Tim-3介导NK细胞功能耗竭导致食管癌免疫逃逸及机制研究

Escape of immune surveillance is a key mechanism of tumor occurrence and malignant progression. NK cells are the most important effectors of the innate immunity and provide the first line of host defense against cancer. Esophageal cancer is one of the common human malignancies, with high incidence and poor prognosis in China. Our preliminary data showed that NK cell infiltration was significantly reduced and defective in esophageal tumor tissue accompanied by up-regulation of Tim-3 expression. In the in vitro study, we further found that esophageal cancer stem cells were resistance to NK cell cytotoxicity partially due to up-regulation of the Tim-3 ligand. However, less is known about the role of Tim-3 in modulating the function of NK cells in tumor sites and the precise molecular mechanisms remain to be identified. In the project, we hypothesize that Tim-3 serve as a crucial checkpoint and promotes NK cell exhaustion in tumor microenvironment, contributing to immune escape of esophageal cancer cells. Based on our clinical observation, next we will identify the role of Tim-3 mediated NK cell exhaustion in immune escape of esophageal cancer by both in vivo and in vivo assays. Using a variety of techniques, we will investigate the molecular mechanisms underlying Tim-3 function in NK cell exhaustion. Finally, we will screen and establish therapeutic interventions to block the Tim-3 pathway for treatment of esophageal cancer in animal models. A better understanding of tumor escape from innate immunity would point to innovative ways for the treatment of esophageal cancer.

免疫逃逸是肿瘤发生进展的关键机制。NK细胞是重要的固有免疫细胞和机体抗肿瘤的第一道防线。食管癌是常见的消化道肿瘤，进展快预后差。我们在预实验发现：食管癌组织中NK细胞明显减少并且功能下降，并伴有Tim-3表达上调，两者呈负相关。体外实验中，我们还发现食管癌干细胞对NK细胞杀伤抵抗，并与Tim-3信号通路相关。肿瘤微环境中NK细胞表达Tim-3导致功能耗竭的研究还较少，相关分子机制尚未见报道。我们推测：Tim-3是肿瘤微环境中NK细胞的重要免疫检查点，参与诱导NK细胞功能耗竭并导致食管癌细胞的免疫逃逸。本课题拟从临床现象入手，通过体内外实验阐明Tim-3表达上调诱导NK细胞功能耗竭在食管癌免疫逃逸中的作用；利用一系列分子生物学实验探究Tim-3诱导NK细胞功能耗竭的分子机制；筛选和建立干预Tim-3通路的食管癌治疗模型。本研究有望发现新的肿瘤免疫逃逸机制，为治疗食管癌提供新思路。

食管癌是常见的消化道恶性肿瘤，全球约半数食管癌发生在中国，其中绝大多数是食管鳞癌且预后不良，亟需开发新的治疗策略。NK细胞属于固有免疫细胞，是机体清除肿瘤细胞的第一道防线。本项目主要研究了Tim-3介导NK细胞功能耗竭导致食管癌免疫逃逸。我们首先解析了食管鳞癌患者肿瘤组织中NK细胞的表型特征，发现了Tim-3是肿瘤浸润NK细胞表达的主要免疫检查点分子。利用多种生物学实验，我们证实了食管鳞癌组织中Tim-3+ NK细胞表现出更高的活化水平，但早期凋亡比率明显上升，且表达IFN-γ、CD107a和颗粒酶B等功能分子显著下降，提示Tim-3+ NK细胞功能耗竭。在体外实验中，我们阐明了TNF-α通过NK-κB通路诱导人NK细胞上调Tim-3的分子机制。通过临床资料分析，我们发现了Tim-3+ NK细胞与肿瘤侵袭、淋巴结转移和临床分期密切相关。此外，我们还发现了食管鳞癌患者肿瘤组织中NK细胞表面的代谢相关靶点CD39明显上调。有意思的是，这些CD39+ NK细胞表现出功能耗竭的特征，但并不表达经典的免疫检查点和代谢相关靶点CD73。我们在体外实验中证明了食管癌细胞通过分泌IL-6诱导人NK细胞上调CD39分子。更重要的是，我们发现了CD39+ NK细胞与肿瘤侵袭和生存期下降显著相关。最后，我们利用体外富集的食管鳞癌干细胞和多种实验模型，发现了食管鳞癌干细胞抵抗NK细胞的杀伤作用。我们还揭示了食管鳞癌细胞表面ULBP-1是介导NK细胞杀伤作用的关键靶点，也是食管癌干细胞逃逸NK细胞杀伤的关键机制。全反式维甲酸能诱导食管鳞癌干细胞分化并显著上调ULBP-1的表达，进而增强食管鳞癌干细胞对NK细胞杀伤的敏感性。本项目的研究具有潜在的应用前景：NK细胞表面的Tim-3和CD39可能成为食管鳞癌的新型标志物和治疗靶点，联合全反式维甲酸可能为NK细胞杀伤食管鳞癌干细胞提供新的治疗策略。