| Objective:The programmed death receptor and its ligand(PD-1/PD-L1)is a common immuno-checkpoint.Aberrant expression of PD-1/PD-L1 significantly affects patients’immuno-therapy.Targeting PD-1/PD-L1 evidently improves more than 50%patients living quality in advanced bladder cancer.However,there are still a large group of patients show poor clinical outcome.Hence,it is of vital importance to further identify how PD-L1 is regulated in bladder cancer.Tumor microenvironment(TME)consists of a group of cells that enable promoting cancer cell survival which including cell proliferation,invasion,metastasis and drug resistance.Cancer associated fibroblast(CAFs)participates in TME the most.CAFs enable to recruit suppressive immuno-regulative cells and elevates immuno-checkpoints expression to mediate immuno-evasion via secreting various cytokines.Whereas the mechanisms that how CAFs affect tumor PD-L1 expression remains less investigated.Therefore,considering the elevated PD-L1 expression that is regulated by CAFs,the study aimed to further clarify how CAFs affect PD-L1 expression in bladder cancer cells,which may provide novel support that will help to identify better and potential tumor target and biomarkers in bladder cancer.Methods:TCGA and GEO bladder cancer datasets were collected to analyze the correlation among CAFs marker proteinα-smooth muscle actin(α-SMA),fibroblast activation protein(FAP)expression and the prognosis of bladder cancer patients;the correlation amongα-SMA,FAP and PD-L1 expression in bladder cancer were analyzed as well.The normal tissue fibroblasts(NFs)and tumor-associated fibroblasts(CAFs)were obtained from patients that received radical resection of bladder cancer.NFs and CAFs were primarily cultured.Cell immune-fluorescence and western blot assay were applied to detectα-SMA,in order to verify the correct isolation of human primary NFs and CAFs.The CAFs and bladder cancer cells were co-cultured via adopting a trans-well culturing chamber.The effects of CAFs on tumor migration,invasion and proliferation of bladder cancer cells were examined by trans-well,wound healing,cell counting kit assay and colony formation assay.After co-culturing with CAFs,real-time quantitative PCR(RT-q PCR)and western blot assay were applied to detect expressive changes on major factors that are associated with glucose metabolism,glutamine metabolism and the PD-L1 in bladder cancer cells.The expression of pathways that are ultimate for PD-L1 expression was detected by western blot.Inhibitors for the above PD-L1 related pathways were used to treat bladder cancer cells,and the effect of CAFs on PD-L1 expression in bladder cancer cells was observed.Supernatant of CAFs were collected and was used to treat bladder cancer cells.After 24h of treatment,bladder cancer cells were co-cultured with CD8~+T cells,and the secretion of IFN-γwas analyzed by ELISA assay.Results:It was observed that higherα-SMA and FAP indicates poor overall survival in TCGA and GEO bladder cancer patients.By taking cell immuno-fluorescence and western blot assay,all primary cells expressed Vimentin but did not express Pan-keratin.The primary isolated NFs and CAFs all highly expressesα-SMA,and the expression ofα-SMA and FAP were extremely higher in CAFs when compared with that in NFs.The results suggest that the primary NFs and CAFs were successfully isolated.To next identify how CAFs affects bladder cancer cells behavior,we adopted a trans-well co-culturing system.By performing trans-well,wound healing,cell counting kit assay and colony formation assay,it was found that CAFS enables promoting migration,invasion and proliferation of bladder cancer cells.Moreover,after co-culturing with CAFs,the expression of key enzymes of gluconeogenesis and glutamine metabolism were all up-regulated in T24 bladder cancer cells.The results suggest that CAFs enable triggering reprogramming of gluconeogenesis and glutamine metabolism in bladder cancer.According to the previous findings in our group and related researches,the reprogramming of gluconeogenesis and glutamine metabolism is highly correlated with PD-L1 expression and is responsible for immune-evasion of cancer cells.Hence,for the immuno-checkpoints,we first analyzed the correlation between CAFs signature and PD-L1 expression on TCGA bladder cancer data-sets.It was found that higherα-SMA and FAP levels suggests positive correlation with PD-L1 expression in bladder cancer.After co-culturing with CAFs,the expression of PD-L1 was significantly elevated in T24 bladder cancer cells.To identify how CAFs affects PD-L1 expression in bladder cancer cells,we observed that EGFR and its downstream pathway AKT/c-JUN were all significantly activated.Inhibiting EGFR,AKT,and c-JUN expression eliminates tumor PD-L1 elevation that was regulated by CAFs.PD-L1 is critical to the cyto-toxical work of CD8~+T cells.We next observed how CAFs elevates PD-L1 expression in bladder cancer to affect CD8~+T cells activity.By performing ELISA assay,it was found that CAFs enable suppressing IFN-γproduction in CD8~+T cells via elevating PD-L1 expression in bladder cancer,whereas inhibiting EGFR,AKT and c-JUN activity abrogate the effect.Taken together,the above findings imply that CAFs elevate PD-L1 expression and mediate immune-evasion in bladder cancer cells by activating EGFR/AKT/c-JUN pathway.Conclusions:In this research,we observed that higher CAFs infiltration indicates poor bladder cancer patients’outcome.Co-culturing bladder cancer cells with CAFs promote migration,invasion and proliferation of bladder cancer cells,and triggering reprogramming of gluconeogenesis and glutamine metabolism in bladder cancer cells.Moreover,CAFs enable elevating PD-L1 expression and mediating immune-evasion in bladder cancer cells by activating EGFR/AKT/c-JUN pathway.Generally,the study provides a novel mechanism that how CAFs affects PD-L1 expression in bladder cancer cells,which may provide novel support that will help to identify better and potential tumor target and biomarkers in bladder cancer. |
PDF Full Text Request