微波消融对肺癌肿瘤微环境的抗肿瘤作用及其机制

Anti-tumor effect and mechanism of microwave ablation on lung cancer tumor microenvironment

目的探讨微波消融(MWA)对肺癌抗肿瘤的影响与作用机制。方法将1×106肺癌细胞系3LL皮下注射到6~8周的雄性C57BL/6J小鼠背部两侧,当肿瘤最大径长到7 mm时,随机分为对照组和MWA组,对一侧肿瘤进行MWA处理,每隔1 d监测对侧肿瘤生长。在MWA后的第8天,运用流式细胞术对肿瘤微环境(TME)中免疫细胞群体比例及功能进行分析。在MWA后的1 d腹腔注射程序性死亡受体1(PD-1)或者细胞毒性T淋巴细胞相关蛋白4(CTLA-4)抑制剂(200μg/只),每隔1 d监测对侧肿瘤生长。采用独立样本t检验比较两组间差异。使用双向方差分析来比较肿瘤生长曲线。结果与对照组比较,MWA有效抑制小鼠对侧肿瘤生长,差异有统计学意义(780.70±239.90比1794.00±195.20,t=3.275,P<0.01)。MWA组小鼠对侧TME中CD3^(+)T细胞(79.02±3.61比62.22±3.59,t=3.300,P<0.05)、CD4^(+)T细胞(33.26±1.73比25.80±1.68,t=2.686,P<0.05)和CD8^(+)T细胞(26.26±1.01比21.64±1.40,t=3.096,P<0.05)比例显著高于对照组。MWA组小鼠肿瘤浸润Ly6G^(+)MDSC细胞群体比例(33.00±3.25比44.86±1.46,t=3.327,P<0.05)显著低于对照组。对CD4^(+)T细胞的功能以及增殖分析表明,MWA组小鼠γ-干扰素(IFN-γ)^(+)CD4^(+)T细胞(20.39±1.84比3.05±0.47,t=7.827,P<0.01)、TNF-α^(+)CD4^(+)T细胞(17.25±2.64比5.61±1.11,t=3.571,P<0.05)和细胞核增殖抗原(Ki-67)^(+)CD4^(+)T细胞(90.68±0.81比81.55±1.37,t=6.108,P<0.01)比例显著高于对照组。此外,MWA组小鼠IFN-γ^(+)CD8^(+)T细胞(37.46±5.09比6.84±1.52,t=4.973,P<0.01)和Ki-67^(+)IFN-γ^(+)CD8^(+)T细胞(35.75±4.62比6.55±1.48,t=5.204,P<0.01)比例显著高于对照组。对T细胞表面免疫检查点分子表达分析表明,MWA组小鼠T细胞免疫球蛋白及黏蛋白结构域分子3(TIM-3)^(+)CD8^(+)T细胞(25.97±2.30比15.57±2.33,t=3.142,P<0.05)和PD-1^(+)TIM-3^(+)CD8^(+)T细胞(22.58±2.32比12.77±2.81,t=2.617,P<0.05)比例显著高于对照组。与MWA单独治疗组比较,将MWA与PD-1(514.50±106.40比883.60±144.80,t=1.911,P<0.05)或CTLA-4(534.40±82.06比961.90±131.00,t=2.941,P<0.05)抑制剂联用显著抑制小鼠对侧肿瘤生长。结论MWA能够有效抑制肺癌肿瘤生长,将MWA联合PD-1抑制剂或者CTLA-4抑制剂产生更强抗肿瘤效果。

Objective To explore the anti-tumor effect and mechanism of microwave ablation(MWA)on lung cancer.Methods Totally,1×1063LL cells(lung cancer cell line)were subcutaneously injected into both sides of the back of 6-8 week old male C57BL/6J mice.When the maximum diameter of the tumor reached 7 mm,the animals were randomly divided into the control group and MWA group.The tumor on one side was treated with MWA,and the growth of the contralateral tumor was monitored every 1 day.On the 8th day after MWA,flow cytometry was used to analyze the proportion and function of immune cell populations in the tumor microenvironment(TME).After intraperitoneal injection of programmed cell death protein 1(PD-1)or cytotoxic T lymphocyte-associated antigen-4(CTLA-4)inhibitor(200μg/mouse),the contralateral tumor growth was monitored every 1 day.The independent samples t-test was used to compare the differences between the two groups.Tumor growth curves were compared using two-way ANOVA.Results Compared with the control group,MWA effectively inhibited the growth of contralateral tumors in mice,and the difference was statistically significant(780.70±239.90 vs.1794.00±195.20,t=3.275,P<0.01).The proportion of CD3^(+)T cells(79.02±3.61 vs.62.22±3.59,t=3.300,P<0.05),CD4^(+)T cells(33.26±1.73 vs.25.80±1.68,t=2.686,P<0.05)and CD8^(+)T cells(26.26±1.01 vs.21.64±1.40,t=3.096,P<0.05)in the contralateral TME of the MWA group mice was significantly higher than that in the control group.The proportion of tumor-infiltrating Ly6G^(+)MDSC cell population(33.00±3.25 vs.44.86±1.46,t=3.327,P<0.05)in the MWA group mice was significantly lower than that in the control group.The functional and proliferative analysis of CD4^(+)T cells in the MWA group mice revealed a significantly higher proportion of interferon-γ(IFN-γ)^(+)CD4^(+)T cells(20.39±1.84 vs.3.05±0.47,t=7.827,P<0.01),tumor necrosis factor-α(TNF-α)^(+)CD4^(+)T cells(17.25±2.64 vs.5.61±1.11,t=3.571,P<0.05)and proliferation cell nuclear antigen(Ki-67)^(+)CD4^(+)T cells(90.68±0.81 vs.81.55±1.37,t=6.108,P<0.01)than the control group.In addition,the proportion of IFN-γ^(+)CD8^(+)T cells(37.46±5.09 vs.6.84±1.52,t=4.973,P<0.01)and Ki-67^(+)IFN-γ^(+)CD8^(+)T cells(35.75±4.62 vs.6.55±1.48,t=5.204,P<0.01)in the MWA group mice was significantly higher than that in the control group.Analysis of immune checkpoint molecule expression on the surface of T cells showed that compared with the control group,the proportion of T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)^(+)CD8^(+)T cells(25.97±2.31 vs.15.57±2.33,t=3.142,P<0.05)and PD-1^(+)TIM-3^(+)CD8^(+)T cells(22.58±2.32 vs.12.77±2.81,t=2.617,P<0.05)in the MWA group mice was significantly increased in the control group.Compared with the simple MWA treatment group,the combination of MWA and PD-1 inhibitors(514.50±106.40 vs.883.60±144.80,t=1.911,P<0.05)or CTLA-4 inhibitors(534.40±82.06 vs.961.90±131.00,t=2.941,P<0.05)significantly inhibited the growth of contralateral tumors,and the difference was statistically significant.Conclusion MWA can effectively inhibit the growth of contralateral lung cancer tumors,and combined use of MWA with PD-1 or CTLA-4 inhibitors produces a more powerful anti-tumor effect.