调控肠道菌群对靶向间皮素的CAR-T细胞抗胰腺癌疗效的影响及机制研究

Pancreatic cancer (PC) is one of the deadliest diseases of human digestive malignancies. Despite the recent advances in surgery and chemotherapy, the 5-year survival rate of PC continues to be less than 10%. As a promising tumor therapy, Chimeric antigen receptor T cell (CAR-T), however, performed poorly in PC treatment and need to be further updated. In our study, on the basis of our previous research, we use anti-MSLN CAR-T as effector cell and explore the different effects and mechanism of gut microbiota (PC or healthy control) on anti-MSLN CAR-T treatment. Firstly, we detect the differences of gut microbiota and T cell cholesterol metabolism in PC and healthy control by means of 16S-rRNA, PCR, western blot and ELISA; explore the different effects of gut microbiota on the subtype of T cells; and analyze the relationships between intestinal flora composition and T cell cholesterol metabolism or subtype changes by means of Spearman’s correlation. Secondly, we also explore the different effects of gut microbiota on the proliferation, migration, subtype, inflammatory cytokines expression and anti-tumor effector function of anti-MSLN CAR-T cells by means of flow cytometry and cytotoxicity assay. Thirdly, we discuss the different expression of cholesterol esterification enzyme 1 (ACAT-1) and other core genes of cholesterol metabolism in anti-MSLN CAR-T. Lastly, we evaluate the effects of different gut microbiota on the treatment of PC by anti-MSLN CAR-T cells in NSG mouse model of subcutaneous PC transplantation and liver metastasis. Through the above experiments, a new theoretical basis is provided in which gut microbiota regulates the subtype and anti-tumor function of anti-MSLN CAR-T by ACAT-1 expression. Furthermore, our findings, which demonstrate the relationship of gut microbiota and CAR-T cell, may be translatable for the treatment of other solid tumors like PC.

胰腺癌是一种恶性程度极高的消化系统肿瘤，生存率极低且缺乏有效的非手术治疗方式。嵌合型抗原受体T细胞（CAR-T）虽具有良好应用前景，但其治疗胰腺癌效果欠佳。本课题以间皮素为治疗靶点，以胰腺癌为研究对象，以肠道菌群调控为切入点，探讨胰腺癌患者和健康人肠道菌群对CAR-T靶向抗肿瘤作用的影响及相关机制。首先，通过高通量基因测序分析肠道菌群差异，Spearman相关性分析明确肠道菌群组成与T细胞胆固醇代谢和亚型改变的相关性。其次，通过CAR-T增殖、杀伤等一系列生物学功能检测，评估肠道菌群对CAR-T抗肿瘤作用的影响，揭示胆固醇转脂酶（ACAT-1）在其中的作用机制。进一步构建鼠胰腺癌皮下移植瘤及肝转移瘤模型，体内检测肠道菌群改变对CAR-T的影响。从而阐明肠道菌群通过ACAT-1调节CAR-T亚型进而影响其靶向杀伤能力，为提高CAR-T细胞对胰腺癌等实体肿瘤的疗效提供新思路和可能的理论依据。

胰腺癌是一种恶性程度极高且诊断和治疗都非常困难的消化系统肿瘤，其五年生存率仅为10%左右而且缺乏有效的治疗方式。嵌合型抗原受体T细胞（CAR-T）是一种具有良好应用前景的恶性肿瘤免疫治疗新方式，然而其治疗胰腺癌等实体肿瘤的效果仍欠佳。本课题以靶向间皮素的CAR-T细胞为研究对象，以体内菌群为切入点，探讨胰腺癌患者和健康人体内菌群对anti-MSLN CAR-T抗肿瘤作用的影响及机制。实验取得结果：①通过16s-rRNA测序分析临床患者粪便及胰腺癌组织，初步证实：胰腺癌患者肠道菌群组成及代谢产物与健康人差异明显；胰腺癌患者术前与术后肠道菌群差异明显；胰腺癌组织内存在菌群。②　代谢组学分析表明：菌群代谢产物中脂质代谢通路（包括胆固醇代谢通路）相关产物较多且富集分析差异明显。③通过体外共培养实验证实：菌群代谢产物能够明显影响anti-MSLN CAR-T细胞ACAT-1表达，从而影响其杀伤能力及亚型比例。④进一步通过体内及体外实验证实：抑制ACAT-1能够提高anti-MSLN CAR-T细胞抗胰腺癌的能力。⑤最后通过对胰腺癌细胞内部菌群的探索，初步验证：胰腺癌细胞内存在菌群并同样可以达到抑制ACAT-1，影响胆固醇代谢及胰腺癌细胞生物学特性的作用。本项目主要探讨了体内菌群与anti-MSLN CAR-T的部分作用机制，对整体上菌群与肿瘤相关免疫机制的探索予以一定的丰富和补充。同时，为后续通过菌群干预（如健康人粪菌移植等）来改善anti-MSLN CAR-T 对胰腺癌的治疗效果提供理论基础，同时也为通过调节菌群改善CAR-T 治疗其他实体肿瘤的可行性策略提供新的理论依据。