肿瘤再生细胞逃逸CTL杀伤的力学机理探究

The mechanisms behind tumor immune evasion remains to be fully elucidated. At the core of anti-tumor immunity are the tumor specific cytotoxic T lymphocytes (CTLs), which kill tumor cells through the Perforin/Granzyme B pathway. Perforin punctures the tumor cell plasma membrane and creates pores which allow Granzyme B to enter and induce apoptosis. Our group recently developed a mechanics-based method to select and amplify a minor population of tumor cells with stem cell like characteristics that grow spheroids in soft 3D fibrin gels. These tumor repopulating cells (TRCs) are highly tumorigenic and we have found them to be significantly softer than their differentiated counterparts. This agrees well with previous publications which consistently have found stem cells to be magnitudes softer than differentiated cells. We developed a CTL killing assay based on ovalbumin (OVA) expressing B16 melanoma cells and OVA-specific CTLs isolated from OT1-mice. Using this assay, we have showed that CTLs can eliminate most of the differentiated melanoma cells but not the stem cell like B16 TRCs, indicating that the softer TRCs can withstand attack from the CTLs. Based on the above findings, we hypothesize that TRCs being softer and having a flexible/elastic plasma membrane, are harder for Perforin to puncture. Alternatively, it may be more difficult for the CTLs to form a tight immune synapse with a soft target cell. The aim of this project is to determine the mechanisms by which soft stem cell like TRCs escape immune killing by CTLs through the Perforin/Granzyme B pathway. This project will help answer how tumors evade the immune system, provide new insights for tumor immune evasion theory and inspire new strategies for tumor immunotherapy.

肿瘤免疫逃逸的机理仍有待阐明。肿瘤特异性T细胞作为抗肿瘤免疫的核心,主要是通过释放穿孔素和颗粒酶来介导对肿瘤细胞的杀伤。然而穿孔素在细胞膜表面打孔有赖于细胞膜的软硬度，软的细胞膜不能够受力，从而阻碍打孔。课题组前期建立了体外筛选扩增干性肿瘤细胞的三维纤维蛋白软胶体系，发现筛选获得的高成瘤性肿瘤再生细胞比分化的肿瘤细胞更软，并且OVA特异性CTL可以高效杀伤OVA-B16细胞，但对于OVA-B16肿瘤再生细胞不能有效杀伤，表明肿瘤再生细胞可逃逸CTL的免疫杀伤。基于此，本项目推测CTL分泌的穿孔素难以在肿瘤再生细胞柔软的细胞膜表面形成孔道结构，从而导致免疫逃逸的发生。本项目拟阐明穿孔素在肿瘤细胞膜表面穿孔的力学机理，并尝试通过改变肿瘤再生细胞的硬度来逆转免疫逃逸。本项目的开展将有助于回答具有干性和高成瘤能力的肿瘤细胞如何逃避机体免疫系统攻击这一基本问题，为肿瘤免疫逃逸理论提供新见解，为肿瘤免