基于TIL-T糖代谢重编程研究参芪扶正调节肿瘤免疫机制

Tumor infiltrating lymphocyte (TILs) immune-suppression mediated by low-sugar tumor microenvironment is one of the important reasons for the cause of tumor immune escape, however, no specific interventions on it. TCM of supplementing Qi and nourishing qi has been showing quite good prospects for application in treatment of tumor by immunomodulation. Our preliminary experiment research shows Shen Qi Fu Zheng injection (SFI) has significant reversal effect on Tumor infiltrating T lymphocyte (TIL-T) immune-suppression mediated by low-sugar tumor microenvironment, but the specific links and the underlying mechanisms are remain to be determined. This project use overall and in vitro level of low-sugar tumor microenvironment models to study the effect of SFI and its monomer composition on the ability of glucose uptake by TIL-T and the level of glycolysis in tumor microenvironment, thus to preliminary reveal the main function links underlying reversal effect on TIL-T immune-suppression by SFI. Then the effect of SFI and monomer compositions were studied pro and con, to elucidate their effect on key molecular and signaling pathway of TIL-T immune-suppression which regulated by reprogrammed of glucose metabolism in vitro, and finally make clear on the targets of SFI and its monomer compositions. This project will not only be helpful in clarifying the effect of SFI on TIL-T immune-suppression mediated by low-sugar tumor microenvironment and the underlying mechanisms of multiple-components and multiple-targets, but contributes to reveal the scientific connotation about Huangqi and Dangshen on regulation of tumor immune.

低糖微环境介导的TILs免疫抑制是导致肿瘤免疫逃逸的重要原因之一，而目前尚无针对性的干预策略。益气扶正法在调节免疫防治肿瘤方面已展示良好的应用前景。课题组前期研究显示，益气扶正代表中药参芪扶正注射液（SFI）对低糖微环境介导的肿瘤浸润T淋巴细胞（TIL-T）免疫抑制具有显著的逆转作用，但具体机制不清楚。本项目拟在整体和离体低糖微环境模型中，通过考察SFI及单体成分对TIL-T摄取葡萄糖能力和糖酵解水平的影响，初步揭示SFI逆转TIL-T免疫抑制的主要作用环节；然后在离体水平，利用正、反两种策略考察SFI及单体成分对糖代谢重编程调控TIL-T肿瘤免疫的关键分子及信号通路的影响，最终明确SFI的作用靶点。该项目研究，不仅能阐明SFI对低糖肿瘤微环境下TIL-T免疫抑制的影响及其多成分多靶点作用机制，为开发针对TIL-T免疫抑制的中药提供新思路；还有助于揭示益气扶正法调节免疫抗肿瘤的科学内涵。

低糖微环境介导的TILs免疫抑制是导致肿瘤免疫逃逸的重要原因之一，而目前尚缺乏有效的干预策略。首先，本项目采用高摄入葡萄糖的黑色素瘤细胞株（B16-PKM2-OE）成功建立整体低糖肿瘤微环境动物模型。在整体模型中通过考察SFI对肿瘤组织里活化性TIL-T的数量，确证了SFI在体内确实能够促肿瘤免疫。.其次，在离体的低糖微环境模型中，通过考察SFI对CD4+T细胞增殖与活化的影响，确证了SFI促进CD4+T细胞的增殖，上调IL-2、IFN-γ和CD40L的mRNA和蛋白表达，其中具体的途径是促进TIL-T的糖酵解水平（包括上调GLUT-1膜转位、上调糖酵解关键酶HK、PFK1和PK的表达）。因此，课题组从体内外两种模型均证实SFI能促进TIL-T的增殖与活化进而促进肿瘤免疫。再次，课题组通过考察了SFI对糖酵解下游与免疫激活相关的分子表达和信号通路活性，明确了SFI促肿瘤免疫的机制是通过激活Ca2+/ NFAT信号、增加GPD2和EZH2蛋白表达,下调miR-101和miR-26a水平来实现的。而且课题组还进一步证实了SFI调节这些关键分子和信号通路活性主要与其中的成分黄芪甲苷激活AKT活性，上调AKT/GSK3β和AKT/mTOR信号有关密切相关。最后，课题组还初步得出结论：SFI不仅能直接影响CD4+T细胞的功能，还能通过干预肿瘤的异常代谢而间接影响肿瘤免疫微环境。该项目研究，不仅能阐明SFI对低糖肿瘤微环境下TIL-T免疫抑制的促进作用及其分子机制，为开发针对TIL-T免疫抑制的中药提供新思路；还丰富益气扶正法调节免疫抗肿瘤的科学内涵。