Tumor-Associated Macrophages Upregulate The Expression Of PSMA In Bladder Cancer Via IL-4/STAT6 Pathway

BackgroundBladder transitional cell carcinoma is the sixth most commonly diagnosed cancer in China.Evidence from recent studies suggests that smoking,industrial pollution,chemical substances and other risk factors are associated with an increased risk of bladder cancer.Although non-muscle-invasive bladder cancer(NMIBC)accounts for the major patients with bladder cancer,there is a high progression rate from NMIBC to muscle-invasive bladder cancer(MIBC)approaching 30%,with a high subsequent metastatic rate despite treatment.Therefore,further study is needed to provide the opportunity to identify potential therapeutic targets for bladder cancer.Macrophages(Mφ)are important parts of immune system,with critical roles in innate and adaptive immunity.Macrophages have the functions not only in phagocy-tosis of debris and pathogens,but also in recognization of tumor cells.Tu-mor-associated macrophages(TAMs),one of the major components of tumor micro-environment(TME),play significant roles in proliferation and invasion of most solid tumors.TAMs adopt 2 phenotypes:the classically activated M1 macrophages induced by LPS and IFN-y and the alternative activated M2 macrophages induced by IL-4 and IL-13.Both Ml and M2 macrophages are highly plastic macrophages in regulation of functions which play two opposite functions of anti-tumor and pro-tumor.However,there are no explicit evidences between the expression of prostate-specific membrane antigen(PSMA)and TAMs.Prostate-specific membrane antigen(PSMA),a type Ⅱ membrane glycoprotein,describes the differential regulation of the molecule,its enzymatic functions,and its potential as a biomarker for in vivo imaging,targeted therapy and immunotherapy.PSMA is strongly expressed in the prostate and is upregulated in prostate cancer and the neovasculature of other tumors,such as thyroid cancer,breast cancer,colorectal cancer and renal cancer.But there are conflicting evidences regarding PSMA in the neovasculature of bladder cancer:Samplaski and colleagues reported the expression of PSMA in the tissues of MIBC is associated with histopathologic grading and tumor stages.There are also evidences that increased tissue expression of PSMA may confer a worse prognosis in patients with high infiltration of neovasculature.However,Campbell and colleagues analyzed the imaging and pathological data on 3 cases with metastatic bladder cancer.Low levels of target PSMA expression within the tumor neovasculature limited clinical value of PSMA for imaging patients with metastatic bladder cancer.But the major limitation is the small sample size of only three patients,which may not be representative of the larger population of individuals with bladder cancer.The objective of this study is to investigate the rate of PSMA expression on tumor neovasculature induced by tumor-associated macrophages(TAMs)or epithelial mesenchymal transition(EMT)in muscle-invasive bladder cancer(MIBC),which demonstrates PSMA has potential significance for diagnostic and prognostic evalua-tion for bladder cancer.PurposeThis project will establish the co-culture model of bladder transitional cell carci-noma T24 together with THP-1 cell line and the models of tumor bearing nude mice with bladder cancer cell lines in combination with the study of the clinical specimens of bladder cancer.The objective of this project is to evaluate this hypothesis:Bladder cancer cells release IL-4 into tumor microenvironment,which activates TAMs polar-ized to M2 macrophages via the phosphorylation of STAT6.M2 macrophages secrete Sema4D to induce tumor neovasculature in tumor microenvironment,which upregu-lates the expression of prostate-specific membrane antigen(PSMA).Method1.Bladder cancer cells release IL-4 to promote TAMs polarization to M2 mac-rophages via the phosphorylation of STAT6.THP-1 cells were cultured in RPMI-1640 and were induced by 5μl PMA(1:30000)for about 6 hours to CD68+/CD11b+M0 macrophages.The M0 macrophages were added up with 5μl LPS(1:1000)and 5μl IFN-y(1:1000)for 18 hours,induced to-wards iNOS+/CD80+M1 macrophages.In reverse,The M0 macrophages were added up with 5μl IL-4(1:500)and 5ul IL-13(1:500)for 18 hours to be polarized towards Arg-1+/CD 163+M2 macrophages.All these different phenotypes cells were cultured in RPMI-1640.2.PSMA inhibitor 2-PMPA prohibits CD31 and Sema4D in the models of tumor bearing nude mice with bladder cancer.All 10 Nude Mice were obtained from Laboratory Animals Center of Shandong University and brought up in specific pathogen free(SPF)experiments of Laboratory Animals Center of Shandong Provincial Hospital affiliated to Shandong University.All experiments were operated in 14-day-old male mice in SPF facilities.Models were