具核梭杆菌通过FGL1诱导TAM浸润参与结直肠癌免疫逃逸的机制研究

In the last few years, the importance of gut microbiota in the carcinogenesis and progression of colorectal cancer has been raised. A variety of pathogenic bacteria have been proved to participate in the malignant progression of colorectal cancer. Previous studies have shown that Fusobacterium nucleatum promotes the development of colorectal cancer by regulating the immune microenvironment, whereas the interaction and mechanism of Fusobacterium nucleatum and immune checkpoints still unclear. We have previously found that Fusobacterium nucleatum can promote the enrichment of immune-suppression cells in the intestinal tumor microenvironment. miR-194 down-regulation and its target gene FGL1 up-regulation after Fusobacterium nucleatum stimulation were confirmed by microarrays and vitro experiments. Fusobacterium nucleatum also increased CCL2 and CCL5 expression, as well as macrophages migration. Therefore, we proposed our hypothesis of Fusobacterium nucleatum promoting immune checkpoint FGL1 expression through inhibiting miR-194, then recruiting tumor-associated macrophages infiltration, which participates in immune escape of colorectal cancer. The present study intends to reveal the role of FGL1 in immune evasion mediated by Fusobacterium nucleatum in vitro, in vivo and in clinical samples, then elucidating the molecular mechanism of FGL1 activation by Fusobacterium nucleatum in colorectal cancer, and support the exploration of potential candidate targets in developing novel therapeutics.

肠道菌群失调在结直肠癌的发生发展中发挥重要作用，多种肠道致病菌被证实促进结直肠癌的恶性进展。既往研究表明具核梭杆菌介导免疫微环境参与结直肠癌的发生发展，但具核梭杆菌与免疫检查点的相互作用及机制尚不清楚。我们前期发现具核梭杆菌可促进肠道肿瘤微环境中抑制性免疫细胞数目增多；高通量测序和细胞学实验表明具核梭杆菌刺激结直肠癌细胞后miR-194下调，其靶基因FGL1表达上调；具核梭杆菌刺激结直肠癌细胞后趋化因子CCL2和CCL5表达上调，对小鼠巨噬细胞的趋化能力增强。据此我们提出假说：具核梭杆菌通过抑制miR-194表达上调免疫检查点FGL1，进而招募肿瘤相关巨噬细胞浸润参与结直肠癌免疫逃逸过程。本项目拟通过体外细胞实验、体内动物模型及临床样本分析等方法揭示FGL1在具核梭杆菌介导结直肠癌免疫逃逸过程中的作用，阐明具核梭杆菌调控FGL1活化的分子机制，为探索结直肠癌防治的新模式提供理论依据。

目前，肠道微生物的代谢产物与免疫治疗之间的联系与机制在结直肠癌中尚不清楚。在这项研究中，我们对接受免疫治疗的晚期结直肠癌患者的粪便和血清进行了多组学分析（粪便宏基因组测序和血清靶向代谢组学测序），结果显示在免疫治疗无反应的患者粪便中具核梭杆菌的丰度升高。此外，代谢组学结果提示具核梭杆菌通过其代谢产物琥珀酸降低了对抗PD-1单抗的敏感性。血清琥珀酸水平较低的结直肠癌患者对免疫治疗的疗效更佳。机制上，具核梭杆菌的代谢产物琥珀酸抑制了肿瘤细胞表面的cGAS-interferon-β通路，降低肿瘤细胞分泌趋化因子ccl5和cxcl10水平，限制肿瘤微环境中CD8+T细胞招募至肿瘤组织中，进而抑制抗肿瘤免疫反应。最后，甲硝唑治疗能降低肠道中具核梭杆菌丰度，进而降低血清中琥珀酸水平，从而提高肿瘤组织对免疫治疗的敏感性。因此，我们的研究结果表明，具核梭杆菌及其代谢产物琥珀酸能降低结直肠癌对免疫治疗的敏感性，为结直肠癌中肠道微生物-代谢产物-免疫治疗扰提供了新的视角。依托本项目，已投稿第一作者SCI论文1篇，已被Cell子刊Cell Host &Microbe杂志（IF=31.13）第二轮修回。