4,CCL5,CXCL9,and CXCL10,and therefore reduced the infiltration level of CD8~+T cells,which finally induced the formation of a non-inflamed TME.Meanwhile,Siglec15 overexpression promoted the invasiveness,proliferation,and migration ability of BLCA cell line.In addition,Siglec15 overexpressed BLCA cells directly inhibited the killing activity of CD8~+T cells.Finally,we successfully validated that Siglec15 overexpression promoted the formation of a non-inflamed TME and resulted in immunotherapy resistance in C57 models and in a real-world neoadjuvant immunotherapy cohort.Conclusions:Siglec15 is a potential immunotherapy target for BLCA.Siglec15 overexpression impairs the ability of CD8~+T cells homing to TME and inhibited the killing activity of CD8~+T cells,which finally contributes to the formation of a non-inflamed TME and immunotherapy resistance.