CD55~+ Cancer-associated Fibroblasts Suppress The Cytotoxicity Of CD8~+t Cells Through JNK Signaling

Bladder cancer is the most common tumor in the urinary system,with about570,000 new cases every year.Bladder cancer is extremely expensive to treat and patients have a low quality of life because of its highest recurrence rate.The current main treatments for bladder cancer are surgical resection,chemotherapy mainly based on platinum-based drugs and the rise of immune checkpoint inhibitors recently.Although substantial progress has been made in cancer biology and treatment,clinical outcomes of bladder cancer patients are still not satisfactory.Previous work in our laboratory has shown that blocking JNK signaling pathway can effectively inhibit the development of bladder cancer in vivo and in vitro.However,as a signaling pathway with complex downstream effects,the tumor inhibitory effect and molecular mechanism of JNK in bladder cancer remain unclear.In order to comprehensively interpret the role of JNK signaling in bladder cancer TME and further validate the role of JNK signaling as a potential target for bladder cancer treatment,we constructed a BBN-induced mouse bladder cancer model in situ,then analyzed the cellular and molecular changes after JNK signal suppression using single-cell RNA sequencing.Our work identified the JNK signaling pathway as a potential therapeutic target for bladder cancer,and SP600125(JNK inhibitor)was able to limit the development of bladder cancer mouse model in situ.Through single-cell RNA sequencing,we found that SP600125 reduced the exhaustion of CD8~+T cells and increased effector and toxicity.The number of genes affected by JNK inhibiting treatment was significant in CAFs among all cell types.Besides,Cell-to-cell communication analysis also suggested that fibroblasts interact most frequently with CD8~+T cells.Using promoter prediction tools,Ch IP database and ligand receptor pair analysis,we hypothesized that CD55(Code by Adgre)on fibroblasts to inhibit effector and toxicity of CD8~+T cells by interacting with CD97 on CD8~+T cells.SP600125inhibited the expression of CD55 on fibroblasts,thus restored CD8~+T cells’killing function and played an antitumor role.To test this hypothesis,we treated CAFs with SP600125 and results showed the expression level of CD55 m RNA in CAFs was reduced.In tumor killing assay,CD8~+T cells were co-cultured with fibroblasts treated with SP600125 or DMSO for 3 days.Later CD8~+T cells were co-cultured with bladder tumor cells for 7 hours,flow cytometry showed the apoptosis of tumor cells increased in SP600125 group,suggesting JNK inhibiting reduced the immunosuppressive ability of CAFs.In subcutaneous tumorigenesis model,C57BL/6 and BALB/C nude mice were subcutaneously inoculated with murine Bca cells(MB49).Then C57BL/6 mice were administrated the JNK inhibitor SP600125 or anti-PD1 by intraperitoneal injection,BALB/C nude mice only received SP600125 treatment.Compared with the untreated group,both C57BL/6 and BALB/C mice in SP600125 group showed reduced tumor volumes.In C57BL/6 mice group,the combination of SP600125 plus anti-PD1 resulted in the greatest tumor reduction.These results indicate that treatment of JNK inhibitors suppress the tumor progression by targeting both cancer cells and TMEs.Our results suggest that activation of JNK signaling pathway in CD55~+CAF cells can inhibit the killing of CD8~+T cells by CD97,confirming that JNK signaling pathway is a potential target for bladder cancer therapy.