Crosstalk Between Lactic Acid Remodeled Tumor-associated Macrophages And Bladder Cancer Promotes TGF-β Positive Feedback Pathway

Background: The application of immune checkpoint inhibitors in bladder cancer is rapidly expanding.In particular,anti-PD-L1/PD-1 immunotherapy has become the most potential drug for bladder cancer patients who fail prior platinum-based chemotherapy.Although anti-PD-L1/PD-1 immunotherapy has improved survival for certain patients with metastatic bladder cancer,a majority of patients have relapsed after a period of response,that is,drug resistance has occurred.It is speculated that some unknown complex network regulatory factors influencing the clinical efficacy of anti-PD-L1/PD-1 immunotherapy in the microenvironment of bladder cancer.The latest studies revealed that tumor glycolysis,tumorassociated macrophages(TAMs)infiltration,and TGF-β were significantly related to the antiPD-L1/PD-1 immunotherapy resistance.However,the interaction between tumor glycolysis,TAMs,and TGF-β in the efficacy of PD-L1/PD-1 inhibitors and the progression of BC still to be elucidated.Methods: The TCGA database was used to evaluate the correlation and prognostic values of TAMs and glycometabolic level in the BC microenvironment.To further study the crosstalk between TAMs and bladder cancer cells,THP-1 cell derived TAMs were treated with lactic acid or the supernatant of bladder cancer cells.A TGF-β ELISA kit was used to detect the TGF-βsecretory level of M2 macrophages.At the same time,bladder cancer cells and TAMs were cocultured to detect the secretory level of TGF-β.In addition,after adding lactic acid inhibitors into the culture system of bladder cancer cells,the cells were co-cultured with TAMs,and the secretory level of TGF-β in the co-cultured system was measured.To explore the mechanism of that,TAMs were incubated with lactic acid to detect the m RNA and protein expression of HIF-1α.Furthermore,TAMs were treated with HIF-1α inhibitor and measured the TGF-βsecretory level of TAMs.To analyzed the effect of TGF-β on bladder cancer cell glycolysis,bladder cancer cells and TAMs were co-cultured to detect the expression of GLUT1,HK2,PKM2,and LDHA and the concentration of lactic acid.In addition,a TGF-β inhibitor was added into the co-cultured system and detect the changes of above factors.Moreover,the expression levels of phosphorylation Smad2/3,Akt,and Erk under a co-cultured system were investigated,and the inhibition of these pathways to analyze the effects of these pathways on bladder cancer cells glycolysis.To study the association between TAMs secreted TGF-β and the PD-L1 expression of bladder cancer cells,a TGF-β inhibitor was added into the co-cultured system to analyzed the PD-L1 expression of cancer cellsResults: Glycometabolic level and TAMs infiltration has a positive correlation and were significantly associated with the prognosis in the bladder cancer microenvironment by analyzing the TCGA database.We further explore the crosstalk between bladder cancer cells and TAMs and found that lactic acid promotes the TGF-β secretion of TAMs by regulating the HIF-1α pathway.Our study also suggested that TAMs promoted the expression of glycolysisassociated genes and the production of lactic acid by secreting TGF-β.In addition,TAMs secreted TGF-β promotes the glycolysis of bladder cancer cells through Smad2/3,Erk,and Aktm TOR signaling pathways.Furthermore,TAMs secreted TGF-β promotes the PD-L1 expression of bladder cancer cells via TGF-β signaling pathway.Conclusions: The crosstalk between metabolic reprogramming of cancer cells and functional remodeling of TAMs in bladder cancer microenvironment,on the one hand,bladder cancer glycolysis can induce the functional remodeling of TAMs through lactic acid and then promote the secretion of TGF-β.In addition,lactic acid-remodeled TAMs promoted the glycolysis and lactic acid production of bladder cancers via TGF-β signaling pathway.Therefore,a TGF-β positive feedback pathway was formed in the microenvironment of bladder cancer.Furthermore,TAMs secreted TGF-β upregulated the PD-L1 expression of bladder cancer cells,which promoting immune escape and anti-tumor immunity of bladder cancer cells.