顺铂诱导的卵巢癌细胞自噬对TAM极化的调控作用及机制研究

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, although important advances in surgical and chemotherapeutic strategy over the past several decades have improved the response rate, but he 5-year survival rate is only 25-30% due to the advanced stage with distant dissemination at diagnosis for most women with this cancer as well as the emergence of resistance to conventional chemotherapy. Therefore, the mechanism of resistance and metastasis of ovarian cancer cell is imperative to be elucidated. The regulation of chemotherapeutic drugs on the immune cells in the tumor microenvironment plays an important role in cancer therapy. Cisplatin is one of the first-line chemotherapy drugs in ovarian cancer treatment, while tumor-associated macrophages (TAM) are main immune cells in epithelial ovarian cancer (EOC) microenvironment. Therefore, to clarify the regulation mechanism of cisplatin on TAMs polarization may have important clinical implications. In the preliminary experiments, we found that there is a significantly positive correlation between TAMs density and autophagy degree in ovarian cancer tissues. Moreover, there is a significantly negative correlation between M1-TAM/M2-TAM ratio and autophagy degree. Furthermore, we found in the previous study that cisplatin could induce autophagy in human ovarian cancer cells in vitro. Finally, it has been reported that tumor autophagy affect macrophage polarization. Thus, we hypothesized that cisplatin-induced autophagy in ovarian cancer cells may regulate TAM polarization. This study, performed using patient tissue samples, cell and animal models, will investigate the relationship between cisplatin, ovarian cancer autophagy and TAM polarization, uncover the role of cisplatin induced autophagy in ovarian cancer cells on TAMs polarization and its related mechanisms. Our study may provide a theoretical basis for the targeted therapy of ovarian cancer.

化疗诱发的抗肿瘤免疫反应在肿瘤治疗效应中发挥决定性作用。我们的临床实践和基础研究提显示：死亡率居妇科恶性肿瘤之首的上皮性卵巢癌（EOC）患者接受顺铂为主的一线化疗时，机体的免疫状态和局部免疫微环境随化疗周期发生动态变化；对顺铂耐药的EOC患者组织标本和细胞株均表现出高水平自噬，后者可分泌多种免疫调节分子如GDF15，进而与免疫微环境中浸润比例最多的肿瘤相关巨噬细胞（TAM）发生着潜在的相互调控；并且TAM极化亚型比例（M1/M2）与EOC患者的分期、病理分级和预后密切相关。本课题拟整合我们在自噬和免疫微环境领域的研究资源，从临床病例、细胞实验及动物试验三个层面，探索顺铂诱导的EOC细胞自噬和TAM极化间的相互调控效应及机制，并从临床角度揭示两者在EOC耐药和预后中发挥的重要作用。预期成果将会对EOC耐药机制的阐明、靶向自噬和免疫微环境的抗肿瘤策略的方案制定等具有重要的指导价值和临床意义。

卵巢癌死亡率位居女性恶性肿瘤前列，严重威胁女性的健康和生命。尽管治疗初期卵巢癌患者对手术后铂类联合化疗的反应率很高，但大多数患者最终对化疗药物产生耐药并导致癌症的复发，因此，卵巢癌的治疗仍然具有很大的挑战性。最新研究表明，化疗过程中化疗药物除了直接作用于肿瘤细胞发挥杀伤性作用外，还能调节肿瘤微环境中的巨噬细胞，从而影响肿瘤细胞的恶性生物学活性，相关机制尚未完全阐明。本课题研究结果显示：（1）经过PMA和M-CSF以及后续LPS/IFNγ的诱导，成功诱导出THP1来源和PBMC来源的未活化巨噬细胞Mφ和经典活化巨噬细胞CAM；经顺铂的CAM（而非Mφ）的CM可明显促进卵巢癌细胞的迁移能力。（2）经Luminex液体悬浮芯片筛选及后续验证，顺铂可明显促进CAM中IL-1β和CCL20的表达。采用抗CCL20中和抗体或采用siRNA敲低卵巢癌细胞中CCL20的受体CCR6，均可显著抑制由Cis-CAM-CM促进的癌细胞迁移。（3）相比于人源永生化卵巢上皮细胞IOSE，卵巢癌细胞株中CCR6的表达明显增加；rhCCL20可显著促进CCR6high卵巢癌细胞迁移能力，而对CCR6low卵巢癌细胞的迁移能力无明显影响，敲除CCR6表达能显著抑制由rhCCL20促进的癌细胞迁移；动物试验显示，rhCCL20可明显促进WT-A2780细胞在裸鼠腹腔中成瘤的个数和重量，而对CCR6 KO-A2780细胞无此效果；卵巢癌组织中CCR6表达较高者，病人的总生存期较短，且其表达谱基因在转移相关基因集“Positive regulation of cell adhesion”和“Positive regulation of epithelial cell migration”中高度富集。综上，我们的研究阐明了顺铂化疗应激下经典活化巨噬细胞（CAMs)促进卵巢癌细胞的转移及分子机制，揭示CCL20-CCR6信号轴在卵巢癌转移过程中发挥的显著促进作用。为卵巢癌转移的机制提供了研究新导向，为卵巢癌联合治疗的药物研发提供了理论根基。