The Study Of Cryoablation Combined With Anti-TGF-? Ab For Prostate Cancer And The Impacts On MDSCs

BackgroudArgon-helium cryoabltion has been widely used an alternative method in treating middle-late prostate cancer patients because of its curative effect and the good local control rate.At the same time cryoablation can induce cryoablation immune response,which can active the immune system of the human body and help eliminate the left tumor cells.However,the strength and last duration is various for different individuals.In addition,there are also some negative cytokines and immune cells that restrict the cryoablation immune response.Currently,how to eliminate the negative effects and enhance the immune response has become a hotspot in this field.TGF-? plays an important role in the invasion,metastasis and the epithelial-mesenchymal transition(EMT).Furthermore it can recruit some immunosuppressive cells such as regulator T cell(Treg),myeloid derived suppressor cells(MDSC)and tumor-associated macrophages(TAM)which leading to the immune escape.In the previous studies we found that the serum TGF-? increased following the cryoablaion,this may be connected with the tumor recurrence and the metastasis.This study tries to invest the effect of cryoablion combined with anti-TGF-? in the treatment of prostate cancer and the change of the immune microenvironment.This may provide theoretical and experimental evidence for whether cryoablation combined with immunotherapy can be a more effective treatment for prostate cancer.Materials and MethodsMaterials and Methods1.The vitro experiments: the cells were divided into four groups,Group A :Control group,Group B:Cryoablation only group,Group C:Anti-TGF-? only group,Group D:Cryoablation+Anti-TGF-? group.Transwell and MTT were performed to assess the function of the cells.2.Making RM-1 prostate cancer xenograft mice model and divided into 4 groups randomly,Group A:Control group,Group B:Cryoablation only group,Group C:Anti-TGF-? only group,Group D:Cryoablation+Anti-TGF-? group.3.At 7 days and 14 days after the operation,the mice were killed.HE staining was used to assess the lung metastasis.The number and proportion of CD4~+T,CD8~+T and MDSC in the spleen were detected by flow cytometry.Number changes of MDSC,CD4~+T,CD8~+T in tumors were detected by immunohistochemistry.The changes of serum IL-10 were detected by ELISA in each group.The protein expression in tumor tissues which was associated with EMT was detected by Western blotting.Results1.The vitro experiments showed that: after the combined treatment the proliferation,migration and invasion of the cell were reduced significantly.2.The tumor volume increased gradually as the time went by.Compare to other groups,the Group D got the smallest tumor volume at 7 day and 14 day after the operation(P<0.05)Furthermore,the rate and number of lung metastasis also increased in each group.However,when compared with other three group,Group D got the lowest rat and number of lung metastasis(P<0.05).3.As time went by,the percent of MDSCs in the spleen of Group A and C have the increased trend,while in the Group B and D the percent is lower than the baseline.Furthermore,Group D is significant lower than other groups(P<0.05).4.As time went by,the percent of MDSCs in the marrow of Group A increased gradually,while Group B and D have the declined trend.Group C increased first then decreased at 14 day.In addition,the content of MDSCs of Group D is higher than the other three group and has a statistical significance(P<0.05).5.As time went by,the percent of CD4~+ T and CD8~+ T cells in the spleen of Group A decreased gradually,while Group B?C and D increased.Moreover,Group D has a higher persent than the other three group and has a statistical significance(P<0.05).6.Tumor-specific cytolytic activity of cytotoxic T lymphocyte(CTL)of spleens of Group D is the highest when compared with other group at 7 day and 14 day after the treatment(P<0.05).7.MDSCs in the tumor were detected by immunohistochemistry.In the group D the ratios of MDSCs in the tumor decreased gradually and has the statistical significance compared with the other three groups(P<0.05).8.Cryoablation + Anti-TGF-? can prevent the occurrence of EMT to some extent,including down-regulation the expression of E-cadherin and up-regulation of Vimentin and N-cadherin.9.Group D serum IL-10(pg/ml)levels were much lower than the other three groups(P<0.05)at 7 and 14 days after the treatment.10.The number of MDSCs in marrow,spleen and the tumor was positively correlated with the serum TGF-?.In addition,the number of CD8~+T cells in tumor were negatively correlated the MDSCs,while the serum IL-10 were positively correlated with the MDSCs.