Glycolysis Could Promote The CSCs And EMT By Stimulating The Secretion Of TGF-β By M2 Macrophages In Bladder Cancer

Objective: Immune checkpoint inhibitors represented by PD-1/PD-L1 have been used to treat a variety of cancer types and can greatly improve the survival rate of patients.However,the low response rate and drug resistance of patients with immunotherapy limit its clinical application.A deeper understanding of the regulatory mechanism of PD-1/PD-L1 axis is essential to solve the above-mentioned bottlenecks in clinical application and develop combined treatment strategies.Existing studies have shown that the level of tumor glycolysis,tumor-associated macrophage infiltration,the expression of TGF-β,tumor stemness and epithelial-mesenchymal transition are closely related to the poor efficacy of tumor PD-1/PDL1 treatment,however the correlation and mutual regulation mechanism between them in the occurrence and development of bladder cancer are still lack of in-depth research.In this study,we aimed to preliminarily investigate the the correlation between the level of glycolysis,the infiltration of M2 macrophages and the expression of TGF-β,and the mechanism of glycolysis promoting the CSCs and EMT by stimulating the secretion of TGF-β by M2 macrophages in bladder cancer.Methods: Seventeen glycolysis-related genes were screened by bioinformatics methods according to the transcriptome and proteomics data of bladder cancer tissues.Combined with the clinical information data of bladder patients in TCGA database and GEO database,we used univariate COX regression analysis to determine the differential expression of glycolysisrelated genes and whether they were prognostic genes.According to the expression of glycolysis-related genes,the patients were classified into 3 clusters using “cluster Profiler” by R software.PCA was used to verify the differences of the three subtypes.Kaplan-Meier survival analysis was used to verify survival differences between different glycolysis subtypes.The heat map was generated by combining the survival status and survival time information of patients with the level of gene expression in the database.The clinical information and bladder tissue of 70 patients with bladder cancer from the Affiliated Hospital of Qingdao University was collected,and the protein expression of glycolysis key gene-PKM2,M2 macrophage marker-CD206 and TGF-β was detected by immunohistochemistry(IHC).The relationship between the expression of PKM2,CD206,TGF-β protein and the clinical characteristics of patients with bladder cancer was analyzed by using SPSS software.Subsequently,the correlation of PKM2,CD206 and TGF-β expression in bladder cancer was analyzed.To further understand the effect of TGF-β secreted by M2 macrophages on the tumor stemness and epithelial-mesenchymal transition(EMT)process of bladder cancer,bladder cancer cells T24/5637 were cocultured with M2 macrophages.We examined the changes in colony formation,migration and invasion ability of bladder cancer cells,and detected the altered expression levels of CD44,E-cadherin and Vimentin in mRNA and protein.Then,we added TGF-β inhibitor in the coculture system of bladder cancer cells and M2 macrophages,and then observed the bladder cancer cell colony formation,migration and invasion ability in the coculture group.The mRNA and protein expression levels of CD44,E-cadherin and Vimentin were detected.Results: Sixteen glycolysis-related genes were screened by bioinformatics methods according to the transcriptome and proteomics data of bladder cancer tissues.According to the different expression levels of the 17 glycolysis-related genes,the TCGA bladder cancer cohort was divided into three different subtypes,which represented different glycolysis levels of bladder cancer.Survival analysis showed that the level of glycolysis was closely related to the poor prognosis of bladder cancer.Meanwhile,glycolysis-related marker PKM2,M2 macrophage markers CD206,TGF-β,were positively correlated in the bladder cancer cohort.The expression of PKM2,CD206 and TGF-β in bladder cancer tissues of 70 patients were detected by immunohistochemistry.PKM2,CD206 and TGF-β proteins were found to be significantly over-expressed in bladder cancer tissues compared with normal bladder tissues.The positive expressions of PKM2,CD206 and TGF-β were closely related to the T stage of patients with bladder cancer,but not to the age,gender and metastasis of lymph node.And the high expression of CD206 is closely related to the distant metastasis of bladder cancer.The expression of PKM2,CD206 and TGF-β were not related to the age,gender,and lymph node metastasis of bladder cancer patients.Correlation analysis showed that PKM2,CD206 and TGF-β expressions were positively correlated with each other.Our previous study showed that enhanced glycolysis in bladder cancer could promote the transformation of macrophages into M2 macrophages and secrete TGF-β,thus we further cocultured bladder cancer cells T24/5637 with M2 macrophages.It was found that the formation of tumor spheres and the expression of CSC marker CD44 were significantly increased in bladder cancer cells,while the addition of TGF-β inhibitor significantly reduced the formation of tumor spheres and the expression of CD44 in bladder cancer cells,indicating that M2 TAMs can promote BC cells to acquire CSC-like characteristics through TGF-β.In addition,the expression of epithelial marker(E-cadherin protein)was significantly down-regulated and the expression of mesenchymal marker(Vimentin protein)was significantly up-regulated in BC cells co-cultured with M2 TAMs.Transwell and Matrigel assay showed that M2 Tams promoted the invasion and migration of BC cells.However,TGF-β inhibitor significantly inhibited the above EMT process in bladder cancer cells,indicating that M2 TAMs can promote the CSCs activity and EMT process of bladder cancer cells through TGF-β and further enhancing the migration and invasion behavior of bladder cancer cells.Conclusion: In the microenvironment of bladder cancer,there is a significant correlation and mutual regulation among glycolysis level,M2 macrophage infiltration and TGF-β,and they are closely related to the prognosis of patients with bladder cancer.Enhanced glycolysis in bladder cancer can promote the transformation of macrophages into M2 TAMs and can further promoting the stemness and epithelial-mesenchymal transition of bladder cancer cells through TGF-β,and enhancing the migration and invasion ability of bladder cancer cells.