CXCL12甲基化修饰介导骨肉瘤免疫逃逸的机制研究

The immune escape plays a critical role in the process of tumorigenesis and tumor progression, but the underlying mechanism by which osteosarcomas escape from immune attack is still largely unknown. The main functions of the immune system including immune monitoring, defence, and elimination are closely associated with the migration and homing of immune cells. It is widely accepted that the chemokine CXCL12 plays a key role in mediating immune cell migration. Our previous works provided the evidence that osteosarcoma downregulated the expression of CXCL12; applying DNMT inhibitor could significantly strengthen the expression of CXCL12 in osteosarcoma, as well as elevate the number and ratio of tumor-infiltrated lymphocytes, indicating that the downregulated expression of CXCL12 might be associated with immune escape in osteosarcoma. Based on our preliminary data, we proposed this project, aiming to further study the epigenetic regulation of CXCL12 expression in osteosarcoma by established techniques and resources including animal models, osteosarcoma clinical samples, multi-color flow cytometry, live imaging, and IHC/IF. This project is supposed to demonstrate the association between epigenetic regulation in CXCL12 and intratumoral lymphocytes in osteosarcoma, and the effects of targeting DNMT against osteosarcoma immune escape, which may unveil the mechanism by which osteosarcoma immune escape and CXCL12 regulation, and consequently provide evidence for further targeted therapy against osteosarcoma.

免疫逃逸在肿瘤发生发展中发挥关键作用，但骨肉瘤如何逃逸机体的免疫监视和攻击仍未清晰。免疫系统的核心功能如免疫监控、免疫防御、免疫清除等均与免疫细胞的迁移归巢紧密关联。已有研究表明，趋化因子CXCL12在免疫细胞的迁移归巢过程发挥重要作用。申请人前期研究发现骨肉瘤CXCL12表达下调，而使用DNA甲基转移酶抑制剂能显著上调骨肉瘤中CXCL12表达，同时肿瘤浸润淋巴细胞的数量和比例显著提高，提示CXCL12表达下调可能介导骨肉瘤免疫逃逸。本项目拟在上述研究的基础上,进一步使用动物模型、骨肉瘤临床样本等，采用多色荧光标记流式细胞分析、活体成像、IHC/IF等技术，深入研究骨肉瘤CXCL12表达的表观遗传调控及其与骨肉瘤内淋巴细胞浸润的关系、靶向抑制DNA甲基化修饰对骨肉瘤免疫应答的影响，旨在揭示骨肉瘤CXCL12表达的表观遗传调控及其介导骨肉瘤免疫逃逸的机制，为后续的骨肉瘤靶向治疗提供依据。

本项目研究对象原发骨肿瘤的免疫微环境及免疫反应，集中在趋化因子CXCL12对骨肿瘤免疫的影响，旨在探讨以CXCL12为代表的趋化因子通过何种方式、影响何种免疫细胞最终导致骨肿瘤的免疫逃逸，为骨肿瘤的免疫治疗提供基础。在项目申请的过程中，骨肉瘤部分率先获得突破，在获得资助之前就成功将论文发表于Cancer Research，明确了骨肉瘤通过高甲基化调控降低了CXCL12的表达，阻止了CXCR4阳性的免疫细胞通过CXCL12浓度梯度向骨肉瘤组织归巢，进而抑制了免疫反应，并根据以上基础，提出对骨肉瘤进行低甲基化治疗可能可以促进骨肉瘤的免疫反应。在项目资助期间，申请人还同时深入研究了软骨肉瘤、骨巨细胞瘤的免疫微环境和趋化因子。在软骨肉瘤的研究中，申请人利用新鲜肿瘤组织标本进行单细胞水平的研究，使用CyTOF技术绘制了普通型软骨肉瘤的免疫图谱，以此为基础在学术界率先对软骨肉瘤进行了免疫分型，结合临床免疫治疗数据，提出“免疫衰竭型”软骨肉瘤是免疫检查点抑制剂的敏感人群，且“免疫衰竭型”软骨肉瘤的分子特征是IDH突变。在机制研究中，申请人发现IDH突变的软骨肉瘤高表达CXCL12、CXCL9、CXCL10，且以上三种细胞因子是“免疫衰竭型”软骨肉瘤病灶中存在大量免疫细胞的关键。IDH突变导致局部CXCL12聚集，而高浓度的CXCL12促使具有抗原递呈活性的树突状细胞DC向软骨肉瘤归巢，进而发挥抗原递呈作用、分泌CXCL9和CXCL10促使杀伤性T细胞向肿瘤归巢，发挥抗肿瘤作用。该研究发表于Clinical Cancer Research，并以此为基础注册、开始临床试验。在骨巨细胞瘤相关研究中，分析了骨巨细胞瘤中破骨样巨细胞的表型，相关研究发表于中华骨科杂志。