| Objective:As an important treatment for prostate cancer,cryoablation can induce anti-tumor immune response while killing tumor cells.A large amount of evidence shows that inhibitive factors(immune cells and immune factors)still exist in the body after cryoablation to restrict the anti-tumor immune response,and it is necessary to further explore the mechanism of inhibitive immune cells(regulatory T cells,myeloid derived suppressor cells)and immune factors(TGF-?,IL-10)in this process.Our previous work found that after cryoablation,the levels of transforming growth factor beta(TGF-?)in peripheral blood and tumor tissues of prostate cancer patients were significantly increased.Literature studies have shown that TGF-?can promote the immune escape of tumor cells by recruiting MDSC(myeloid derived suppressor cells),Treg(regulatory T cells)and other immunosuppressive cells,and MDSC and Treg can secrete negative regulatory factors such as TGF-?and IL-10 to further enhance their immunosuppressive function.Previous studies of the research group have preliminarily confirmed that cryoablation combined with TGF-?block in the treatment of prostate cancer can reduce the number of MDSC cells in the body,and the specific mechanism remains to be further explored.Therefore,this study focused on the analysis of the effect of cryoablation combined with TGF-?blockade on MDSC cell function,and discussed its related mechanism,in order to provide experimental basis and theoretical basis for the removal of inhibiting factors in the process of cryoimmunity and the improvement of comprehensive efficacy mainly based on cryoablation..Methods:1.MDSC and Treg cell ratios in peripheral blood were detected by flow cytometry.The expression levels of TGF-?in peripheral blood were detected by enzyme-linked immuno sorbent assay(ELISA).Meanwhile,the expression of TGF-?,CD11b(CD11b-positive mononuclear cells representing MDSC)and FOXP3(FOXP3-positive T cells representing Treg)in prostate cancer tissues was detected by tissue microarray technology,and the correlation between the expression levels of the three in prostate cancer tissue was analyzed.2.MDSC cells were selected from the spleen of mice bearing prostate cancer using magnetic bead sorting method.MDSC cells were co-cultured with prostate cancer cells(RM-1)in vitro and was performed with different treatments,no treatment was given to group A(control group),TGF-?monoclonal antibody was added to group B(monoclonal antibody group),RM-1 frozen necrosis solution was added to group C(frozen group),and both RM-1 frozen necrosis solution and TGF-?monoclonal antibody were added to group D(combined group).After co-culture for 24h and 48h,the supernatant of cells were collected,and the expression levels of TGF-?and IL-10in the supernatant of each group were detected by ELISA.Meanwhile,MDSC cells were collected for flow cytometry to detect the differentiation trend of of M1-type(CD11b,f4/80,CD16/32,CD86)and M2-type(CD11b,F4/80,CD206)macrophages.In addition,the chemotaxis of MDSCs were simulated by transwell system.MDSC cells were placed above the chamber,and RM-1 cells were laid on the lower chamber.The proportion of MDSC in the lower chamber was detected by flow detection,and the number of MDSC in the subchamber was observed under the microscope.3.CD4~+T cells in the spleen of normal mice were separated by magnetic beads and co-cultured with MDSC cells derived from prostate cancer.The cells were treated with different treatments(same group as above),and 24h and 48h after co-culture were collected for flow staining of Th1-type(CD4,IL-4)and Th2-type(CD4,IFN-?)T cells to detect the differentiation trend of CD4~+T cells after co-culture.Western Blot was used to detect the expression level of immunosuppressive protein of Arg-1 and iNOS in MDSC,and the expression level of mRNA was verified by RT-PCR.4.The effects of the supernatant on the proliferation,migration and invasion of RM-1cells were examined by using plate cloning assay,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and Transwell assay.Results:1.The expression level of TGF-?in peripheral blood of patients with prostate cancer was positively correlated with the number of MDSC and Treg,respectively(r=0.6710,P=0.0012;r=0.6906,P=0.0007,respectively),the number of Treg was also positively correlated with the number of MDSC(r=0.9418,P<0.0001).2.The