肿瘤浸润调节性T细胞在肝癌循环肿瘤细胞释放中的作用及相关机制研究

We previously reported that circulating tumor cells (CTCs) were significantly correlated with postoperative recurrence and metastasis of hepatocellular carcinoma (HCC). Our recent research indicated that tumor infiltrating regulatory T cells intimately correlated with CTC release from hepatocellular carcinoma. However, the role and mechanism of Treg in promoting dissemination of CTC in HCC are still unclear. In this study, we plan to establish the Treg-HCC cell co-culture system to investigate the role of Treg in stimulating migration, invasion and epithelial-mesenchymal transition of HCC cells in vitro. Establish Treg-depletion mouse model to evaluate the impact of Treg on intravasation and dissemination of CTC in vivo. Using gene microarray technology to explore and validate key genes and activated signaling pathways that regulate Treg promoted CTC release. Prospectively evaluate the clinical utility of key genes and activated signaling pathways that regulate Treg promoted CTC release in predicting postoperative recurrence/metastasis in HCC. In summary, the role and underlying mechanism of Treg initiated CTC dissemination in HCC are expectable from this project. This study offer new intervention strategies or new therapeutic targets for preventing metastasis/recurrence of hepatocellular carcinoma.

我们前期研究表明循环肿瘤细胞（circulating tumor cell, CTC）在肝癌转移复发中扮演重要角色，进一步研究提示肿瘤浸润调节性T细胞（regulatory T cell, Treg）与CTC释放关系密切。目前肿瘤浸润Treg在肝癌CTC释放中的作用及分子机制不明。本课题在大量前期工作基础上建立Treg-肝癌细胞共培养体系，探讨Treg 在体外对肝癌细胞侵袭迁移及上皮间质转化的影响；建立Treg耗减小鼠模型，观察Treg在体内调控CTC释放促转移的作用；利用基因芯片比较Treg共培养组和对照组肝癌细胞基因表达谱，筛选并验证Treg促肝癌CTC释放关键基因及信号通路；在临床标本中验证关键基因与CTC释放的关系及预测转移复发的临床价值。本研究将进一步阐明Treg促肝癌CTC释放的非免疫学作用及相关机制，奠定免疫微环境促转移的理论和实验基础，为肝癌转移复发防治提供新思路和新靶点。

本研究揭示了肝癌浸润Treg在驱动CTC释放入血中的非免疫学作用，阐明了肝癌浸润Treg介导CTC释放入血的分子机制。外周血CTC负荷与肝癌浸润Treg数量呈正相关。Treg-肝癌细胞共培养实验表明Treg可在体外可促进肝癌细胞发生EMT，进而增强肝癌细胞的迁移和侵袭能力。CBA技术和抗体中和实验发现肝癌浸润Treg通过分泌TGF-β调控肝癌细胞EMT、迁移和侵袭。Treg-depletion C57BL/6J 小鼠模型结果显示Treg在体内具有促进肝癌CTC释放和转移的作用。基因表达谱芯片检测结果表明Treg共培养组和对照组肝癌细胞在基因表达谱上存在显著差异。进一步的生物信息学分析表明这些基因与信号转导、细胞黏附、趋化、抗凋亡、细胞运动等功能密切相关。Kegg信号通路分析表明这些基因与JAK-STAT、p53、TGF-β、MAPK、Wnt等肿瘤相关信号通路有关。我们随后选择了Treg共培养组和对照组差异最明显的JAK-STAT通路及其中的关键分子白细胞抑制因子（leukemia inhibitor factor, LIF），进一步揭示Treg促进肝癌CTC释放的分子机制。免疫共沉淀实验表明Treg分泌TGF-β作用于肝癌细胞，导致SMAD2/3复合物入核结合LIF的启动子区，激活肝癌细胞表达LIF，LIF通过自分泌的方式作用于肝癌细胞，促进STAT3活化，进而促进肝癌细胞EMT、侵袭和转移。肿瘤浸润Treg与肝癌CTC释放密切相关；Treg可能通过分泌TGF-β，激活肝癌细胞LIF-JAK-STAT信号通路，促进肝癌细胞发生EMT，增强肿瘤细胞运动和侵袭能力，进而导致CTC的大量释放；Treg及其介导的TGF-β-LIF-JAK-STAT信号通路轴可能会成为今后干预肝癌CTC释放的新靶点。