肿瘤相关性巨噬细胞通过外泌体激活糖代谢通路促进胃癌转移的作用和机制

Metastasis is the major cause of death in patients with gastric cancer. In tumor microenvironment, tumor associated macrophages (TAM) crosstalk with tumor cells to promote tumor metastasis. However, the detailed mechanism of TAM on gastric cancer metastasis need to be clarified. Recently, exosome has been shown to mediate communication within the tumor microenvironment. Our data also demonstrated that TAM can secrete exosome, which promote the migration of gastric cancer cells. Our preliminary data showed that TAM-derived exosome increase the PKM2 activation, which is the key kinase of glucose metabolism. Based on these critical findings, we propose that tumor associated macrophage-derived exosome reprograms glucose metabolism to promote gastric cancer metastasis. In this project, we will intend to dissect the mechanism of TAM-derived exosome to regulate glucose metabolism by coculture system and microRNA array assay, and identify the functional microRNA or protein carried by exosome, which will explain the mechanism of TAM promote metastasis of gastric cancer cell. Using in vitro over-expression/silencing model and transwell/migration assay, we will determine the role of reprogrammed glucose metabolism on gastric cancer invasion and metastasis. We will perform an immunoprecipitation assay to identify the upstream and downstream proteins of migration signaling pathway by glucose metabolism induced by TAM exosome. Using in vivo imaging technology, we will dynamically observe the metastatic tumor development in the gastric cancer tumor-bearing mice with TAM derived exosome. This project will characterize the detailed molecular mechanism of TAM in gastric cancer metastasis through the regulation of glucose metabolic signaling pathway via TAM derived exosome.

侵袭转移是导致胃癌患者死亡的最重要原因，肿瘤微环境中肿瘤相关巨噬细胞（TAM）可促进转移，但具体机制未知。最新研究表明外泌体（exosome）是肿瘤微环境细胞间信息传递的重要方式，TAM可分泌大量外泌体并促进胃癌细胞转移。我们前期研究进一步证实TAM源性外泌体可影响胃癌细胞糖代谢关键激酶。因此提出假设TAM可能通过外泌体调控胃癌细胞糖代谢重编程促进转移。本项目拟首先通过体内外模型证实TAM源性外泌体调控胃癌细胞糖代谢重编程的关键作用；利用miRNA/蛋白芯片结合功能实验鉴定调控糖代谢的TAM外泌体中关键靶分子。然后体外过表达/沉默实验确定该外泌体靶分子介导糖代谢异常对胃癌侵袭转移的作用，并鉴定TAM外泌体介导糖代谢重编程调控胃癌转移的上下游分子机制；最后利用活体成像技术动态观察TAM外泌体对胃癌荷瘤小鼠转移的影响。该研究有望阐明肿瘤微环境中TAM外泌体调控糖代谢促进胃癌侵袭转移的新机制。

肿瘤微环境中相关巨噬细胞在胃癌进展中起到重要的调控作用，然而其详细的作用机制并不明确。外泌体作为基质细胞与肿瘤间信号传递的重要介质，近年来发现其在肿瘤生展中起到重要作用。本研究主探讨肿瘤相关巨噬细胞来源外泌体在胃癌转移和耐药中的作用及其机制。.本课题采用①流式细胞术、免疫组化和实时荧光定量PCR技术对胃癌组织标本中肿瘤相关巨噬细胞进行表型鉴定，并分析其与胃癌进展的相关性。②建立体外诱导肿瘤相关巨噬细胞模型，并通过体外细胞共培养及动物体内实验研究肿瘤相关巨噬细胞对胃癌转移和耐药的影响。③分离并鉴定体外诱导肿瘤相关巨噬细胞来源外泌体，利用蛋白质质谱技术分析巨噬细胞来源外泌体蛋白组分，体内外实验研究其在胃癌转移中的作用及其分子机制，并对临床标本进行相关验证。④利用miRNA芯片技术分析体外诱导肿瘤相关巨噬细胞来源外泌体miRNA组分，研究肿瘤相关巨噬细胞来源外泌体在胃癌细胞化疗耐药中的作用及其分子机制。结果表明①胃癌组织中肿瘤相关巨噬细胞大多数为M2型巨噬细胞，并且其数量与胃癌进展呈正相关。②成功构建M2型巨噬细胞体外诱导极化模型，体内外实验证实体外诱导M2型巨噬细胞促进胃癌细胞的转移和对顺铂的耐药。③M2型巨噬细胞来源外泌体富含ApoE，并通过外泌体的传递增加胃癌细胞中ApoE的水平，ApoE分子通过激活P13K-AKT通路相关蛋白表达影响胃癌细胞的细胞骨架重塑促进了胃癌细胞的转移。④M2型巨噬细胞通过外泌体传递miR-21至胃癌细胞，通过调控PTEN及BCL-2的表达水平，增强胃癌细胞的抗凋亡能力，从而促进胃癌细胞的对顺铂的化疗耐药。综上所述，本实验研究表明肿瘤相关巨噬细胞通过外泌体传递功能性的蛋白及miRNA分子至周围的胃癌细胞，从而促进胃癌的转移及耐药，靶向肿瘤相关巨噬细胞的新型免疫治疗手段或将有助于临床胃癌患者的治疗。