前列腺癌冷冻消融联合抗CTLA-4抗体增强小鼠肿瘤引流淋巴结免疫反应的实验研究

Enhanced immune response of cryoablation combined with anti-CTLA-4 mAb against murine tumor draining lymph nodes of prostate cancer

目的:评价前列腺癌冷冻消融联合抗细胞毒性T淋巴细胞抗原-4(cytotoxic T lymphocyte antigen-4,CTLA-4)抗体对小鼠肿瘤引流淋巴结T细胞抗肿瘤免疫反应的影响。方法:建立前列腺癌小鼠模型并随机分为空白对照(A)组、冷冻消融治疗(B)组、冷冻消融联合抗CTLA-4抗体治疗(C)组、抗CTLA-4抗体治疗(D)组。各组分别于治疗前及治疗后7、14、21天四个时间点取5只小鼠获取肿瘤引流淋巴结标本,测量各组不同时间点肿瘤大小变化,流式细胞术检测调节性T细胞(regulator T cell,Treg)和细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)比例变化,乳酸脱氢酶(LDH)法检测CTL对肿瘤细胞的杀伤活性,记录各组小鼠总生存期。结果:治疗后14天与A组的Treg比例相比,B组(9.78%±1.88%vs.6.02%±0.44%,)、C组(9.78%±1.88%vs.6.03%±0.45%)均下调(均P<0.05);与A组的CTL比例相比,B组(27.34%±2.13%vs.34.23%±1.15%)、C组(27.34%±2.13%vs.52.21%±2.53%)、D组(27.34%±2.13%vs.33.99%±1.21%)均上调(P<0.05、P<0.001、P<0.05);与A组的CTL杀伤活性相比,B组(13.32%±3.39%vs.26.36%±2.95%)、C组(13.32%±3.39%vs.45.25%±3.27%)、D组(13.32%±3.39%vs.25.31%±3.24%)均升高(P<0.05、P<0.001、P<0.05)。同时小鼠生存分析显示,B组的生存时间(34.20±6.98)天和C组的(43.60±2.88)天较A组(25.60±1.52)天明显延长(P<0.05和P<0.001),C组的生存时间(43.60±2.88)天较B组的(34.20±6.98)天明显延长(P<0.05)。结论:冷冻消融联合抗CTLA-4抗体可降低肿瘤引流淋巴结Treg比例,提高CTL比例及其杀伤活性,明显延长小鼠生存期,其具体机制尚需进一步研究。

Objective： To evaluate the effect of immune response of cryoablation combined with anti-CTLA-4 mAb against murine tumor draining lymph nodes（TDLNs） of prostate cancer（PCa）. Methods： PCa-bearing murine models were established and mice were divided randomly into 4 groups-control（A）, cryoablation treatment（B）, cryoablation combined with anti-CTLA-4 mAb treatment（C）, and anti-CTLA-4 mAb treatment（D）. TDLN specimens were obtained from 5 mice of each group at 4 time points before treatment, and 7, 14, and 21 days after treatment. Tumor size was measured at each time point. Flow cytometry was utilized to detect changes in Treg and CTL proportions in TDLNs. LDH was utilized to detect killing activity for tumor cells of CTL. Overall survival（OS） of each group was recorded. Results： Fourteen days after treatment, compared with that of group A, Treg proportion in group B（9.78%±1.88% vs. 6.02%±0.44%） and C（9.78%±1.88% vs.6.03%±0.45%） was decreased（both P〈0.05）; compared with that of group A, CTL proportion of group B（27.34%±2.13% vs. 34.23%±1.15%）,C（27.34%±2.13% vs. 52.21%±2.53%）, and D（27.34±2.13% vs. 33.99±1.21） was increased（P〈0.05, P〈0.001, P〈0.05）; compared with those of group A, CTL killing activities of group B（13.32%±3.39% vs. 26.36%±2.95%）, C（13.32%±3.39% vs. 45.25%±3.27%）, and D（13.32%±3.39%vs. 25.31%±3.24%） were improved（P〈0.05, P〈0.001, P〈0.05）. Survival analysis showed that the OS of PCa-bearing mice were prolonged（P〈0.05, P〈0.001） in group B（25.60 d±1.52 d vs. 34.20 d±6.98 d） and C（25.60 d±1.52 d vs. 43.60 d±2.88 d） compared with that of group A; OS was prolonged（P0.05） in group C compared with that of group B（34.20 d±6.98 d vs. 43.60 d±2.88 d）. Conclusions： Cryoablation combined with anti-CTLA-4 mAb can reduce the proportions of Tregs in the TDLNs, increase the proportions of CTL as well as killing activities,and significantly prolong OS of mice. However, the specific mechanism needs further study.