Research On The Immune Microenvironment Changes Induced By Cryoablation In Prostate Cancer

Prostate cancer (PCa) is the most common malignant tumor in male urogenital system. Recently, the incidence of prostate cancer shows a rising trend in China. Because of the absentce of effective screening schedule, middle-late prostate cancer is the majority in China. For moderate or advanced prostate cancer, little effective treatment is available. How to control the tumor metastasis; improve the overall survival (OS) and quality of life (QOL)? It is still an important issue at home and abroad. With the application and development of cryosurgery and transrecctal ultrasound, cryoablation is an important alternative in the treatment of prostate cancer. Cryoablation can be used as primary treatment or salvage treatment for the failure of radiotherapy or chemotherapy, showing a vital value in clinical treatment.Besides of locally controlling the tumors, cryoablation can induce tumor cells to release a huge amount of tumor antigens, which can result in the specific anti-tumor immune response. Furthermore, going deeply into the related mechanisms of cryoablation immune response, and seeking a method for enhacing the cryoablation immune response could provide a novel strategy for the comprehensive treatment of moderate or advanced prostate cancer.Regular T cell (Treg, TR) is the main inhibitive immune cell involed in tumor immunosurveillance escape. Exploring the change of immune microenvironment including Treg after cryoablation and illustrating the regulation mechanisms will be helpful for futher inhibiting immune suppressor cells and enhancing the anti-tumor immune response. It’s also important for the combination of tumor immunotherapy and other treatments.As a pro-inflammatory material, arachidonic acid is involved in the development of cancers. Cyclooxygenase-2(COX-2) is the main limiting velocity enzyme in the metabolism of arachidonic acid. And prostaglandin E2(PGE2) is one of the important metabolic products. Both of them play an important role in the progression of prostate cancer. Recently, a wide variety of evidences showed that COX-2/PGE2was involed in the tumor immunosurveillance escape. It’s supposed to be a target of immunosurveillance regulation. The tumor-drived CO-2/PGE2may induce the non-regulatory T cells to transform into Tregs, and cryoablation can result in the variations of immune microenvironment. Therefore, it’s an important issue for us to explore the effects of cryoablation on the COX-2/PGE2signal and tumor immune microenvironment in vivo and in vitro.Objectives:To confirm the efficacy of cryoablation for prostate cancer, observe the immune microenvironment viriation after cryoablation, analyze the change of PGE2and the related fators during cryoablation, explore the possible mechanisms of COX-2/PGE2involed in the immune regulation, and provide the decision basis for clinical treatment.Methods:Firstly, we observed the changes of tumor microenvironment after cryoablation in vivo and in vitro. Then we adopted enzyme linked immunosorbent assay (ELISA) to test the plasma PGE2, TGF-β1and IL-2in proste cancer patients and RM-1tumor-bearing mice. Western blot assay was used to test the E-cadherin, MMP-2and MMP-9protein changes after cryoablation, Transwell matrigel invasion assay and migration experiments were adopted to evaluate the adhesive and invasive abilities of tumor cells. We also detected the PGE2, TGF-β and IL-2in the culture supernatant of PC-3cell line. Realtime PCR and western blot was used to compare the mRNA and protein change of COX-2in PC-3cell line before or after cryoablation. The mixed culture of tumor cell and human peripheral blood lymphocyte was used to imitate the vivo environments. Then we detected the PGE2, TGF-β and IL-2in the culture supernatant by ELISA, evaluated the Foxp3+T lymphocyte proportion by flow cytometry, and compared the Foxp3mRNA and protein of T lymphocyte by realtime-PCR and western blot. All in all, we evaluated the effect on the Treg cell in the tumor environments by cryoablation directly.Quantity-One software was used to analyze the gray value of the stip. Graphpad prim5, Excel and Adobe Photoshop were used to draw and operate the figures. SPSS16.0software was used to analysis. For continuous variables, x±S was used to depict their central tendency. Paried t test was used to compare their differences between two paried groups. T test was used to compare their differences between two independent groups. For the comparison among several groups, variance analysis or rank sum test was used. The overall survival between two groups was analyzed by Kaplan-Meier.P＜0.05was respresentive of statistical significance. Results:The positive rate of COX-2was77.2%in the pathological tissue of prostate cancer.33cases showed a high-expressed, while24cases were loew-expressed. There was no siginificant difference in the overall survival between two goups (high-expressed group and low-expressed group). One month after cryoablation, the peripheral CD4+T cell group and NK cell was higher than before the operation, while the Treg cell was lower than before. CD8+T cell was not different before and afer cryoablation. Three days after cryoablation, the plasma PGE2declined obviously, but showed a recovery at7day. However, the plasma TGF-β1was lower at3day and7day than before, with a step by step decline trendency. For IL-2, the plasma level ascended at3day but descended at7day, with a trendecy of "saddle pattern".7day and14d after cryoablation, in prosate cancer-beared RM-1mice, the plasma PGE2and TGF-β1level declined significantly, but the IL-2level rised. The Treg proportion of spleen in RM-1model was significantly declined after cryoablation. The exotic celecoxib, with a dose of25μM and50μM, was added into the culture medium. Then the PGE2level in the cell culture supernatant decreased siginificantly. However, the TGF-β1in the cell culture supernatant was not affected by the exotic celecoxib. After two freezes thawing cycles, the prosate cancer cell line (PC-3cell) showed a siginificant increase in the proportion of apoptosis and necrosis. The invasion and migration abilities of PC-3cell line declined after cryoablation. Correspondingly, the E-cadherin protein was up-regulated, while the MMP-2and MMP-9proteins were down-regulated, which resulted in a declined invasion and migration ablities of tumor cell. Similarly, the mRNA and protein level of COX-2was also declined. After cryoablation, the PGE2and TGF-β1level in the PC-3cell culture supernatant decreased significantly. The co-cultrue of cryoablated PC-3cell and T cell didn’t show a significant effect on the T cell survival and apopotosis. After co-cultrue, the number of Foxp3positive T lymphocyte was increased, and the mRNA and protein level of Foxp3was higher than that in co-cultrue of control PC-3cell and T cell. The PGE2level in the culture supernatant of co-cultrue of cryoablated PC-3cell and T cell was lower than that in co-cultrue of control PC-3cell and T cell. However, the IL-2showed a contrary change. The TGF-β1level in the co-cultrue system was not different between cryoablated PC-3cell group and control PC-3cell group.Conclusions:(1) The rate of apoptosis/necrosis increased sharply after cryoablation. The invasion and migirtion ablility was weakend by cryoablation, with an upregualtion of E-cadherin and a downregulation of MMP-2and MMP-9.(2) The peripheral CD4+T cell group and NK cell were higher than before cryoablation, while the Treg cell was lower than before.(3) The COX-2mRNA and protein and PGE2concerntration declined obviously after cryoablation, which reversed the immune inhibition by PGE2.(4) Cryoablation may suppress the TGF-β1production and weaken its effect on IL-2through COX-2/PGE2pathway. Cryoablation also may lead to the decline of Treg cells through the pathway, which play a role on the tumor immune microenvironments.