GDF-15诱导巨噬细胞向M2亚型极化促进肿瘤免疫逃逸及其机制研究

Functional changes of immune cells in the tumor microenvironment are key factors leading to tumor immune escape. In the previous study, we found that, GDF15 inhibits DC maturation and promote Treg differentiation, suggesting that the GDF15 is closely related with immune cells function regulation. It has been reported that macrophages can polarize to M2 subtype thereby promoting tumor immune escape. Preliminary experiments also found that GDF15 upregulated IL-10 and downregulated IL-12, suggesting that GDF15 may mediated colon cancer immune escape by promote M2 polarization in mouse bone marrow-derived cells, but its regulatory role and the specific mechanism is not clear. The project intends to adopt in vivo and in vitro experiments such as flow cytometry, ELISA, mixed lymphocyte reaction, phagocytosis experiments to confirme the regulation of GDF15 on M2 macrophage polarization and colon cancer immune escape. By using co-immunoprecipitation, immunoblotting, lentiviral transfection methods to investigate the receptor of GDF15 and its downstream signaling pathways. We intent to provide a new mechanism for the M2 polarization, provide new targets and experimental basis for colon cancer immunotherapy.

肿瘤微环境中免疫细胞的功能改变是导致肿瘤免疫逃逸的关键因素。我们在前期研究中系统筛选了结肠癌中能与DC及na?ve CD4+ T细胞相互作用的分子，发现其中GDF15能抑制DC成熟并促进Treg分化，提示其与免疫细胞功能调节密切相关。文献提示，巨噬细胞可通过发生向M2极化从而促进肿瘤免疫逃逸。随后的实验还发现，GDF15能够在小鼠骨髓源性细胞中上调IL-10并下调IL-12这两个M2 亚型标志性分子，提示GDF15可能通过诱导M2极化而介导结肠癌免疫逃逸，但其调控作用及具体机制尚不明确。本课题拟通过流式细胞术、ELISA、淋巴细胞共培养等方法从体内、外证实GDF15对巨噬细胞M2极化及结肠癌免疫逃逸的调控作用；采用免疫共沉淀、免疫印迹、慢病毒转染等方法寻找GDF15诱导M2极化的受体、下游信号通路及调控分子机制。以期为M2 极化提供新的机制，为结肠癌的免疫治疗提供新的靶点和实验依据。

肿瘤微环境中免疫细胞的功能改变是导致肿瘤免疫逃逸的关键因素。我们在前期研究中系统筛选了结肠癌中能与DC及naïve CD4+ T细胞相互作用的分子，发现其中GDF15能抑制DC成熟并促进Treg分化，提示其与免疫细胞功能调节密切相关。文献提示，巨噬细胞可通过发生向M2极化从而促进肿瘤免疫逃逸。那么，GDF15是否可影响巨噬细胞的极化呢？.本课题为明确GDF-15对巨噬细胞极化及功能的影响，通过体外磁珠分离培养CD14+细胞并建立体外诱导巨噬细胞的体系，进行流式细胞术，ELISA,Real-time PCR等方法结合体内外实验，证实GDF15直接诱导CD14+单核细胞向M2样巨噬细胞极化，在CD14+单核细胞来源巨噬细胞极化阶段可抑制 M1型巨噬细胞极化，诱导M2样巨噬细胞极化；GDF15 刺激后的 M2 样巨噬细胞能够诱导Treg形成；GDF15可显著诱导CD14+单核细胞及其来源巨噬细胞在极化阶段表达TGF-β1。利用慢病毒感染构建 GDF15 过表达的小鼠结肠癌CT26 稳转细胞系；通过Real-time PCR、FCM 和 MLR 等研究方法发现过表达 GDF15 可促进肿瘤体内生长，增加腹腔M2样巨噬细胞比率及 M2 样 TAM 浸润；血浆GDF15水平、CD206+ TAM 浸润比率及TAM的TGF-β1 表达水平，三者之间成两两正相关；GDF15可诱导THP-1细胞及其极化阶段表达TGF-β1，其为受GDF15影响最显著的M2相关细胞因子。我们进一步研究了GDF15调控TGF-β1表达的分子机制。通过双荧光素酶报告基因系统、Real-time PCR、ELISA、Western blot、转录因子活性芯片、激光共聚焦、ChIP和RNA干涉等方法方法证实GDF15通过K-Ras-JNK/ERK-c-Myc通路调控TGF-β1表达。.本研究提示了肿瘤细胞分泌的GDF-15是诱导巨噬细胞产生M2样表型和功能的一个关键分子，GDF15在诱导肿瘤免疫逃逸中起到重要作用。这为肿瘤诱导巨噬细胞产生免疫耐受提供新的机理，也为阻断肿瘤的免疫逃逸提供新的靶点，为肿瘤的临床诊治提供新的思路和策略。