局部分割放疗联合PD-1/PD-L1阻断治疗食管癌的模式探索及免疫机制分析

As the main treatment of esophageal cancer, local fractionated radiotherapy can not only directly kill cancer cells, but also activate the anti-tumor immunity, thus the patients may exhibit the abscopal effect. The combat between the tumor and the immune system is of great importance in generation and development of cancers. Recently, the immune checkpoint blockade immunotherapy targeting PD-1/PD-L1 has attracting more and more attention. The combination of PD-1/PD-L1blockade and physical therapy has become the new trend of oncotherapy. Based on our previous results and published data, we speculate that the combination of radiotherapy and PD-1/PD-L1 blockade immunotherapy should be an effective part of future multimodal anti-tumor strategies, which may help improve the prognosis of esophageal patients. In this project, we will explore the appropriate combination mode of radiotherapy and PD-1/PD-L1 blockade and the underlying mechanisms involved in this process with the help of the tumor-bearing animals and the specimens from the clinical patients. We will explore the dynamic effect of the long-time fractionated radiotherapy to the host anti-tumor immunity. We aim to elucidate the immune characteristics of the patients with a good response to PD-1/PD-L1 and identify new biomarkers predicting the patients’ response to PD-1/PD-L1 blockade. Results of this project will help not only to provide novel treatment strategies of esophageal patients, but also identify patients suitable for PD-1/PD-L1 blockade immunotherapy.

局部分割放疗是食管癌的重要治疗手段，杀伤癌细胞的同时激活抗肿瘤免疫，产生远隔效应。肿瘤和免疫的博弈在肿瘤生发中起关键作用，以靶向免疫检查点PD-1/PD-L1的免疫治疗近年来表现瞩目，其与物理治疗的联合应用已成为肿瘤治疗新趋势。我们基于预实验及文献报道提出设想，放疗的免疫激活效应使其适合与PD-1/PD-L1阻断治疗联用，这是高效食管癌治疗新模式。本课题将利用小鼠模型及患者标本，从细胞及分子生物学、组织病理学、预后分析等多个层次探究分割照射和PD-1/PD-L1阻断治疗的最佳联合模式，并揭示其背后的免疫学机制，进而阐明PD-1/PD-L1阻断发挥疗效的机体背景免疫状态，最终找到可预测PD-1/PD-L1阻断疗效的免疫学指标。该研究结果将有助于拓宽局部分割放疗对肿瘤免疫微环境影响的认知，发现适用PD-1/PD-L1治疗的患者人群的免疫学特征，为食管癌治疗提供合理高效的综合治疗新策略。

放疗是食管癌主要治疗方法之一，放疗对抗肿瘤免疫具有激活作用，联合免疫治疗可以诱导出更强更持久的免疫反应。研究证实大分割放疗的免疫激活效应明显强于常规分割放疗，因此单次大剂量放疗模式联合免疫治疗是实质性器官肿瘤未来治疗的发展趋势。但食管是特殊的薄壁管状结构，大分割放疗导致食管穿孔风险远高于常规分割放疗，多数患者还是采用常规分割放疗模式，阐明食管癌常规分割放疗对肿瘤微环境的调控十分必要。.申请人构建了小鼠食管癌常规分割及大分割放疗模型，发现常规分割放疗小鼠肿瘤中CD8+ T细胞数量明显增多，且不低于大分割放疗组。我们继续利用GFP小鼠制作GFP+CD8+ T细胞，并回输入接受放疗的普通食管癌小鼠中，免疫荧光的结果显示常规分割放疗组小鼠肿瘤中GFP+CD8+T细胞数量明显高于对照组小鼠和大分割放疗组小鼠，进一步证实了常规分割放疗对CD8+ T细胞浸润的持续促进作用。后续流式质谱技术检测肿瘤浸润免疫细胞分群和细胞功能，首次发现和未放疗及大分割放疗组相比，常规分割放疗的小鼠肿瘤特异性富集Tigit+、Lag-3+、Tim-3+、PD-1+、CTLA-4+、TNF-α-、IFN-γ-、Ki-67-的耗竭性T细胞。常规流式细胞术再次验证了该结果。上述研究结果提示，常规分割放疗可特异性诱导耗竭态T细胞。进一步分析流式质谱的结果，我们发现，和对照组及大分割放疗组相比，常规分割放疗组小鼠肿瘤Tex细胞特异性高表达细胞因子CCL7，不仅如此，RNA-seq的分析显示，常规分割放疗组小鼠食管癌中特异性高表达CCL-7。免疫组化的结果再次验证了该结果。我们用健康小鼠的脾脏制作CIK细胞，同时给予CCL-7刺激，首次发现CCL-7可以促进CD8+T细胞表达PD-1、CTLA-4等抑制受体，同时，抑制IFN-γ分泌及Ki-67的表达，提示CCL-7可以诱导T细胞耗竭。.耗竭T细胞形成是抗肿瘤免疫的主要障碍之一。放疗诱导肿瘤微环境中T细胞耗竭未有人报道，申请人此次发现常规分割放疗可通过CCL-7特异性诱导食管癌微环境中T细胞耗竭，尚属首次。研究结果丰富了食管癌免疫治疗的理论基础，提示CCL-7可能是全新的食管癌免疫治疗靶点。