紫杉醇对胃癌中PDL1表达及免疫微环境的调控机制及联合治疗模式探索

The overall response rate of combinational chemotherapy with immune checkpoint blockade was over 70% in the first line treatement of gastrric cancer, however it is still little known how does chemotherapy influence PDL1 expression and immune microenvironment in gastric cancer. Our previous study of 1014 cases of gastric cancer specinmens revealed that PDL1 expression was closely correlated with tumor infiltrating lymphocytes (Tils), predicted better outcome and also influenced by chemotherapy. Using the whole genome CRISPR/cas9 high throughput screening on gastric cancer cell line N87, we found knocking out of TSC2 positively regulated PDL1 expression while knocking out of HIF1α inhibits PDL1 expression. The mTOR pathway was activitated and PDL1 was upregulated when treated with paclitaxel. We would further investigate and validate whether and how paclitaxel regulated PDL1 expression via TSC2/MTOR/HIF1α pathway and its molecular mechanism. Meanwhile, the gastric cancer specimens would be sequenced and multistained to analyzed the dynamic altearations of the immune subgroup. The combinational therapy with chemotherapy and PDL1 antibody would be performed in vivo and the resistance mechanism would be detected, so that the study would provide systemic solid basis for combinational therapy.

临床研究发现化疗联合免疫节点PD1/PDL1抗体一线治疗晚期胃癌可达到70%缓解率，大大优于单一治疗，但其相关协同机制尚不清楚。.前期大样本研究显示PDL1表达显著提示胃癌预后良好。紫杉醇能够引起多种胃癌细胞系内PDL1表达上调和AKT/mTOR通路活化。通过CRISPR全基因组文库筛选，发现N87细胞中TSC2敲除正性调节PDL1，HIF1α敲除抑制PDL1表达,提示紫杉醇可能通过TSC2/MTOR/HIF1α通路上调PDL1表达。本课题将进一步从分子、细胞、临床样本和动物模型等方面探索和验证紫杉醇是否通过mTOR通路调控PDL1表达及其具体机制。同时系统分析紫杉醇及PDL1高表达对胃癌免疫微环境中各细胞亚群的动态影响及二者的交互作用。在此基础上，开展紫杉醇联合PDL1抗体治疗的体内实验，探索最佳组合方案，分析耐药机制。这将为联合治疗提供深入的理论依据，打开新的治疗思路。

本课题一方面深入观察了化疗药物对于胃癌免疫微环境的影响，对接受新辅助化疗的胃癌患者手术标本中肿瘤浸润免疫细胞密度和克隆扩增进行多维度分析。新辅助化疗后，FOXP3+Treg细胞的浸润比例明显下调，且新辅助化疗前肿瘤微环境中FOXP3+Treg细胞浸润比例高的胃癌患者对新辅助化疗较为敏感。相比之下，CD8T+细胞的浸润比例在新辅助化疗后明显增高，在接受XELOX方案及新辅助化疗获益的胃癌患者中该变化尤为显著，并伴随着T细胞受体多样性的上调。与未经化疗样本相比，经过新辅助化疗后残余胃癌组织中CD68+巨噬细胞的浸润比例显著降低。此外残余肿瘤组织中CD8+T细胞和CD68+巨噬细胞的浸润比例与新辅助化疗疗效显著相关。新辅助化疗后肿瘤组织中CD8+T细胞浸润比例仍是胃癌患者生存的独立预后因素。在此基础上，构建并验证了一组包含EMT相关分子标志物（CDH1）和免疫相关分子（CD3,CD4, PDL1, GZMB, PAX5）的新型胃癌预后预测模型。该模型在术前未经治疗的胃癌患者中可弥补TNM分期对预测预后的局限性，进行风险评估，预测化疗受益人群。另一方面，对化疗药物对于PDL1表达的分子机制进行了深入探索，发现化疗药物紫杉醇可引起SIRT1/TRIM28复合体的活化，导致PDL1水平上调，引起化疗耐药。TRIM28可通过K63位泛素化修饰TBK1，并激活TBK1-IRF1和TBK1-mTOR信号通路，进而上调PD-L1的转录水平。进一步我们将对SIRT1/TRIM28复合体在化疗药物影响下，是如何发挥调控PDL1表达和免疫微环境调控的再行深入探索。