Caspase-8功能缺失重塑肿瘤微环境致免疫治疗耐药机制及扭转策略研究

Immune checkpoint inhibitors (ICIs), as remarkable breakthrough in cancer therapy, its clinical application is limited due to low response rate among tumors. Combination therapy strategies with immune genetic cell death is a potential method to overcome resistance. Caspase 8 is the key regulator of extrinsic cell death, which determines cell fate. Normally, activation of caspase 8 induce a rather immune tolerant apoptosis, when deleted or malfunction induces a more immunogenic necroptosis. Whether caspase-8 loss of function improve the sensitivity in ICIs therapy is largely unknown. We found when caspase 8 were deleted in B16F10 melanoma mice models, the sensitivity of ICIs were not enhanced but became more resistant, coming with less CD8+T cell infiltration, less dendritic cells (DCs) activation and down regulated calrecticulin (CRT) transportation from endoplasmic reticulum (ER) to cell membrane. As reported, the transportation of CRT were crucial for DCs to recognize and engulf dying tumor cells, which inducing antigen presenting. Malfunction of antigen presenting and aberrant T cell infiltration were regarded as the most important factors leading to ICIs resistance. Based on these findings, we proposed that caspase 8 function loss limited CRT transport from ER to cell membrane, affect DCs to uptake the tumor associated antigen which down regulated DC maturation and activation, thus less T cells were primed and migrated to tumor microenvironment, leading to ICIs resistance. To test our hypothesis, we plan to use the knock-out/knock-in system, ICIs therapy animal models and cell membrane iTRAQ technology to uncover the role of caspase 8 in tumor immunogenicity and sensitivity of ICIs. After that, we would recover the core molecular CRT expression on cell membrane by irradiation or over-expression, then combined with ICIs to test whether over comes resistance induced by caspase-8 deletion. Our research not only unveiled the function and mechanisms of caspase 8 in ICIs resistance, but also provided preclinical data in determine therapeutic/combination strategies to the caspase-8 mutant melanoma patients.

客观反应率低限制免疫检查点抑制剂(ICIs)临床应用，诱导免疫原性细胞死亡可增敏。caspase-8功能缺失细胞死亡形式以强免疫原性坏死为主，其对ICIs疗效影响未知。我们发现B16F10细胞敲除caspase-8致ICIs耐药、T细胞和树突状细胞(DC)浸润减少、细胞表面钙网蛋白(CRT)表达降低，提示肿瘤微环境重塑或为其耐药机制。结合进展，DC吞噬抗原需依赖细胞膜上CRT表达，抗原递呈障碍和T细胞浸润缺乏导致ICIs耐药，我们提出假说：caspase-8缺失下调CRT细胞膜表达，影响抗原递呈使DC活化和T细胞浸润减少致ICIs耐药。拟用基因敲除/过表达、iTRAQ等方法探讨caspase-8对CRT转运、肿瘤微环境和ICIs敏感性调控作用；联合放疗上调CRT观察对耐药改善情况。本研究阐明caspase-8缺失致ICIs耐药机制，为caspase-8突变患者提供新治疗靶点和扭转耐药策略。

近年来免疫检查点抑制剂(ICIs)为多种实体瘤均带来了临床获益，多种单药或联合方案被批准用于临床治疗，如非小细胞肺癌、胃癌、黑色素瘤、膀胱癌、肝癌等治疗中。然而，客观反应率低限制临床应用，目前已知的生物标志物对少部分获益患者有提示作用，但并不足以满足临床需求。免疫微环境是ICIs治疗反应的重要影响因素，热肿瘤往往预示更佳的疗效。caspase-8是细胞凋亡信号通路中的使动关键分子，然而其在肿瘤免疫微环境塑造中、以及ICIs治疗中的作用尚不清楚。我们通过分析TCGA数据库发现，在多种实体瘤种，Casp8表达与热肿瘤的免疫微环境相关。免疫治疗人群中同样证实，治疗有效的患者有更高的Casp8表达。为进一步明确Casp8在这两个生物学过程中的作用，我们在B16F10细胞中敲除caspase-8，并通过免疫正常小鼠皮下荷瘤模型观察到ICIs耐药的现象，同时肿瘤内T细胞和树突状细胞(DC)浸润减少。通过比较Casp8功能缺失细胞与对照细胞的免疫源性细胞死亡相关分子的表达，发现Casp功能缺失细胞表面钙网蛋白(CRT)表达降低，且这种降低可被放射治疗逆转。在小鼠皮下荷瘤模型中，放疗可重塑肿瘤微环境重塑且改善ICIs耐药。综上，我们提出：caspase-8缺失下调CRT细胞膜表达，影响抗原递呈使DC活化和T细胞浸润减少致ICIs耐药。联合放疗上调CRT，可改善ICIs耐药改善情况。从临床角度，一方面提出Casp8低表达人群可能较难从单独的免疫治疗中获益，但联合升高Casp8表达的治疗方案如放疗则是潜在的扭转耐药策略。

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