【作者】 朱进；

【导师】 王水；

【作者基本信息】 南京医科大学 ， 外科学（普通外科学）， 2018， 博士

【摘要】 微波消融(microwave ablation,MWA)是一种广泛应用于肝脏肿瘤、甲状腺肿瘤、肺部肿瘤及肾脏肿瘤等疾病的一种新兴的微创治疗方法。微波消融不仅可以在原位灭活肿瘤,还能激活机体产生适应性的抗肿瘤免疫反应,但是这种免疫反应还比较弱,不足以阻止肿瘤复发。免疫检查点(immune checkpoint)是指免疫细胞膜表面表达的传导抑制信号的分子,在正常情况下保护机体避免产生剧烈的自我免疫反应,但在肿瘤微环境中却能导致肿瘤细胞发生免疫逃逸。程序性死亡分子1(programmed death-1,PD-1)和细胞毒性T淋巴细胞相关抗原4(cytotoxic T lymphocyte associated antigen-4,CTLA-4)都是 T 细胞表面重要的免疫检查点分子,介导负性调节信号,能有效抑制T细胞的功能和增殖。研究发现阻断PD-1或CTLA-4信号通路可恢复T细胞的活化及增殖能力,增强机体抗肿瘤的适应性免疫反应。免疫检查点抑制剂能否增强和维持微波消融所产生的适应性免疫反应?两者联合使用是否能产生一个协同的抗肿瘤作用呢?我们通过对4T1荷瘤小鼠进行微波消融联合PD-1及CTLA-4拮抗治疗,分析治疗后荷瘤小鼠外周血及肺转移灶中免疫细胞和细胞因子的变化,以及比较对荷瘤鼠总生存的影响,来探讨微波消融与免疫检查点抑制剂的协同抗肿瘤作用。结果显示,微波消融在早期明显升高荷瘤小鼠外周血的CD8+T细胞及IFN-γ,但是在中后期,微波消融所致的适应性免疫反应逐渐消退,小鼠外周血CD8+T细胞比例及IFN-γ浓度下降至无处理组荷瘤鼠水平。然而,微波消融后行PD-1及CTLA-4拮抗治疗能维持这种适应性免疫反应至微波消融后期,该组小鼠外周血CD8+T细胞比例及IFN-γ浓度在微波消融后期仍偏高,并通过肿瘤负荷减少降低了外周血MDSC及Treg细胞的比例。另外,单独PD-1及CTLA-4拮抗治疗4T1荷瘤鼠不论在总生存还是小鼠外周血免疫细胞及因子均未见明显改变,再次证实了免疫检查点抑制剂对弱免疫原性肿瘤效果较差。研究还发现,微波消融治疗虽然早期抑制了肺转移灶的生长,但是却诱导肺转移灶免疫抑制性肿瘤微环境的形成,加速了肿瘤的生长及扩散,最终未能明显延长荷瘤小鼠的生存时间。微波消融联合PD-1及CTLA-4拮抗治疗不仅明显抑制肺转移灶的形成,还明显改善了肺转移灶肿瘤微环境,抑制了残余肿瘤的生长及转移,显著延长了荷瘤小鼠的生存时间。故该研究为开展微波消融联合免疫检查点抑制治疗的临床研究提供了理论依据。更多还原

【Abstract】 As a minimally invasive therapy for focal cancer,microwave ablation(MWA)is widely used in the treatment of liver,thyroid,lung,and kidney tumors.In addition to directly destroy tumor tissues,MWA can lead to the release of large amounts of cellular debris in situ,which can serve as a source of tumor antigens to elicit hosting adaptive immune responses against tumors.However,the responses induced by MWA are weak and not sufficient to prevent tumor recurrence.Immune checkpoints are a series of inhibitory pathways that are crucial for modulating the intensity and duration of immune responses.Among these checkpoints,cytotoxic T-lymphocyte-associated protein 4(CTLA-4)and programmed cell death protein 1(PD-1)have shown to be two key regulators of the activation and proliferation of T cells.CTLA-4 is rapidly up-regulated on activated T-cell surface following immune activation,and binds to B7 molecules on antigen presenting cells(APCs)with a higher affinity than does CD28 molecules on T cells.PD-1 is also increasingly expressed on T-cell surface following activation,and binds to its ligands PD-L1 and PD-L2 on tumor cells or other immune cells,which promotes T-cell anergy,apoptosis and exhaustion.Whether immune checkpoints blockade can enhance and mantain the adaptive immune responses induced by MWA,and whether a combination of immune checkpoints blockade and MWA can synergistically enhance antitumor efficacy.In order to testify the hypothesis,we treated 4T1 tumor-bearing mice with MWA,PD-1 and CTLA-4 blockade,MWA plus PD-1 and CTLA-4 blockade.Immune cells were identified by immunohistochemistry and flow cytometry,and the concentrations of plasma interferon-γ(IFN-γ)were identified by enzyme-linked immunosorbent assay(ELISA).The results showed that the frequencies of CD8+ T cells and the concentrations of IFN-y in the peripheral blood of tumor-bearing mice were significantly increased in the early stage after MWA,but reduced in the middle and late stages after MWA,which implys that the adaptive immune responses induced by MWA weaked gradually.However,PD-1 and CTLA-4 blockade maintained the adaptive immune responses induced by MWA to the late stage after MWA,with increased CD8+ T cells frequencies and IFN-γ concentrations in the peripheral blood.In addition,administration of anti-PD-1 and anti-CTLA-4 without MWA showed no change in overall survival,peripheral blood immune cells and IFN-y of 4T1 tumor bearing mice,which confirmed that weak immunogenicity tumors were refractory to immune checkpoints blockade.Although MWA inhibited the lung metastases of 4T1 tumor bearing mice in the early stage after MWA,it induced the tumor immunosuppressive microenvironment,accelerating the growth and invasiveness of tumors,which failed to prolong overall survival of tumor-bearing mice.MWA combined with PD-1 and CTLA-4 blockade not only significantly inhibited the lung metastases,but also remodeled the tumor microenvironment,which significantly prolonged overall survival of tumor-bearing tumors.Therefore,this study provides a theoretical basis for the clinical study of microwave ablation combined with immune checkpoints blockade.更多还原