aPKC-ι介导的间质表型细胞与CD4+CD25-Foxp3+T细胞以CCL5为信使形成正反馈环路促进胆管癌侵袭转移

Epithelial-Mesenchymal Transition is a core aspect of the invasion and metastasis in tumor initiation stage, meanwhile the tumor immunity is also an important factor. In the previous studies, the applicant firstly found that aPKC-ι could regulate the epithelial-mesenchymal transition in Cholangiocarcinoma, and the number of CD4+CD25-Foxp3+T cells was increased in vitro. Then antibody array showed that the level of CCL5 was higher in the culture supernatant for the cells with highly expressed aPKC-ι. Furthermore, the applicant also find that CD4+CD25-Foxp3+T cells could promote the expression of N-cadherin in cholangiocarcinoma, a mesenchymal marker. Based on the above statements, we infer that there is a positive feedback loop between the mesenchymal cells mediated by aPKC-ι and CD4+CD25-Foxp3+T cells formed by the messenger of CCL5, then it can promote the invasion and metastasis in cholangiocarcinoma. Our project intends to demonstrate the molecular mechanism of aPKC-ι regulating the secretion of CCL5 in cholangiocarcinoma cells, construct the knockout and transgenic mouse targeted at aPKC-ι, using molecular intervention strategies and co-culture system to demonstrate the mechanism of positive feedback loop between the mesenchymal cells mediated by aPKC-ι and CD4+CD25-Foxp3+T cells. This project aims to provide new clues for the therapy in cholangiocarcinoma.

癌细胞上皮-间质细胞转化（EMT）是肿瘤侵袭转移的核心环节，肿瘤免疫也是重要的影响因素。申请者前期研究发现aPKC-ι调控胆管癌EMT并诱导CD4+CD25-Foxp3+T细胞数目的增加；并利用Raybio芯片技术，发现aPKC-ι促进胆管癌细胞分泌趋化因子CCL5，以及CD4+CD25-Foxp3+T细胞诱导胆管癌间质表型标志物N-cadherin表达。申请者据此推测：aPKC-ι介导的胆管癌间质表型细胞通过趋化因子CCL5与CD4+CD25-Foxp3+T细胞形成正反馈环路，进而影响胆管癌侵袭转移。本项目拟阐明aPKC-ι促进胆管癌细胞分泌CCL5的分子机制；构建aPKC-ι基因敲除和转基因小鼠，利用分子干预策略及共培养体系，探讨aPKC-ι介导的胆管癌间质表型细胞与CD4+CD25-Foxp3+ T细胞之间的正反馈环路形成机制。本项目可望从新的角度为胆管癌治疗的新策略提供依据。

胆管癌是一种源于胆管上皮细胞的恶性肿瘤，而免疫抑制肿瘤微环境为胆管癌恶性进展创造有利条件，与预后不良密切相关。申请者前期研究发现aPKC-ι诱导CD4+CD25-Foxp3+T细胞数目的增加，但具体机制尚未阐明。本项目通过研究实施，首次利用10X单细胞转录组测序技术，发现胆管癌组织中CD4+CD25-FOXP3+T细胞主要分布于调节性T细胞亚群（重要的免疫抑制性CD4+T细胞之一），并通过分析转录组数据，发现CD4+CD25-FOXP3+T细胞的转录信息与调节性T细胞相似，CTLA4、BATF和TNFRSF4/9/18等免疫抑制分子表达上调，提示CD4+CD25-FOXP3+T细胞具有肿瘤免疫抑制功能。发现aPKC-ι 过表达胆管癌细胞通过促进TGF-β1分泌，诱导CD4+CD25-FOXP3+T细胞数目增多及免疫抑制。利用转录组测序技术，联合生物信息学方法分析aPKC-ι调控TGF-β1表达的信号通路，发现HIF-1信号通路调控aPKC-ι/TGF-β1介导的胆管癌CD4+CD25-FOXP3+T细胞变化及免疫抑制，并提出通过阻断HIF-1信号通路抑制胆管癌CD4+CD25-FOXP3+T细胞浸润的可能性。通过相关实验，发现aPKC-ι与HIF-1α存在相互结合关系，aPKC-ι经去泛素化途径调控胆管癌HIF-1α蛋白表达，并促进HIF-1α在胆管癌细胞核内积累。收集胆管癌组织及相应临床病理资料及预后，发现aPKC-ι与HIF-1α、TGF-β1蛋白表达上调，与胆管癌患者病理分化程度、TNM分期及预后相关。上述研究提示，aPKC-ι/HIF-1α/TGF-β1信号轴促进胆管癌CD4+CD25-FOXP3+T细胞数目变化及免疫抑制。本实验揭示了aPKC-ι诱导胆管癌CD4+CD25-FOXP3+T细胞变化及免疫抑制的相关机制，提示aPKC-ι/HIF-1α/TGF-β1信号轴可能是改善胆管癌免疫抑制微环境的潜在靶点。