constructed by injecting subcutaneous T24 cells into right upper limb.The mice of experiment group were injected with 2-PMPA,and control group were injected with the same volume of heat-inactivated NS.Weight and tumor volume of mice were collected every 5 days before injection for 5 times.Tumors were harvested from the mice at 5 days after the last 2-PMPA or NS injection.No mice or tumors were elimi-nated from this analysis.3.Clinical specimens of PSMA and CD31 in TNM stages and prognosis of blad-der cancer.Bladder cancers and adjacent normal tissues were obtained from Shandong Pro-vincial Hospital Affiliated to Shandong University between 2013 and 2018 in 50 pa-tients with pathologically proven muscle invasive bladder transitional cell carcinoma.Every tissues obtained from these 37 patients were made into tissue microarray and 10 sections from each sample were chosen randomly.All pathology samples were examined independently by two pathologists from Shandong Provincial Hospital Af-filiated to Shandong University.Result1.Bladder cancer cells release IL-4 into tumor microenvironment to promote TAMs polarized towards M2 macrophages via the phosphorylation of STAT6.Under the high power field,THP-1 cells showed suspension cells with small and equal-sized round,regularity and no pseudopod.M0 macrophages became huge,ir-regular and polygonal adherent cells.M1 macrophages were huger spindle in shape.M2 macrophages became branches-shaped edges of cells.Western blot and RT-PCR revealed that the expressions of iNOS and CD80 were higher in M1 macrophages and the expressions of Arg-1 and CD 163 were higher in M2 macrophages and T24+M0 co-culture group.The expressions of CD 163 were lower after adding IL-4R or STAT6 inhibitor AS1517499.ELISA showed that IL-4 from M2 macrophages and T24+M0 co-culture group culture supernatant were higher than M0 and M1 macrophages after exchanging of non-revulsant supernatant for 48 hours.2.M2 macrophages secrete and express Sema4D in tumor microenvironment.RT-PCR and ELISA showed that Sema4D from M2 macrophages and T24+M0 co-culture group culture supernatant were higher than M0 and M1 macrophages.While,the expressions of Sema4D were lower after adding IL-4R or STAT6 inhibitor AS1517499.Immunohistochemical analysis and immunofluorescence data showed the expression and positive rates of Sema4D on M2 macrophages were higher in tu-mor tissues than in para-tumor tissue samples.3.2-PMPA decreases CD31 through inhibiting M2 macrophages infiltration and Sema4D expression.The tumor volumes became smaller in 2-PMPA groups after injection.The strip-ping tumor weights in 2-PMPA groups were less than control groups.Immunohisto-chemical analysis showed the expression of CD31,Plexin-B1,CD163,Sema4D were lower in 2-PMPA group than in control group.Immunofluorescence data showed the positive rates of PSMA on CD31+cells and Sema4D on M2 macrophages were lower in 2-PMPA groups than in control groups.4.PSMA expression rates on CD31+ cells are correlated with pathological T stages and WHO grades in bladder cancer.Immunohistochemical data revealed that the mean counts per cubic millimeter of CD31+cells in tumor tissues were higher than in normal adjacent tissues.And for WHO pathological grades,the results showed that the counts in high grade were higher than low grade.As for pathologic T stages,the mean counts per cubic millime-ter of CD31+cells were the highest in T4 stage,next to T3 stage and the lowest in T2 stage.Immunofluorescence data revealed that the mean expression rates of PSMA were higher in tumor tissues than those in normal adjacent tissues.While,the mean expression rates of PSMA were the highest in patients of T4 stage,following by T3 stage and the lowest in T2 stage.And the similar outcomes reflected in the features of WHO pathological grades that the mean expression rates of PSMA were higher than low grade.5.PSMA expression on CD31+cells might be induced by TAMs in T2 and T3 stages,and induced by EMT in T4 stage of bladder cancer.Immunohistochemical data revealed TAMs infiltration was the highest in T3 stage and lower in T4 and T2 stage.Immunofluorescence data showed the ratio of N-cadherin/ZO-1 of EMT on CD31+cells were the higher in T4 and T3 stages and the lowest in T2 stage.Therefore,PSMA expression on tumor neovasculature might be induced by TAMs in T2 and T3 stages,and induced by EMT in T4 stage of bladder cancer.Conclusion1.Bladder cancer cells release IL-4 into tumor microenvironment to promote TAMs polarized towards M2 macrophages to secrete Sema4D via the phosphorylation of STAT6.2.PSMA expression rates on CD31+cells are correlated with CD31 expression to prodict the prognosis of bladder cancer.3.PSMA expression on CD31+cells might be induced by TAMs in T2 and T3 stages,and induced by EMT in T4 stage of bladder cancer.