改变炎性微环境构建新型自发结直肠癌小鼠模型及巨噬细胞Act1调控癌化机制研究

Colorectal cancer (CRC) is the world's third most common malignant tumour. Inflammatory microenvironment plays an important role in the development of CRC, but its part in the initiation of colorectal cancer still remains to be elucidated. Act1(NF-κB activator 1) is primarily involved in the physiological and pathological processes of inflammation and autoimmune diseases. However, there are limited researches focusing on the role of Act1 in carcinogenesis of CRC. In the present project, anti-Act1 genetically engineered mouse, whose macrophages are deprived of Act1 expression, was crossed with Apc(Min/+) mouse to investigate the impact of macrophage-derived Act1 on the carcinogenesis process of CRC. Our data shows that repression of macrophage-derived Act1 can significantly promote the initiation of cancerization in Apc(Min/+) mouse model, thereby we successfully established a novel model of spontaneous colorectal cancer. The preliminary data indicate that specific repression of Act1 in macrophages can affect macrophage polarization and T cell differentiation, resulting in an elevated ratio of M2 type macrophages and CD4+T cells as well as increased secretion of cytokines IL-17 and TNF-α in the tumour microenvironment, henceforth the promotion of neoplasia in CRC. This project is to to further evaluate the advantages of modulating the inflammatory microenvironment along with the pathological diagnostic criteria in characterizing a novel spontaneous colorectal cancer mouse model, and to elucidate the molecular mechanism for regulation of M1/M2 polarization and T cell differentiation and promotion of carcinogenesis in CRC, with the aim of providing insight into the development of novel anticancer therapies by targeting tumour-associated inflammatory microenvironment.

结直肠癌是世界第三大恶性肿瘤。炎性微环境在结直肠癌化启动中的关键作用仍不明确，至今尚没有合适的反映炎性微环境影响结直肠癌化的自发小鼠模型。Act1基因对结直肠癌作用未见报道。利用自建的巨噬细胞Act1表达抑制的小鼠与自发肠道腺瘤Apc(Min/+)模型小鼠杂交，发现巨噬细胞Act1表达抑制后结直肠肿瘤数目和体积明显增加，生存周期明显缩短；并在早期明显促进结直肠癌化发生；我们通过改变炎性微环境成功构建了新型自发结直肠癌小鼠模型。前期工作提示，抑制巨噬细胞Act1表达能影响巨噬细胞极化及T细胞亚群的分化，造成M2型巨噬细胞和CD4+T细胞增多，分泌IL-17和TNF-α水平升高。本项目拟进一步确定改变炎性微环境创建的自发结直肠癌小鼠模型的优势及病理诊断标准；阐明巨噬细胞Act1调控M1/M2极化及促进癌化的免疫分子机制，为以炎性微环境为靶点防治结直肠癌提供新策略。

结直肠癌是世界第三大恶性肿瘤。炎性微环境诱导腺瘤转化是结直肠癌化的关键。在本项目中利用巨噬细胞Act1下调小鼠（anti-Act1）以及自发肠道腺瘤小鼠（ApcMin/+），我们目前已完成如下工作：1）利用TCGA数据库分析及临床结直肠癌病人组织病理切片，发现Act1在结直肠癌病人癌组织间质中中呈低表达且与癌组织中CD68+巨噬细胞浸润数量呈反比；2）利用构建anti-Act1; ApcMin/+转基因小鼠(AA小鼠)证实肿瘤微环境中，巨噬细胞Act1下调促进自发肠道腺瘤的癌化和进展并促进大量巨噬细胞及CD8T细胞在癌组织间质浸润；3）利用皮下移植瘤模型，证实巨噬细胞Act1下调促进皮下移植瘤的生长；4）利用耗竭巨噬细胞方法证实炎症微环境中的巨噬细胞是诱导结直肠癌化以及CD8+T细胞失活的关键免疫细胞；5）利用细胞共培养体系证实Act1下调的巨噬细胞促进结直肠癌细胞EMT及迁移；6）利用RNAseq技术揭示Act1下调的巨噬细胞与结直肠癌细胞对话后可活化IL-6/STAT3；IFNg-NF-kB信号途径且高表达CXCL9/10，PD-L1，IDO1及Arg1；7）利用siCXCR3技术及细胞共培养证实Act1下调的巨噬细胞通过调控CXCL9/10-CXCR3信号轴，影响结直肠癌细胞的EMT和迁移；8）利用siSTAT3及细胞共培养方法证实Act1下调的巨噬细胞通过活化STAT3促进与NF-kB相互作用及上调CXCL10和PD-L1的表达，并促进对结直肠癌细胞的EMT和迁移；9）利用荧光共定位技术，明确在AA转基因小鼠中，Act1下调的巨噬细胞与PD-L1呈共定位；而CD8+T细胞则与PD-1共定位；10）利用PD-L1抑制剂，体内动物实验证实PD-L1抑制剂可有效逆转巨噬细胞Act1下调诱导的结直肠癌化及进展。.在本项目中，我们不仅完成了构建自发结直肠癌化的小鼠模型，而且进一步证实巨噬细胞中Act1下调可通过活化CXCL9/10-CXCR3信号轴以及PD-L1/PD-1信号轴分别与肿瘤细胞以及CD8T细胞对话进而促进自发腺瘤的癌化作用机制，更重要的是我们的研究提示检测结直肠癌病人癌组织内巨噬细胞Act1的表达可作为临床结直肠癌病人PD-L1免疫治疗有效性的预测指标。