The Study On The Regulatory Effect Of Polyporus Polysaccharide On Tumor Microenvironment Of Bladder Cancer Based On The Analysis Of Macrophage Subtype

ObjectiveBladder cancer is the leading cause of cancer-related deaths in urinary system.The role of immune cells in tumorigenesis and development is further recognized in solid tumors,including tumor associated macrophages.Macrophages can promote the deterioration of tumors,increase the invasiveness of tumors,and evade host immune response and anti-treatment response.However,under certain conditions,they can be involved in host immune surveillance and killing in tumor.Anti-inflammatory type-2(M2 macrophage)can promote the tumorigenesis,development,metastasis,and drug resistance of solid tumors,including bladder cancer,while pro-inflammatory type-1(M1 macrophage)in turn inhibits these effects.It has been pointed out that TAMs(M2 macrophage)are closely related to the poor prognosis of bladder cancer.In this study,we studied the relationship between TAMs and tumor malignancy.Increasing the proportion of M1-subtype cells in TME is a promising strategy for cancer treatment.Previous studies found that homogenous polyporus polysaccharide(extracted from Polyporus polysaccharide with better purity,named HPP)could alleviate the adverse reactions of Bacille Calmette-Guerin(BCG)and improve its efficiency,and effectively inhibit the progression of N-butyl-N-(4-hydroxybutyl)-nitrosamine(BBN)-induced bladder cancer in rats.In additional,HPP regulated the immune activity of macrophages and promoted them to M1-like macrophages in vitro.In this study,we explored the direct effect of PPS activated macrophages in bladder cancer cells and the potential molecular mechanism.The regulatory effect of HPP on macrophages in tumor microenvironment of bladder cancer rats was also studied.By analyzing the subtypes of macrophages,we further discussed the mechanism of HPP on bladder cancer.MethodsSection 1.The basic clinical data and pathological sections of bladder were collected from 29 patients with urothelial carcinoma.The expression of CD163,CD206 on the membrane surface of M2 macrophage and cell proliferation index Ki-67 was detected by immunohistochemistry,then the relationship between the infiltration degree of M2 macrophages and the malignant degree of tumor cells was analyzed.Section 2.The paraffins of BBN bladder cancer model rats treated with HPP were collected,and we take the blank group,model group,HPP group and BCG group.Immunohistochemistry was carried out after the section to detect the expression of CD 163,CD206,CD 16,CD86 and Ki-67 in the tumor microenvironment of bladder cancer rats.Then we discuss the change of macrophages subtype in the tumor microenvironment of bladder cancer rats with HPP intervention.Section 3:Human monocyte line THP-1 cells were induced into macrophages by PMA with different concentrations(0,10,20,50,100,200 ng/mL).The optimal concentration was determined by the changes of cell morphology,cell adhesion and the expression of CD 14 and CD68 on the surface of cell membrane.Different concentrations of HPP(1,10 and 100?g/mL)were used to interfere with THP-1-derived macrophages.RT-PCR was used to study the effect of HPP on the expression of IL-1 ? mRNA,IL-6 mRNA,TNF-? mRNA and iNOS mRNA in THP-1-derived macrophages.Flow cytometry was used to detect the expression of CD86,CD 16,CD40 and CD23 on the surface of cell membrane.ELISA was used to detect the expression of IL1? and TNF-?.After HPP intervened with macrophages for 72 H,the fresh medium was replaced and cultured for 24 H.The supernatant of activated macrophages was collected and then human bladder cancer cells were treated with the conditional medium according to the ratio of macrophage supernatant:fresh medium 1:1.The cell viability of human bladder cancer cell lines T24 and EJ was detected by MTT method.The proliferation of bladder cancer cells was detected by EDU proliferation test.The cell cycle and apoptosis of T24 and EJ cells were detected by flow cytometry.Transewll was used to detect the change of cell migration ability.Western blot was used to detect the expression of apoptotic associated protein,EMT related protein and JAK2-STAT5/NF-?B pathway protein.AZD1480,an inhibitor of JAK2,was treated with T24 and EJ to detect the expression of p-JAK2 and the changes of cell viability and apoptosis.Section 4:Macrophages derived from THP-1 were induced with different stimulants,which were grouped as follows:HPP 10 ?g/mL,the supernatant of T24 tumor cells:fresh medium 1:1.After 72 H of incubation,the cells were collected to detect the expression of CD209,CD301,CD172?,CD36,CD163,CD16,CD23 and CD206 on the surface of cell membrane.The fresh medium was replaced to continue to culture for 24 H,and cell supernatant was collected.The contents of TNF-?,IL-6,CXCL10,IL-10,CCL2,IL-1?,CLL18,CCL24,CCL22,CXCL9,CCL17,IL-23p40,IL-18,IL-12p70 and IL-23 were detected by liquid suspension chip technology,and the expression of TGF-? was detected by ELISA.According to the expression of the above cytokines and cell membrane