携带ANGPTL1的外泌体改造结直肠癌肝转移前微环境的研究

Liver metastasis accounts for much of the colorectal cancer mortality. It is well established that cancer-derived exosomes can transform the pre-metastatic liver mircoenvironment and support colonization of cancer cells in liver, but the molecular compositions and mechanisms are not fully understood. Our previous study found that ANGPTL1 was significantly downregulated in colorectal cancer tissues, and animal studies implied that ANGPTL1 overexpression attenuated liver metastasis and prolonged overall survival, while ANGPTL1 knocking down promoted mouse liver metastasis. What’s most interesting is that ANGPTL1 is enriched in colorectal cancer-derived exosomes. Colorectal cancer-derived exosomes are abundantly presented in liver, and uptaken by Kupffer cells. We proposed that ANGPTL1 influences formation of pre-metastatic niche, thus inhibiting liver metastasis via exosomes. The study aims to analyze the effect of exosomes carrying ANGPTL1 on pre-metastatic liver niche by exosomes’ education on liver. Conditional liver Kupffer cells knockout mice will be employed to illustrate the significance of liver Kupffer cells. And we also aim to select and confirm the changed signaling pathways in liver Kupffer cells and the molecules interacting with ANGPTL1 directly. It will be the first study to reveal the functions of ANGPTL1 carried by exosomes, further, to elucidate and optimize the significance of mechanisms of colorectal cancer liver metastasis.

肝转移是结直肠癌患者死亡的最重要原因。已知肿瘤特异性外泌体能改造肝转移前微环境，影响癌细胞定植于肝脏，而外泌体携带的物质及其作用机制是一盲区。课题组前期工作发现肠癌组织中ANGPTL1表达显著下降；动物实验证实ANGPTL1过表达降低肝转移率并提高生存；而ANGPTL1敲低后肝转移能力明显增强。最关键的是发现：ANGPTL1在肠癌细胞的外泌体中富集，而肠癌分泌的外泌体在肝脏中高度富集，并主要被库普弗细胞摄取。故假设：ANGPTL1通过外泌体引起肝转移前微环境的改变，从而抑制肠癌肝转移。本研究拟通过外泌体肝脏“教育”，分析携带ANGPTL1外泌体对肝转移前微环境和肠癌细胞定植的影响；通过库普弗细胞条件特异性敲除小鼠，明确库普弗细胞的作用；并进一步筛选及验证库普弗细胞中变化的信号通路及与ANGPTL1相互作用的分子。本项目拟揭示外泌体ANGPTL1的功能，完善和拓展肠癌肝转移的相关新机制。

肝转移是结直肠癌患者死亡的主要原因。目前研究认为肿瘤来源外泌体能改造肝转移前微环境从而促进癌细胞在肝脏定植，而外泌体携带的物质及其功能尚不明确。课题组前期研究发现血管生成素类似蛋白1（ANGPTL1）能够抑制结直肠癌肝转移，并且发现ANGPTL1存在于外泌体中。本研究旨在探索携带ANGPTL1的外泌体对结直肠癌肝转移的作用及机制。首先，本研究通过超速离心法分离结直肠癌组织及细胞来源的外泌体，并检测外泌体中ANGPTL1的表达水平。我们发现相较于正常肠粘膜组织，结直肠癌组织来源的外泌体中ANGPTL1的表达水平明显降低。随后，我们通过小鼠眼球后静脉注射构建外泌体教育模型，结合经脾肝转移模型探究携带ANGPTL1的外泌体对结直肠癌肝转移及转移前微环境的作用。我们发现携带ANGPTL1的外泌体能够抑制小鼠肝脏血管渗漏情况，并且减轻肝脏转移瘤负荷。为了探究携带ANGPTL1的外泌体对肝脏微环境的作用机制，我们通过外泌体示踪实验明确了外泌体主要被肝脏Kupffer细胞吞噬。进一步转录组测序的结果提示，Kupffer细胞的分泌谱显著改变，其中MMP9差异最明显。体内外血管通透性实验也证实，携带ANGPTL1的外泌体通过下调Kupffer细胞MMP9表达抑制了肝脏血管渗漏。通过KEGG富集分析，我们发现并验证了外泌体ANGPTL1通过抑制JAK2-STAT3信号通路下调Kupffer细胞MMP9表达。本研究发现携带ANGPTL1的外泌体通过抑制JAK2-STAT3信号通路下调Kupffer细胞MMP9表达，从而抑制肝脏血管渗漏，减弱结直肠癌肝转移。本研究拓展了ANGPTL1蛋白的功能，丰富了结直肠癌肝转移机制研究，为外泌体ANGPTL1在肝转移治疗中的应用提供了理论依据。