微粒-TACE治疗肝癌诱导抗肿瘤特异性免疫反应及机制研究

In our institute, transcatheter arterial chemoembolization with gelatin sponge microparticles (GSMs-TACE) have demonstrated sufficient necrosis inside the tumor tissue of advanced hepatocellular carcinoma(HCC). During the one year follow up,we observed some special clinical phenomena. No patient with HCC showed the lymph metastasis after GSMs-TACE. Moreover, abdominal lymph node metastases and multiple lung metastases have decrease or disappear in some HCC patients. Previous studies have reported that necrotic tumor cell can induce antigen-specific T-cell mediated immune response. We speculated the possible mechanism related to above special clinical phenomena. One of the most important feature of GSMs-TACE is obvious necrosis inside tumor 3 hours after GSMs-TACE as soon as possible. Necrotic tumor tissue could become antigens that carried on by antigen presenting cells (dendritic cells) and migrate to the surrounding lymph node. Hence, we make assumption that necrotic tumor tissues inside liver have a protential ability of inducing and activating the anti-tumor specific immune response to tumor,which could lead to the positive clinical effect, including of disapperance of lymph node and lung metastasis in HCC patients. Our preliminary experiments showed that the T lymphocyte subsets and related cellular factors changes after GSMs-TACE in some HCC patient. The results confirmed the GSMs-TACE could cause Th1/Th2 drift and improve immune function in patients with HCC. Based on the preliminary experimental results, we prepare to detect the changes of tumor antigen-presenting cells, specific cytotoxic T lymphoid cell (CTL) and immunosuppressive cells through the application of double color immunofl uoirescence staining, Tetramer and ELISPOT methods. From both tumor immunity and interventional medical engineering interdisciplinary angle, to explore the inhibition mechanism of lymph node metastasis after GSPs-TACE and reveal whether GSMs-TACE could stimulate specific anti-hepatoma cell immune response.

经导管肝动脉化疗栓塞术（TACE）是中晚期肝癌有效治疗手段，但术后复发和转移是临床难题。微粒-TACE具有术后肿瘤坏死显著特点。我们应用明胶微粒-TACE治疗411例肝癌病人，术后1年内未出现淋巴结转移，多例已发生淋巴结或肺转移病人，术后转移灶逐渐消失，分析原因可能与肿瘤坏死诱导抗肿瘤特异性免疫反应有关。预实验证实，明胶微粒-TACE可逆转外周血Th1向Th2漂移，并正向调节外周血树突状细胞（DCs）频数和功能。本项目拟采用流式细胞术、流式液相多重蛋白定量技术、Tetramer、ELISPOT等技术观察术后不同时间点：①外周血、肿瘤微环境内DCs表面标志变化；②外周血和肿瘤微环境内炎症细胞及相关细胞因子变化；③治疗前后外周血GPC3-特异性细胞毒T淋巴细胞（CTLs）率及其特异性抗原诱生细胞因子变化，明确微粒-TACE→肿瘤坏死→DCs活化→激活CTLs的证据链条，为HCC治疗提供新思路。

肝细胞癌（HCC）是全球性疾病，我国HCC患者数量占全世界近50%，死亡率高居恶性肿瘤第2位。经导管肝动脉化疗栓塞术（TACE）是中晚期肝癌有效治疗手段，但术后复发和远处转移是临床难题。我们应用明胶微粒-TACE（GSMs-TACE）治疗肝癌患者，术后肿瘤客观反应率高，进一步提高了患者生存期。分析原因可能与GSMs-TACE阻断肿瘤供血动脉“致肿瘤坏死”，暴露和释放更多的肿瘤抗原，同时肿瘤局部微环境也将发生适应性改变，并诱导抗肿瘤特异性免疫反应有关。本研究证实1）GSMs-TACE致肿瘤坏死能降低肝癌患者外周血MDSCs、Treg 细胞等免疫抑制细胞，GSMs-TACE治疗前、术后10天及术后30天外周血MDSC频率分别为36.56±9.17%、17.99±7.61%及14.23±5.93，（P<0.0001）；GSMs-TACE治疗前、术后10天及术后22天外周血Treg频率分别为11.52±3.78%、9.07±1.94%及9.96±1.48%，（P＜0.05）。证实GSMs-TACE治疗对肝癌患者的机体免疫功能具有正向调节作用。2）GSMs-TACE致肿瘤坏死能促进肝癌患者外周血GPC3-CTLs频数及百分率增加，GSMs-TACE治疗前、术后10天及术后30天外周血MDSC频数分别为120±15、316±17及230±14（P<0.05）；证实GSMs-TACE可以激活机体内源性抗肿瘤特异性免疫反应。3）目前免疫治疗及靶向治疗为肝癌患者提供新的治疗手段。GSMs-TACE治疗后诱导机体抗肿瘤免疫反应，术后联合细胞免疫治疗及靶向治疗有望进一步提高了患者生存期。4）肿瘤新抗原相关治疗应用的开发是当今肿瘤免疫治疗领域的热点和难点。该研究结果为研究团队与清华大学基础医学院林欣教授合作奠定理论基础。利用林欣教授团队已开发搭建的肿瘤新抗原筛选预测平台、TCR文库多样性分析技术和TCR-T细胞治疗平台，研究GSMs-TACE治疗对患者TCR多样性的影响，并筛选鉴定肝癌肿瘤特异新抗原及TCR。进一步将系统阐明GSMs-TACE治疗如何激活免疫系统，为实现GSMs-TACE治疗联合肿瘤免疫疗法用于肝癌的个体化精准“根治性”治疗提供坚实的临床前基础。