“益气除痰方”促TAM凋亡的抗肿瘤免疫功能及机理研究

Tumor-associated macrophages (TAMs) derived from immature monocyte in the circulatory system, which biological properties is similar to M2 macrophages and can inhibit anti-tumor immune response, and therefore participate in the development and progression of tumorigenesis, angiogenesis, invasion and metastasis. Anti-tumor immunotherapy, which has advantages of high efficiency, high selectivity, low toxicity, and low drug resistance, is a major theme of the research in current cancer treatment. In this context, Yiqi Chutan Formula (YQCTF), a China's traditional herbal medicine agent that has been previously shown, as such, to suppress Akt signaling, is expected to increase the efficacy of therapy. In addition to inhibition of tumor proliferation, the involvement of YQCTF in the regulation of specific anti-tumor immunity is one of the hot scientific issues. This study explored the function and underlying mechanisms of YQCTF on TAMs by using this primary culture model. The current study showed that YQCTF can efficiently induce the apoptosis of TAM cells. These works could indicate that YQCTF can promote anti-tumor immunity response by inducing apoptosis of TAMs, together with the directly anti-tumor function of YQCTF, strongly indicating that YQCTF might be a novel potential therapy target for targeted cancer therapy. Meanwhile, for the first time, this study will demonstrate that YQCTF can block Akt\FOXO3 signaling in human lung cancer cells and TAM, revealing the molecular mechanism and pathway for YQCTF-induced TAM apoptosis. In conclusion, this study carries out in-depth study in antitumor activity of YQCTF, and YQCTF’s ability to induce TAMs apoptosis. Our data suggest that use of YQCTF might represent a potentially promising approach to the treatment of lung cancer as anti-tumor immunotherapy. Thus, this study has an important academic significance and potential clinical application value.

中医药治疗肿瘤的主要优势在于改善机体的内环境，重塑肿瘤相关微环境。肿瘤微环境中肿瘤相关巨噬细胞（TAM）可抑制机体的抗肿瘤免疫应答，其异常聚集是肿瘤重要的恶性表型，可用于不良生存预后的判断标志，因此靶向TAM的免疫疗法已成为肿瘤治疗新策略。本课题组前期临床研究表明益气除痰方可以延长非小细胞肺癌患者生存期，并通过体内外实验证明了其具有较强的抗肺癌作用。同时特别发现在小鼠肺癌移植瘤模型中，益气除痰方能较强的抑制荷瘤小鼠体内TAM的产生。本项目拟应用TAM体外及体内模型，系统性开展益气除痰方诱导TAM凋亡的新功能研究；进而在分子水平从调节TAM细胞内Akt\FOXO3通路而促凋亡方面对其抗肿瘤免疫机理进行深入探讨。本项目将揭示益气除痰方通过诱导TAM凋亡而促进机体抗肿瘤免疫的新功能及机制，展示了中医药抗癌治疗的优势体现，为中医药在肺癌免疫调节个体化治疗和现代化进程提供新思路和新方向。

为了探讨“益气除痰方”的抗肿瘤免疫活性，并阐明其诱导肿瘤相关巨噬细胞凋亡的功能及分子机制，本课题结合了体内外实验进行研究。.体外实验部分，首先我们用不同浓度的益气除痰方含药血清作用于人肺腺癌顺铂耐药细胞株A549/DDP后发现随着益气除痰方含药血清浓度增加，A549 /DDP细胞增殖抑制率升高，这可能与其阻滞A549/DDP细胞从G1期进入S期有关。接着，我们采用Annexin V/PI双染结合流式细胞术、Caspase分光光度法及Western blotting等多种方法探讨了“益气除痰方”诱导顺铂耐药肺癌细胞凋亡的作用及其分子机制，结果表明“益气除痰方”通过抑制Akt/FoxO信号转导而激活内源性凋亡通路，诱导肿瘤细胞凋亡，从而逆转肺癌细胞顺铂耐药。.体内实验部分，我们构建鼠肺癌细胞LLC的小鼠肺癌移植瘤模型，以检测益气除痰方对小鼠体内移植瘤的影响，同时应用流式细胞分选技术，以CD11b-PE和F4/80-FITC标记抗体测定移植瘤中TAMs，以测定益气除痰方对小鼠肺癌移植瘤中肿瘤相关巨噬细胞产生的影响。结果发现益气除痰方能较强地抑制小鼠体内移植瘤的生长，作用可能与抑制肿瘤微环境中肿瘤相关巨噬细胞从而达到抗肿瘤免疫效应有关。肿瘤相关巨噬细胞可作为临床治疗肺癌的新靶点。