Notch信号通路抑制剂对胰腺癌患者PD-1/PD-L1免疫治疗的增敏作用及机制研究

Pancreatic cancer is a malignant disease with poor prognosis. The early diagnosis is difficult, the treatment is limited and the mortality is high. Notch and PD-1 / PD-L1 signal pathway play an important role in the development and progression of pancreatic cancer, as well as the immunosuppression in tumor microenvironment (TME)..Based on former studies, we found that pancreatic cancer can express both PD-1 and PD-L1 proteins, while Notch signal pathway not only promotes the progression of pancreatic cancer, but also enhances PD-1 expression. It is speculated that Notch signal pathway inhibitors can suppress the progression of pancreatic cancer, and inhibit the classical and intrinsic PD-1 / PD-L1 pathway respectively, so as to sensitize PD-1 / PD-L1 related immunotherapy..First of all, we are going to clarify the time, space and tissue specificities of PD-1 and PD-L1 expression in pancreatic cancer. Then, to proof the effects of intrinsic PD-1 / PD-L1 pathway on biological behavior of pancreatic cancer in vivo and in vitro. We are also going to study the sensitization effect of Notch inhibitors on PD-1 / PD-L1 treatment via drug intervention experiment, and the modification of tumor-related immune cells’ functions before and after the intervention. At last, using correlation analysis of molecular co-expression and sequence analysis of DNA binding domain, we want to investigate the specific mechanism of the sensitization process..This study will provide experimental evidences for the sensitization effect of Notch inhibitors on PD-1 / PD-L1 immunotherapy. And provide a theoretical basis for the application of both signaling pathways’ inhibitors on patients with pancreatic cancer.

胰腺癌是预后极差的恶性肿瘤，早期诊断困难、治疗效果欠佳。Notch与PD-1/PD-L1(后简称“PD”)通路对胰腺癌的发生、进展及肿瘤的免疫抑制均有重要作用。本课题组前期发现：胰腺癌可同时表达PD-1及PD-L1，Notch通路不仅促进胰腺癌进展，亦增强PD-1表达。故推测：Notch抑制剂不仅能延缓肿瘤进展，亦可抑制经典及内源性PD通路，对PD相关免疫治疗产生增敏作用。本项目拟明确PD相关蛋白在胰腺癌中表达的时间、空间、组织特异性；通过体内、体外实验研究内源性PD通路对胰腺癌恶性程度的影响；通过药物干预实验研究Notch抑制剂对PD相关免疫治疗的增敏作用，及对肿瘤相关免疫细胞功能的修饰；通过分子共表达分析及结合域序列分析，探索Notch通路对PD通路直接调控的机制。为探究Notch抑制剂对胰腺癌患者PD相关免疫治疗的增敏作用提供实验证据，为临床中联合应用两信号通路抑制剂提供理论基础。

胰腺癌恶性程度高，治疗效果欠佳，预后差，其免疫微环境在肿瘤发生中起重要作用。Notch通路与PD1/PD-L1通路对胰腺癌的发生及进展有重要的作用。目前免疫治疗在胰腺癌中治疗效果有限，需进一步探索联合治疗。本课题使用胰腺癌细胞系、人胰腺癌单细胞测序数据及生信数据库cBioPortal初步分析了PD1、PD-L1及Notch通路关键分子HES1的表达情况，并进一步探索Notch通路与PD-L1通路的机制关系。通过动物实验探索双通路抑制的治疗效果。研究结果发现PD-L1及Notch通路关键分子HES1在胰腺癌细胞系及人胰腺癌组织中均存在表达。在人胰腺癌细胞系中PD-1的表达不高。离体及在体实验均发现Notch通路抑制剂DAPT可上调胰腺癌中PD-L1的表达，进一步研究发现HES1基因的敲低能上调胰腺癌细胞系MIA PaCa2和SW1990中PD-L1的表达。在Pan02胰腺癌皮下小鼠模型中，DAPT及PD-L1抗体联合组胰腺癌肿瘤的重量、Ki-67的表达明显低于单药组及对照组。流式细胞分析结果显示，在联合用药组，肿瘤细胞中CD8+ T细胞的比例明显高于单药组。总的来说，Notch通路抑制剂可以增强PD-L1抗体对胰腺癌的抗肿瘤作用，为进一步临床上病人的用药提供了一定的理论依据。