Mechanism Of PD-1 Regulating Tumor Associated Macrophages Polarization And Promoting Bladder Cancer Progression Through IL4/pSTAT6 Pathway

Background:Bladder urothelial carcinoma is one of the most common malignancies of the urinary system.In 2018,the incidence of bladder cancer ranked 10th among all malignancies worldwide,with about 549,000 new cases and 200,000 deaths.The majority of the patients were non-muscle invasive bladder cancer(NMIBC),of which approximately 30%progressed to muscle invasive bladder cancer(MIBC),with 5-year and 10-year survival rates of 50%and 36%,respectively.Cisplatin combined with gemcitabine chemotherapy was the first choice for patients with metastatic bladder cancer,but the median survival time was only 15 months and the 5-year survival rate was only 15%.Currently,immunotherapy targeting the microenvironment of bladder cancer has become a research hotspot.PD-1/PD-L1 inhibitor has been approved by the FDA as a second-line treatment for patients with progression and metastasis after receiving platinum chemotherapy,but the effective rate is only 15.0-36.1%.Therefore,it is of great clinical significance to further study the mechanism affecting immune escape of bladder cancer and to find new targets for immunotherapy of bladder cancer.Programmed cell death-1(PD-1)can be expressed in T cells,NK cells,B cells.monocytes and macrophages.PD-1 and its ligands.PD-L1 and PD-L2,can regulate the activation and suppression of T cells as well as immune-mediated tissue damage.Although the role of PD-1 in adaptive immunity has been well documented,its role in the innate immune system has only recently been studied.lt was found that PD-1 could inhibit the Ml polarization of tumor-related macrophages(TAM)by reducing the phosphorylation of STAT1,and promote the M2 polarization by increasing the phosphorylation of STAT6.In addition,nearly all PD-1+TAM showed the M2 phenotype,while PD-1-TAM showed M1 phenotype.The infiltration of PD-1+M2 macrophages increased with the disease stage,suggesting that PD-1+M2 macrophages may accumulate in the tumor microenvironment over time.Therefore,M2 macrophages can mediate the immune escape of tumor cells through PD-1.It was found that the expression of PD-1 in TAM was negatively correlated with the phagocytosis of macrophages to tumor cells.Blocking the PD-1/PD-L1 pathway could promote the phagocytosis of macrophages and inhibit the proliferation of tumor cells.It was suggested that PD-1/PD-L1 inhibitors could also inhibit tumor progression by improving macrophage phagocytosis.Besides weakened phagocytosis,M2 macrophages also secrete immunosuppressive factors such as TGF-p,IL-4,IL-10,Arg-1 and NO to suppress the immune response of T cells to tumor antigens.IL-4 and IL-10 can directly act on tumor cells and produce different effects.IL-4 is a pleiotropic cytokine that promotes T cells,B cells and non-hematopoietic cells to proliferate,and differentiate.IL-4 binding to IL-4R can activate two different signaling pathways,the STAT6 pathway and the insulin receptor substrate pathway,respectively.In the STAT6 pathway,STAT6 collects phosphorvlated tyrosine residues and is phosphorylated to pSTAT6,which is translocated into the nucleus and binds to specific DNA elements of the IL-4 response gene?mediating the relevant biological effects.In the insulin receptor substrate pathway,phosphorylated IL-4Ra recruits the insulin receptor substrates-1 and insulin receptor substrates-2 proteins.activates the PI3K-Akt pathway,and leads to cell proliferation and anti-apoptosis effects.The biological effects of IL-4 vary according to tumor cells.In most tumor cells,IL-4 promotes proliferation,metastasis,and expression of anti apoptosis genes.However,in breast cancer,gastric cancer,colon cancer,hepatocellular carcinoma and renal clear cell cancer cells,IL-4 induces apoptosis and inhibits the proliferation and invasion of tumor cells.At present,the biological effect of IL-4 in bladder cancer is unknown.It has been found that PD-1 expression in TAM in the colorectal cancer tissues can cause TAM to polarize toward M2 phenotype,and inhibit macrophage phagocytosis and tumor immunity.Tumor-derived exosomes can induce PD-1 expression in TAM and promote tumor progression in