BCAT2 Shapes A Noninflamed Tumor Microenvironment And Induces Resistance To Immunotherapy

Background and purpose:Bladder cancer(BLCA)is one of the most common malignancies of urinary system.In view of the outstanding performance of immunotherapy,many domestic and foreign guidelines have recommended it for muscle-invasive or advanced stage bladder cancer patients.However,with the deepening of large-scale clinical trials,the results show that only a part of patients are sensitive to immunotherapy,there is still a lot of patients with immunotherapy resistance,which has become a difficult problem to be solved.We found that BCAT2 has a close connection with tumor microenvironment(TME)of BLCA,and the type of TME largely determines the efficacy of immunotherapy.Therefore,we attempted to explore the underlying mechanism of BCAT2 regulating TME and its potential of being a combination therapy target.Methods:Through analysis of multiple public BLCA cohorts and Xiangya BLCA cohort,the overall relationship between BCAT2 and pro-inflammtory indicators in the TME was preliminatively evaluated.Then,on the single cell level,multiple public single cell BLCA datasets and Xiangya single cell BLCA dataset were used to explore the regulatory mechanism of BCAT2 in TME.After pinpointing the core signaling pathways of regulatory mechanism(lymphocyte related chemokine signaling pathway),high throughput liquid-phase protein chip,q RT-PCR and Elisa were utilized to further ascertain which kind of tumor infiltrated immune cell(TIIC)related chemokines has a close correlation with BCAT2.Chemotaxis assay,T cell-mediated tumor cell killing assay,multi-color immunofluorescence semi-automatic panoramic analysis and immunohistochemistry were employed to demonstrate the direct regulatory relationship between this type of TIIC and BCAT2.In vivo,BCAT2 overexpression(BCAT2 OE)and BCAT2 knockdown(BCAT2KD)cell lines were applied to construct subcutaneous BLCA model in MB49 mice.After that,anti-PD-1 immunotherapy was applied to bearing tumor mice.By comparing the outcomes of tumor volume,survival time,flow cytometry and immunofluorescence,we further explored the regulatory mechanism of BCAT2 on tumor progression and assessed its potential of being a combination immunotherapy target.Finally,Xiangya BLCA immunotherapy cohort and public BLCA immunotherapy cohort were applied to evaluate the potential of BCAT2 on predicting immunotherapy efficacy in the real world.Results:In multiple BLCA cohorts,BCAT2 has a broad immunosuppressive effect on inflammatory indicators in the TME and shapes a non-inflamed TME in BLCA.For further revealing the underlying mechanism,we analyzed multiple BLCA single cell datasets and found that BCAT2 is mainly expressed in tumor cell subset rather than immune cell subsets.Moreover,it formed a non-inflamed TME by inhibiting the activities of lymphocyte related chemokine signaling pathways.Then,high throughput liquid protein chips were used for detecting secretion levels of cytokines and chemokines of BCAT2 stable transfection bladder cancer cells,and found that CD8~+T cell related chemokines(CCL3,CCL4,CCL5,CXCL9,CXCL10)have a more intimatly negative correlation with BCAT2 than other indicators.More importantly,both on the level of vitro and tissue,we verified that expression of BCAT2 can significantly restrain chemotaxis and cytoxicity abilities of CD8~+T cell.In vivo,tumor model with decreased BCAT2expression has a better efficacy of anti-PD-1 immunotherapy.Finally,in BLCA immunotherapy cohorts,we found that the expression of BCAT2is negatively correlated with survival outcome of patients,and it is competent to be a joint marker of predicting the efficacy of immunotherapy.Conclusion:BCAT2 shapes a non-inflammed TME by inhibiting chemotaxis and cytoxicity abilities of CD8~+T cell.It is not only a potential combination immunotherapy target,but also a biomarker of forecasting the efficacy of immunotherapy.