molecules,cluster analysis and discriminant analysis were used to explore the classification of HPP activated macrophages and tumor related macrophages.According to the similarity of the data,the cell subtypes closest to the two were judged.ResultsSection 1.There was no correlation between the basic data(age,gender)and pathological grade of bladder cancer(P>0.05).The expression of CD 163,CD206 and Ki-67 in invasive bladder cancer was higher than that in non-invasive bladder cancer(P<0.05).The expression of CD 163,CD206 and Ki-67 in bladder tissue of high-grade bladder cancer was higher than that of low-grade bladder cancer(P<0.05).The infiltration degree of CD 163 and CD206 in bladder cancer was positively correlated with the expression of Ki-67(rs>0).Compared with CD206,CD 163 and Ki-67 showed stronger correlation(P<0.001).Section 2.Compared with the blank group,the invasion degree of BBN bladder cancer in the model group was significantly higher than that of CD 163 and CD206(P<0.05).Compared with the model group,the expression of CD 163 and cd206 in BCG group and HPP group decreased,but showed statistical significance in CD 163(P<0.05).2.Compared with the blank group,CD16 and CD86 in the tumor tissue of BBN bladder cancer rats in the model group showed a significantly increased trend.Compared with the model group,the expression of CD86 and CD 16 in BCG group and HPP group showed an increasing trend,but there was no significant statistical significance(P>0.05).3.The expression of Ki-67 in tumor tissue of model group was significantly higher than that of normal rats(P<0.05).After BCG and HPP intervention,Ki-67 in tumor tissue decreased significantly(P<0.05).Section 3.10 ng/mL PMA could induce THP-1 cell morphological changes,increase adhesion ability and the expression of CD 14 and CD68 on cell surface(P<0.05).50ng/mL PMA was the best concentration for THP-1 to induce macrophage.HPP increase the expression of IL-1 ?.IL-6,INOS,TNF-? mRNA in THP-1-derived macrophages.HPP upregulated the expression of CD86,CD23,CD40,CD16 on the surface of THP-1-derived macrophage cells.HPP induce the increase of IL-? and TNF-? in THP-1-derived macrophages(P<0.05).HPP activated macrophages could inhibit the cell viability of human bladder cancer cell line T24 and EJ with time-dependent(P<0.05).HPP activated macrophages could inhibit the proliferation and the clone formation of human bladder cancer cell line T24 and EJ(P<0.05).Cell cycle arrest in bladder cancer cells in the G0/G1 phase when treated with the PPS-polarized macrophages(P<0.05).HPP activated macrophages can promote the apoptosis of human bladder cancer cell line T24 and EJ cells.mainly in the late stage,accompanied by cell volume shrinkage.The apoptotic protein Bax in T24 and EJ Cells has no obvious change,and the level of apoptosis inhibitory protein Bcl-2 decreases(P<0.05).HPP activated macrophages can inhibit the migration of human bladder cancer cell line T24 and EJ Cells.HPP activated macrophages inhibited the expression of N-cadherin and vimentin,and up reg ulated the expression of E-cadherin.HPP activated macrophage downregulated the expression of JAK2-STAT5/NF-?B in EJ and T24 cell lines(P<0.05).AZD1480,a JAK2 inhibitor,inhibit the expression of JAK2 phosphorylated protein p-JAK2(P<0.05).It also inhibited the cell viability and promoted apoptosis of human bladder cancer cell line T24 and EJ(P<0.05).Section 4.HPP upregulated the expression of TNF-?,IL-1?,IL-23 P40,IL-10,CD 16,CD86,CD23,CD40 and CD36,while downregulated the expression of CCL2,CCL24,CD209,CD301 and SIRP ?,but had no significant effect on TGF-?,IL-6,CCL22,CCL17,CCL18,CXCL9,CD 163 and CD206;the supernatant of tumor cells upregulated the expression of cytokines TGF-?,IL-6,IL-10,CCL2,IL-1?,CCL18,CCL22,CCL24 and SIRP?,CD36,CD 16,CD86,CD209,CD 163,CD23 on the surface of cell membrane,and down regulate the expression of TNF-?,which has no significant effect on the expression of IL-23,CXCL9,CCL17,CD40,CD206,CD301.Conclusion1.In the tumor microenvironment of bladder cancer patients,M2 macrophages increased abnormally,and the degree of M2 macrophage infiltration was related to the degree of myometrial invasion,pathological grade and malignant degree of bladder cancer.2.HPP downregulated the expression of M2 macrophages and upregulate M1 macrophages in tumor environment of bladder cancer rats,and inhibit the proliferation of tumor cells.3.HPP activated THP-1-derived macrophages inhibited the cell viability and proliferation,block cell cycle,promote apoptosis,inhibit cell migration and epithelial stromal transformation of human bladder cancer cell lines T24 and EJ.HPP activated macrophages play an anti-tumor role through JAK2-STAT5/NF-?B pathway.4.The macrophages activated by Polyporus umbellatus can kill tumor cells by secreting inflammatory factors,and play an anti-bladder cancer role by alleviating the immunosuppression in tumor microenvironment;tumor cells can induce TAMs to secrete inflammatory factors and immunosuppressive molecules to induce the deactivation of the immune system,so as to promote the progress of tumor.