EGFRE746-A750缺失突变肺腺癌细胞外泌体诱导耐受性DC形成及机制研究

Patients with EGFR mutations showed unfavorable response to immunotherapy such as programmed cell death-1 (PD-1) blockade immunotherapy in non-small cell lung cancer (NSCLC). Yet the underlying association between EGFR mutation and immune resistance remains largely unclear. Previous literature and our preliminary result showed that tumor tissue containing EGFR mutation was in a status of severe immunosupression. We still don’t know why this type of cancer showed an uninflamed tumor microenvironment..Exosomes are present in nearly all human body fluids. They have diameters within the range of 30-100 nm and have spherical to cup-shaped nanoparticles with specific surface molecular characteristics, such as CD9 and CD63. The main function of exosomes is to participate in cell-to-cell communication by transferring bioactive molecules to recipient cells close to or distant from the original cells; exosomes subsequently alter the content and behavior of the recipient cell. Exosomes are important conveyors of immune response and present antigen to the antigen-presenting cell (APC). We thought tumor cells containing EGFR mutation may shed exosomes to regulated the immune state of whole tumor tissue. .Our preliminary results showed that exosomes from EGFR mutation cells can induce mice unanswed to cytokine therapy. Exosomes derived from EGFR E746-A750 deletion mutation lung adenocarcinoma cells can carry EGFR mutation protein and induce formation of tolerogenic DC. More importantly, this induction was probably achieved by EGFR E746-A750 deletion mutation pathway. This positive feedback loop might confer the crosstalk between lung cancer cells and tolerogenic DC though the machanism is still unclear. In the following study, we will verify the induction ability of tolerogenic DC by mutaion exosomes in the spleen cells of mice. Then we will adopt RNA interference/rescue test, PCR, western blot, flow cytometry, ELISA to test the EGFR-PKC-STAT3 pathway and its vital role in the formation of tolerogenic DC. We will adopt mRNA chip to test the different expression of mRNA in DC with and without IL-10 and try to find how IL-10 affect tumor cell to shed more exosomes. .Our study for the first time show the vital role of exosomse in the immunoregulation in lung adenocarcinoma with EGFR mutation and will provide theoretical foundation for immunotherapy for lung caner with EGFR mutation.

EGFR突变（简称突变）肺腺癌对免疫检查点抑制剂疗效不佳。既往文献及前期工作证实突变肺腺癌肿瘤组织处于严重免疫抑制状态且机制不明。前期工作发现突变外泌体可携带突变蛋白诱导耐受性DC形成且极有可能通过EGFR-PKC-STAT3-IL-10正反馈通路参与肿瘤细胞与DC的cross talk，导致抑制性肿瘤微环境的形成，以上发现有待进一步验证且具体作用机理不明。后续将在小鼠脾细胞中进一步验证突变外泌体诱导耐受性DC的能力，RNA干扰挽救过表达实验、PCR、WB、流式细胞术、ELISA实验验证以上通路在耐受DC形成中的作用。RNA芯片检测IL-10对突变细胞基因表达的影响，寻找IL-10促进细胞外泌体分泌的可能机制并进行验证。课题首次从外泌体的角度阐明EGFRE746-A750突变细胞外泌体在肺腺癌免疫抑制微环境形成的重要作用，为EGFR突变肺腺癌的免疫治疗发展方向提供新的理论基础和研究方向。

EGFR突变肺癌对免疫治疗尤其是免疫单药治疗疗效不佳，其原因很可能与该类肿瘤处于T淋巴细胞浸润较少的免疫“荒漠”状态有关。其免疫抑制状态的形成机制不明，探索EGFR突变肺癌免疫抑制状态的形成机制并寻找可能的解决方案对我国肺腺癌患者非常重要。本项目主要围绕EGFR突变肺癌临床免疫治疗疗效、肿瘤免疫抑制微环境形成机制和新的免疫检查点蛋白筛选三个方面开展研究。第一部分研究通过回顾性收集EGFR突变、使用EGFR-TKI耐药后并使用以免疫药物为基础的治疗方案的肺癌患者，观察分析其免疫治疗疗效，确认EGFR突变肺癌使用免疫治疗疗效欠佳。第二部分通过构建EGFR-19del和EGFR-WT基因转导LLC细胞模型，进行体内外实验证实EGFR-19del肺癌细胞通过分泌携带EGFR-19del蛋白的外泌体介导DC细胞处于免疫耐受状态，耐受DC无法诱导T细胞增殖，导致该类肿瘤处于T细胞减少的免疫抑制状态。第三部分通过检测较大量的EGFR突变和野生型肺腺癌患者B7家族第三类分子（B7-H3、B7-H4、VISTA、B7-H6、HHLA2）以及IDO-1、PD-L1、CD8的表达，试图寻找EGFR突变与野生型肺腺癌差异表达明显的蛋白。结果发现EGFR突变肺腺癌患者B7-H4和HHLA2的表达较野生型患者表达明显升高，而IDO-1和PD-L1在野生型肺腺癌中的表达较EGFR突变肺癌患者表达明显升高，提示B7-H4和HHLA2可能在EGFR突变肺腺癌中发挥重要作用。本项目对于EGFR突变肺癌靶向耐药后免疫治疗疗效、免疫治疗耐药原因及可能的治疗对策提供了较好的临床及临床前依据，为EGFR突变肺癌患者的治疗带来新的探索方向。