溶瘤病毒荷载TRAIL诱导非小细胞肺癌细胞凋亡和巨噬细胞极化逆转抗PD治疗耐药的中的机制研究

Anti-PD therapy is profoundly changing the clinical practice of non-small cell lung cancer, and the primary drug resistance of most patients has greatly limited its clinical application. Therefore, it is an important scientific issue to study the mechanism of anti-PD resistance and develop anti-drug technology. Combination therapy is an important means to overcome drug resistance. Previously, our team found that the combination of PD-L1 and oncolytic virus AD/E1-TRAIL could effectively reverse the primary resistance of NSCLC mice to anti-PD therapy. Further studies showed that oncolytic virus increased macrophages in tumor microenvironment, and the proportion of anti-tumor M1 cells increased significantly. Therefore, we propose the following scientific hypothesis: TRAIL not only directly promotes tumor apoptosis, but also changes the immune microenvironment through macrophage recruitment and M1 polarization, thus reversing resistance to anti-PD therapy. In order to verify this hypothesis, we intend to observe the apoptotic and M1-polarizing effects of TRAIL in the microenvironment on human and mouse NSCLC animal models established in the earlier stage, and then establish a culture system in vitro using human peripheral blood and mouse bone marrow-derived monocytes, observe the changes of cell phenotypes and pathway proteins, and determine the effect of TRAIL on macrophage polarization and its molecular mechanism. Seeking more effective joint strategies to provide theoretical basis.

抗PD治疗正深刻改变着非小细胞肺癌的临床实践，原发耐药极大地限制了其临床应用，因此研究抗PD耐药机制与发展抗耐药技术是重要的科学问题。而联合治疗是克服耐药的重要手段。本课题组前期发现PD-L1与溶瘤病毒AD/E1-TRAIL联用有效逆转NSCLC小鼠抗PD治疗原发耐药。进一步研究发现溶瘤病毒使肿瘤微环境中巨噬细胞增多，且抗肿瘤M1型细胞比例明显升高。由此我们提出以下科学假说：TRAIL不仅直接促进肿瘤凋亡，还通过对巨噬细胞的招募和M1型极化改变免疫微环境，从而逆转抗PD治疗耐药。为验证该假说，本课题拟在前期已建立的人及鼠NSCLC动物模型上观察微环境中TRAIL的促凋亡及M1极化作用；进而利用TRAIL重组蛋白刺激人外周血及鼠骨髓来源的单核细胞，发现TRAIL诱导巨噬细胞极化状态的细胞学效应及其分子机制。探索发现对肺癌、尤其是PD耐药的冷肿瘤具有双重靶向作用的技术手段和细胞与分子机制。

抗PD治疗在肺癌治疗中的应用受到原发耐药的极大限制，既往研究提示肿瘤微环境中M2型TAM的免疫负性作用是重要的耐药机制。本课题组前期发现溶瘤病毒AD/E1-TRAIL可能通过招募并改变动物模型中M2型TAM的极化状态逆转PD1耐药。为进一步证实并完善科学假设，本研究首先进行了方法学的优化，建立了11色免疫细胞分类方法，对肿瘤微环境中的T、NK、M1及M2型TAM进行更准确的鉴定及分型。在此基础上采用AD/E1-TRAIL干预，发现干预后的免疫细胞各个分型间差异具有统计学意义。在该方法学的基础上在小鼠模型中证实了AD/E1-TRAIL对TAM的诱导作用，且在对进行抗PD治疗的患者外周血进行AD/E1-TRAIL处理后观察到了同样的现象，进一步证实了本研究的科学假设。本课题建立的11 色免疫细胞的流式荧光抗体搭配方案可以对肿瘤微环境中的多种免疫细胞进行精确分类,为研究不同免疫细胞和肿瘤细胞之间的相互作用提供了有效的手段,并可以应用于大量的肿瘤微环境研究中；对AD/E1-TRAIL溶瘤病毒作用机制的证实，为临床转化研究提供了坚实的理论基础。

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