Experimental Study On The Effect Of Cryoablation Combined With TGF-? Blockade On Treg Cells In Mice Of Prostate Cancer

Objective The incidence of prostate cancer is increasing at home and abroad.The patients with prostate cancer are mostly in the middle or late stage and lose the opportunities for surgical treatment.Cryoablation is one of the most important treatments for these patients.The cancer cells release antigens to stimulate the immune response during cryoablation for prostate cancer.At the same time,these cancer cells also release TGF-?1.TGF-? signals not only promote the migration of cancer cells,but also recruit a variety of inhibitory immune cells including Treg,leading to the formation of immune tolerance and weakening the anti-tumor immune response.As cryoablation for prostate cancer,if we use TGF-? neutralizing antibody to block the signal pathway at the same time,how will the TGF-?1 change? how will the Treg cells change? is there correlation between TGF-?1 and Treg cells? To solve these problems,this study built model of prostate cancer in mice,then to detect TGF-?1 level and Treg cells after cryoablation combined with TGF-? block,to analyze the correlation between TGF-?1 and Treg cells,to discuss the impact of TGF-?1 on Treg cells and the related mechanism.Materials and Methods1.To detect the migration and invasion ablitity of RM-1 under TGF-? neutralizing antibody by respectively the scratch test and the Transwell test.2.To build model of prostate cancer in mice.All the mice are randomly divided into four groups,including Group A(Control Group),Group B(Cryoablation Group),Group C(Antibody Group)and Group D(Combination Group).3.To collect the specimen before the treatment,7 days and 14 days after the treatment respectively.The secretions of TGF-?1 and IL-2 in peripheral blood are detected by ELISA.The proportions changes of Treg,CD4+T and CD8+T cells are detected by flow cytometry.The killing activity of CTL in spleen are detected by the LDH method.The numbers of Treg,CD4+T and CD8+T cells and the lung metastasis are detected by HE staining.The size of the tumors of all mice are measured.4.To analyze the correlation between TGF-?1 and Treg cells,and to discuss the impact of TGF-?1 on Treg and the related mechanism.Results1.The scratch test and the Transwell test show that TGF-? neutralizing antibody reduces the migration and invasion ability of RM-1,indicating TGF-?1 takes a part in the migration and invasion of RM-1.2.The TGF-?1 level in peripheral blood of Group A shows a trend of increase as tumor growing.Compare with that before treatment,the levels at 7 days and 14 days after treatment increase significantly with statistical difference(P<0.05).Compared with Group A,the levels of Group B,Group C and Group D at 7 days and 14 days after treatment decrease significantly with statistical difference(P<0.05).The IL-2 level in peripheral blood of Group A shows a trend of decrease as tumor growing.Compare with that before treatment,the levels at 14 days after treatment increase significantly with statistical difference(P<0.05).Compared with Group A,Group B and Group C,the levels of and Group D at 7 days and 14 days after treatment decrease significantly with statistical difference(P<0.05).3.The Treg cells in peripheral blood of Group A shows a trend of increase as tumor growing.Compare with that before treatment,the Treg cells at 14 days after treatment increase significantly with statistical difference(P<0.05).Compared with Group A,the Treg cells of Group B and Group D at 14 days after treatment decrease significantly with statistical difference(P<0.05).There is a positive correlation between Treg cells in peripheral and TGF-?1 level(P < 0.001,R=0.931).The Treg cells in spleen of Group A shows a trend of increase as tumor growing.Compare with that before treatment,the Treg cells at 14 days after treatment increase significantly with statistical difference(P < 0.05).Compared with Group A,the Treg cells of Group B and Group D at 14 days after treatment decrease significantly with statistical difference(P<0.05).There is a positive correlation between Treg cells in spleen and TGF-?1 level(P<0.001,R=0.889).The Treg cells in tumor tissue of Group A shows a trend of increase as tumor growing.Compare with that before treatment,the Treg cells at 14 days after treatment increase significantly with statistical difference(P < 0.05).Compared with Group A,the Treg cells of Group B and Group D at 14 days after treatment decrease significantly with statistical difference(P<0.05).There is a positive correlation between Treg cells in tumor tissue and TGF-?1 level(P<0.001,R=0.912).4.The CD4+T and CD8+T cells in spleen of Group A shows a trend of decrease as tumor growing.Compare with that before treatment,the CD4+T and CD8+T cells at 14 days after treatment decrease significantly with statistical difference(P<0.05).Compared with Group A,Group B and Group C,the CD4+T and CD8+T cells of Group D at 7 days and 14 days after treatment increase significantly with statistical difference(P<0.05).There is a negative correlation between CD4+T and CD8+T cells in spleen and TGF-?1 level(P<0.001,R=-0.813;P<0.001,R=-0.839).The CD4+T and CD8+T cells in tumor tissue of Group A shows a trend of decrease as tumor growing.Compare with that before treatment,the CD4+T and CD8+T cells at 14 days after treatment decrease significantly with statistical difference(P<0.05).Compared with Group A,Group B and Group C,the CD4+T and CD8+T cells of Group D at 14 days after treatment increase significantly with statistical difference(P<0.05).There is a negative correlation between CD4+T and CD8+T cells in spleen and TGF-?1 level(P<0.001,R=-0.852;P<0.001,R=-0.914).5.The killing ability of CTL cell in spleen of Group A shows a trend of decrease as tumor growing.Compare with that before treatment,the killing ability at 14 days after treatment decrease significantly with statistical difference(P<0.001).Compared with Group A,Group B and Group C,the killing ability of Group D at7 days and 14 days after treatment increase significantly with statistical difference(P<0.001).There is a negative correlation between CD4+T and CD8+T cells in spleen and TGF-?1 level(P<0.001,R=-0.866;P<0.001,R=-0.875)?6.The pulmonary metastasis rates in mice show a trend of increase with disease progression.Compared with Group A,Group B and Group C,the rates of Group D at 14 days after treatment decrease significantly with statistical difference(P< 0.05).The tumor diameter in mice show a trend of increase with disease progression.Compared with Group A,the rates of Group B,Group C and Group D at 7 days and 14 days after treatment decrease significantly with statistical difference(P<0.05).Conclusion TGF-?1 takes part in the process of migration and invasion of RM-1 cells.TGF-? neutralizing antibody reduces the migration and invasion ability of RM-1 cells.As tumor growing,the TGF-?1 levels in peripheral blood rise,IL-2 levels falling,Treg cells increasing,CD4+T and CD8+T cells decreasing,and the killing ability of CTL cells in spleen falling.There is a positive correlation between Treg cells and TGF-?1 levels,a negative correlation between CD4+T and CD8+T cells and TGF-?1levels,and a negative correlation between the killing ability of CTL cells in spleen and TGF-?1 levels.After the combined treatment of cryoablation and TGF-?blockade,the TGF-?1 levels in peripheral blood fall,IL-2 levels rising,Treg cells decreasing,CD4+T and CD8+T cells increasing,the killing ability of CTL cells in spleen rising,the pulmonary metastasis rates decreasing,and the tumor diameter reducing.