patients with gastric cancer.Anti-PD-1 therapy can redirect macrophages from an M2 to an M1 phenotype inducing regression of osteosarcoma lung metastases.Previous studies have shown that inhibition of PD-1 expression in TAM can inhibits the proliferation of tumor cells,but there is no specific research on whether tumor progression is inhibited by inhibiting the IL-4/STAT6 pathway of tumor cells.This study is to investigate the mechanism that high expression of PD-1 in M2 macrophages promoting the release of IL-4 activates the IL-4/STAT6 pathway in bladder cancer cells and promotes the progression of bladder cancer,and the potential therapeutic methods.Purpose:To investigate the mechanism of high expression of PD-1 in TAMs in bladder cancer cells.To establish a subcutaneous tumor model in nude mice and study the mechanism of nivolumab inhibiting the tumor growth.To investigate M2 macrophages infiltration and PD-1 expression in M2 macrophages in bladder tumor tissues and evaluate their prognostic values in patients with bladder cancer.Method:1.Study on the relationship between the polarization status of tumor-related macrophages and PD-1 expressionTHP-1 cells were induced into M0,M1,M2 macrophages by different cytokines,and cell morphology was recorded in different polarization states,Western blotting,PCR and ELISA were used to detect the biomarkers of M0,M1 and M2 macrophages.Changes in biomarkers of M2 macrophages after siRNA-PD-1 interference and nivolumab addition were detected,as well as effects on their secretion of IL-4.2.The mechanism of Nivolumab inhibiting proliferation and invasion of bladder cancer cells by inhibiting TAM release of IL-4Bladder cancer cells T24 and 5637 were divided into 6 treatment groups:T24/5637 group,T24/5637 co-culture group with M2 macrophages,T24/5637 co-culture group with(M2 macrophages+nivolumab),T24/563 7 co-culture group with(M2 macrophages+anti-IL-4 neutralizing antibody),T24/5637+anti-IL-4 neutralizing antibody.The invasion and proliferation of bladder cancer cells under different treatment conditions were detected by CCK-8,transwell experiment,Ki67 immunofluorescence and Western blotting3.Study on subcutaneous tumor model of nude mice in which Nivoluma inhibited TAM activation of IL-4/STAT6 pathway and inhibited the proliferation and invasion of bladder cancerTo establish subcutaneous transplantation tumor model of bladder cancer cell 5637 in nude mice.Sixteen nude mice were randomly divided into experimental group and control group.The experimental group was given nivolumab 10mg/kg/time intraperitoneal injection,once every 5 days,5 times in total.After the fifth injection for 5 days,the nude mice were killed and the tumors were removed.The body weight and tumor volume of the nude mice were measured before each administration and before the execution.The growth curve was plotted.Immunohistochemistry and immunofluorescence double staining were used to evaluate the infiltration density of TAM and the PD-1 and IL-4 positive rates of TAM4.The effect of M2 macrophage infiltration density and PD-1 positive rate of M2 macrophages on the prognosis of patients with bladder cancerThe infiltration density of M2 macrophages and PD-1 positive rates of M2 macrophages in bladder cancer tissues and adjacent tissues were detected by immunofluorescence and immunohistochemistry in 71 cases undergoing TURBt or radical resection of bladder cancer.The relationship between M2 macrophages and clinicopathological characteristics was evaluated,as well as their influence on the overall survival and prognosis5.Statistical analysisAll data reading and other detection steps were repeated three times,and R(3.6.1 version)was used for statistical analysis.Normally distributed measurement values are expressed as X±S.T test was used to compare the differences between two groups of data.Univariate or multivariate analysis of variance was used to compare the differences among groups of data.In the clinical study,chi-square test and Fisher's accurate test were used to determine the correlation betweenM2 macrophage infiltration density and PD-1 positive rate of M2 macrophages and clinicopathological parameters.Survival rate was calculated by Kaplan-Meier curve and Log-rank test.The significance of univariate and multivariate Cox proportional risk models for assessing prognostic factors.P<0.05 was considered statistically significant.Results:1.PD-1 is highly expressed in M2 macrophages and can promote the polarization of M2 macrophages.