活化肝星状细胞通过上调肝内胆管癌中转录因子NR4A2表达促进肿瘤免疫逃逸的机制研究

Intrahepatic cholangiocarcinoma (ICC) is a highly mortal malignancy with limited therapeutic options. Previously, we identified that ICC samples had abundant tumor stroma characterized by plentiful fibroblasts. High density of cancer associated fibroblasts (CAFs) marked by alpha-smooth muscle actin was associated with poor survival and sparser CD3+, CD8+ tumor infiltrating lymphocytes (TILs), thus indicating that CAFs may promote ICC progression through constructing an inhibitory tumor microenvironment (TME). In our preliminary experiments, NR4A2 and a series of immune checkpoints, such as CD47 and PD-L1, were up-regulated in ICC cells co-cultured with hepatic stellate cells, which were the major precursors of fibroblasts in the liver. The expression of NR4A2 in resected ICC samples was positively correlated with the density of activated hepatic stellate cells (aHSCs). ICC patients with high NR4A2 expression had poor survival and inhibitory TME characterized by decreased counts of CD8+ TILs and higher proportions of CD4+Foxp3+ TILs. Moreover, upregulation of NR4A2 in ICC cell lines enhanced the expression of CD47 and helped ICC cells to evade from the phagocytosis of macrophages. We hypothesized that aHSCs participated in the immune evasion of ICC through upregulating the expression of NR4A2. In this proposal, we will validate this hypothesis and unravel the underlying molecular basis of how aHSCs upregulate the NR4A2 expression in ICC, immune regulatory target genes of NR4A2 in ICC and how aHSCs-NR4A2 axis works to promote the immune evasion of ICC by using protein assay, ELISA, ChIP assay, Flow cytometry and etc. The study may provide valuable clues for the development of novel therapeutic targets for ICC.

肝内胆管癌是预后极差且缺乏有效治疗手段的恶性肿瘤。我们的前期研究发现人肝内胆管癌标本中有丰富的间质及成纤维细胞沉积，其中成纤维细胞的数量与患者的预后及CD3,CD8阳性的肿瘤浸润性T细胞数量均呈负相关，这提示肿瘤相关成纤维细胞在肝内胆管癌中可能参与构成抑制性免疫微环境。预实验中，我们发现与肝星状细胞共培养后，肝内胆管癌细胞系中转录因子NR4A2及CD47、PD-L1等免疫检查点均明显上调; NR4A2高表达提示预后不良；NR4A2高表达的肿瘤样本中, 杀伤性T细胞数量明显减少而调节性T细胞比例增高；NR4A2表达上调的肝内胆管癌细胞中CD47表达增强且逃避巨噬细胞吞噬作用的能力也增强。我们推测：肝星状细胞可通过上调肝内胆管癌中NR4A2的表达促进肿瘤免疫逃逸及肿瘤进展。本课题拟采用抗体芯片、ChIP等技术，验证该假说并鉴定参与这一过程的关键分子和信号通路，为肝内胆管癌的治疗提供新的思路。

肝内胆管癌（ICC，intrahepatic cholangiocarcinoma）是预后极差的恶性肿瘤。我们前期研究发现大量成纤维细胞浸润的肝内胆管癌患者预后更差，通过建立共培养体系我们发现，活化肝星状细胞（HSC, hepatic stellate cell）可诱导ICC中转录因子NR4A2大量表达。本项目中，通过抗体芯片筛选，我们发现血管内皮生长因子（VEGF, vascular endothelial growth factor）及前列腺素E2 (PGE2, prostaglandin E2)为促进NR4A2表达的主要介质。通过体外实验我们发现HSC可通过产生PGE2促进ICC中NR4A2表达，同时还存在NR4A2-骨桥蛋白-VEGF正反馈通路，维持NR4A2持续高表达。通过数据库预测和ChIP实验我们证实，NR4A2作为转录因子可与CD47、IDO1等免疫检查点的启动子区域结合，促进ICC细胞免疫逃逸特征。此外，ICC临床样本中，我们观察到NR4A2与CD47、IDO1表达正相关。同时CD47及IDO1高表达的临床样本，存在巨噬细胞数目减少，M2巨噬细胞占比较多、调节性T细胞占比增多而杀伤性T细胞占比减少等提示免疫抑制微环境的特征。综上，我们的研究揭示了HSC-ICC相互作用可通过PGE2及VEGF等介质发挥作用，促进NR4A2持续高表达。NR4A2可进一步转录激活CD47、IDO1增强ICC的免疫逃逸特征。