神经肽激素受体Gpr54调节巨噬细胞极化在肿瘤发生发展中的功能和机制研究

The functional status of tumor-associated macrophages (TAMs) is closely related to tumor formation and development. Polarizing TAMs toward the M1 phenotype can significantly ameliorate the tumor microenvironment that resulted in the inhibition of tumor growth and immune escape of tumor cells. However, the key regulatory genes of TAMs and their specific mechanisms still need to be further studied. We previously found that expression of the neuropeptide hormone receptor Gpr54 was significantly up-regulated in M1 macrophages through high-throughput screening, while the M1 polarization of macrophages was reduced by knocking out Gpr54 and led to the enhanced growth of tumors. In this study, we will further reveal roles and mechanisms of Gpr54 in the process of macrophage polarization by Q-PCR, flow cytometry and Western blot with Gpr54 knockout and overexpressed cell lines. Then we will focus on establishing transplanted tumor models with macrophage-specific Gpr54 knockout mice to evaluate their tumor growth as well as the ratio of M1/M2 macrophages and infiltrated CD8+T cells by using flow cytometry and immunofluorescence techniques. On this account, we will preliminarily carried out the tumor treatment by targeting Gpr54 , which may probly provide new targets and ideas for the development of tumor immunotherapy strategies based on the regulation of macrophage polarization and the reconstruction of tumor microenvironment.

肿瘤相关巨噬细胞（TAMs）的功能状态与肿瘤发生发展密切相关。调控TAMs功能使其朝向M1表型极化，可显著改善肿瘤免疫微环境，抑制肿瘤的生长和免疫逃逸。而调控TAMs功能的关键基因及其作用机制仍有待深入研究。本项目前期工作通过高通量筛选发现：神经肽激素受体Gpr54在M1型巨噬细胞中的表达显著上调，而敲除Gpr54引起巨噬细胞M1型极化减弱并促进肿瘤的生长。因此本项目拟使用Gpr54敲除及过表达细胞株，以Q-PCR、流式细胞术及蛋白免疫印迹法进一步探究Gpr54对巨噬细胞极化的调控作用和机制；重点利用Gpr54巨噬细胞特异性敲除小鼠建立体内移植瘤模型，综合利用流式细胞术和免疫荧光技术，评价肿瘤的生长以及TAMs中M1/M2比例和肿瘤中CD8+ T细胞的浸润，以此为线索初步开展靶向Gpr54的治疗方法，为基于巨噬细胞极化及肿瘤微环境调控的肿瘤免疫疗法开发提供新靶点和新思路。

本项目发现了kisspeptin/GPR54信号在神经、内分泌以及免疫系统交互调控肿瘤发生、发展中的关键作用。GPR54及下游ERK5信号缺失，可以抑制慢性压力诱导的肿瘤生长，减弱T细胞耗竭，增强T细胞介导的抗肿瘤免疫治疗效果。本研究为深入理解神经、内分泌、免疫稳态调控以及肿瘤免疫逃逸的分子机制提供了重要的理论依据，也为基于T细胞的肿瘤免疫细胞治疗效果的优化提供了新的靶点和思路。在机制上，T细胞特异性缺失Gpr54不影响T细胞激活相关信号通路lymphocyte protein tyrosine kinase（LCK）、Rous sarcoma oncogene（Src）和zeta-chain (TCR) associated protein kinase（Zap70）的磷酸化，但可以显著降低T细胞耗竭相关基因nuclear receptor subfamily 4 group A member 1（NR4A1）的表达。Kisspeptin可以促进离子霉素诱导的mitogen-activated protein kinase 7（ERK5）磷酸化，而这种激活在2-APB螯合胞内Ca2+后受到抑制。此外，T细胞特异性缺失Gpr54可显著降低离子霉素诱导的ERK5磷酸化。与此同时，ERK5特异性抑制剂XMD8-92可以显著降低CD8+ T细胞的耗竭，增强CD8+ T细胞抗肿瘤功能从而抑制肿瘤生长。这些结果表明ERK5/NR4A1激活对kisspeptin/GPR54介导的T细胞功能障碍至关重要。