expression levels of MDSC,Treg and TGF-?in prostate cancer tissues were significantly higher than those in adjacent tissues,with statistically significant differences(P<0.0001).Correlation analysis showed that the expression level of TGF-?in prostate cancer tissues was positively correlated with the expression of MDSC and Treg(r=0.6729,P<0.0001;r=0.6729,P<0.0001,respectively),and Treg expression level was positively correlated with MDSC expression level(r=0.3140,P=0.0026).3.After 24h of co-culture of MDSC and RM-1,the expression level of TGF-?in the supernatant of the group D was significantly lower than that of the group A and the group C(P<0.001 and P=0.009,respectively),while there was no statistical difference between the group D and group B(P=0.780).After co-culture for 24h,the expression level of IL-10 in the supernatant of the group D was significantly lower than that of the group A and the group B(P=0.001 and P=0.027,respectively),while there was no statistical difference between the group D and group C(P=0.247).After co-culture for 48h,the expression levels of TGF-?and IL-10 in the supernatant of the group D was significantly lower than those of the other three groups(P<0.05).4.After co-culture MDSC and RM-1 for 24h and 48h,the differentiation trend of MDSC toward M1-type macrophages in the group D was more significant than that of the other three groups(P<0.05),and the M1/M2 ratio was significantly higher than that of the other three groups(P<0.05).Pairwise comparison of the other three groups showed no significant difference(P>0.05).Compared with the group A,the chemotaxis of MDSC cells to prostate cancer cells in the group D,group C and group B was significantly reduced(P<0.05).In addition,the chemotaxis of MDSC in the group D was lower than that in the group B and group C(P<0.05),while there was no significant difference between the group C and group B(P>0.05).5.After co-culture of MDSC and CD4~+T for 24h and 48h,the expression of protein Arg-1 and iNOS in the group D was significantly lower than that in the other three groups(P<0.05).After co-culture of MDSC and CD4~+T cells for 24h,the proportion of Th1/Th2 in the group D was significantly higher than that in the group A and the group B(P<0.001),but there was no statistical difference in comparison with group C.The proportion of Th1/Th2 in the group C was significantly higher than that in the group A(P=0.042),and there was no statistical significance compared with that in the group B.After co-culture for 48h,the proportion of Th1/Th2 in the group D was significantly higher than that in the other three groups(P<0.05).Th1/Th2 ratio in the group C was significantly higher than that in the group A and the group B(P<0.05).However,the proportion ofTh1/Th2 in the group B showed no statistical significance compared with that in the group A at 24h and 48h after co-culture(P>0.05).6.After co-culture of RM-1 cells with MDSC cells,the proliferation,migration and invasion ability of RM-1 cells in the co-culture supernatant of the group D was significantly decreased compared with the co-culture supernatant of the other three groups(P<0.05).Conclusion:1.There is a close correlation between TGF-?,MDSC and Treg in peripheral blood and tumor tissues of prostate cancer patients,TGF-?is closely related to immunosuppressive cells in tumor microenvironment and may exert immunosuppressive effect by affecting MDSC and Treg cells.2.Cryoablation combined with blocking of TGF-?can reduce the levels of immunosuppressive factors(TGF-?and IL-10),thereby promoting the dominant transformation of MDSC into M1-type macrophages and weakening the MDSC-mediated immunosuppressive effect.In addition,cryoablation combined with blocking of TGF-?can weaken the chemotaxis ability of MDSC cells,which weakening the immunosuppressive effect of MDSC in tumor microenvironment.3.Cryoablation combined with blocking of TGF-?can down-regulate the expression of Arg-1 and iNOS in MDSC,and promote the differentiation of CD4~+T cells into Th1 type dominance,which helps to enhance the anti-tumor effect of CD4~+T cells.4.After treatment with cryoablation combined with blocking of TGF-?,the ability of proliferation,migration and invasion of RM-1 cells in tumor microenvironment was significantly reduced,which was helpful to control tumor growth. |
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