(1)THP-1 cells can be induced into MO,M1 and M2 type macrophages and undergo corresponding morphological changes.(2)Western blotting,PCR and ELISA confirmed the high expression of CD80 and iNOS in M1 macrophages and CD 163,Arg-1,PD-1 and IL-4 in M2 macrophages.(3)After siRNA-PD-1 or nivolumab,M2 macrophages showed M1 macrophages phenotype and reduced IL-4 secretion.2.After co-culture of T24 and 5637 cells with M2 macrophages,their proliferation and invasion abilities were enhanced;Nivolumab or anti-IL-4 neutralizing antibodies can inhibit the proliferation and invasion of T24 and 5637 cells in the co-culture system.(1)CCK8-8 experiment and Ki67 immunofluorescence detected the proliferation ability of bladder cancer cells in different treatment groups.M2 macrophages could significantly promote their proliferation.After adding nivolumab or anti-IL-4 neutralization antibodies into the co-culture system,their proliferation ability was significantly reduced.(2)Transwell assay detected the migration and invasion abilities of bladder cancer cells in different treatment groups.M2 macrophages could significantly promote migration and invasion.After nivolumab or anti-IL-4 neutralization antibodies were added to the co-culture system,their migration and invasion abilities were significantly reduced.(3)Western blotting detected the expressions of pSTAT6,Bcl-2 and Bax in bladder cancer cells in different treatment groups.The expressions of pSTAT6 and Bcl-2 were significantly increased,while the expressions of Bax were significantly decreased in the co-cultured systerm.When nivolumab or anti-IL-4 neutralizing antibodies were added to the co-culture system,Bax expression was significantly increased,pSTAT6 and Bel-2 expressions were significantly decreased.3.Nivoluma inhibited TAM activation and bladder cancer proliferation through IL-4/STAT6 pathway.(1)Tumor volume and tumor weight in the experimental group were significantly lower than those in the control group.(2)In the control group,the infiltration density of M2 macrophages was significantly higher than that of Ml macrophages,the PD-1 positive rate of M2 macrophages was significantly higher than that of Ml macrophages.(3)The infiltration density of M2 macrophages and positive rate of M2 macrophages were significantly higher in the control group than in the experimental group.(4)The intensity of pSTAT6 and Bcl-2 in the control group was higher than that in the experimental group,while the intensity of Bax was lower than that in the experimental group.4.Influence of the infiltration density and PD-1 positive rate of M2 macrophages on prognosis of bladder cancer(1)The infiltration density of M2 macrophages in MIBC was significantly higher than that in NMIBC,and PD-1 positive rate of M2 macrophages was also significantly higher than that in NMIBC.(2)M2 macrophage infiltration density and PD-1 positive rate of macrophage were significantly correlated with T stage and pathological grade of bladder cancer.(3)M2 macrophage infiltration density and PD-1 positive rate of macrophage are independent prognostic factors for bladder cancer.Conclusion:1.PD-1 was highly expressed in M2 macrophages and IL-4 was highly produced.PD-1 inhibitor nivolumab could polarize M2 phenotype to Ml phenotype and reduce IL-4 release.2.M2 macrophages released IL-4 and promoted proliferation,migration and invasion of bladder cancer cells through IL-4/pSTAT6 pathway.PD-1 inhibitor nivolumab or anti-IL-4 neutralizing antibodies could inhibit the effect of M2 macrophages on proliferation,migration and invasion of bladder cancer cells.3.In the subcutaneous tumor model in nude mice,nivolumab could polarized macrophages to Ml phenotype,reduce IL-4 secretion,and inhibit the growth of subcutaneous tumors by inhibiting IL-4/pSTAT6 pathway.4.M2 macrophage infiltration density and PD-1 positive rate of macrophage were significantly correlated with T stage and pathological grade of bladder cancer,and were independent prognostic factors for bladder cancer.