DNA甲基转移酶抑制剂影响结肠癌GVAX疫苗抗肿瘤免疫应答机制的研究

Tumor microenvironment is important for anti-tumor immune response. In our preclinical and clinical data showed GM-CSF secreting allogenic whole tumor cell vaccine (GVAX) is safe and effective on colorectal cancer, but need to further improve the its efficacy. Our study on DNA methyltransferase inhibitors (DNMTi) found that DNMTi may enhance colon cancer cells immunogenicity.We little know about DNA methylation in tumor's immune microenvironment. Based on these data, this project aims to investigate whether DNMTi treatment can improve the antigenicity of colon tumor vaccine and, as a result, whether DNMTi treated GVAX is more effective in inhibiting liver metastasis formation in a hemisplenectomy mouse model of colon cancer and prolonging the lives of the tumor bearing mice. Next, this project will study whether DNMTi treatment can increase the infiltration of effector and memeory T cells but deplete the immunosuppressive cells in tumor microenvironment, thereby enhance the GVAX induced antitumor immune response in the same liver metastasis model of colon cancer. Finally, In colon cancer patients specimen, we will compare the immune cell subset and their related genes expression changes in tumor infitrating lymphocytes(TILs) between these tumor specimens from the biopsies prior to and those post DNMTi treatment. This project will allow us to dissect the basic mechanism of methylation regulation in antitumor immune response and provide the rationale for improving the efficacy of immunotherapy through modulating epigenetic codes in tumor microenvironment.

肿瘤微环境对肿瘤疫苗的抗肿瘤免疫应答至关重要。我们前期动物和人体试验显示结肠癌GM-CSF分泌型肿瘤全细胞疫苗即GVAX疫苗安全有效；对DNA甲基化酶抑制剂（DNMTi）的研究发现，DNMTi可能增强结肠癌细胞的免疫原性，但对其影响肿瘤免疫微环境的机制了解甚少。为进一提高结肠癌GVAX疫苗的抗肿瘤效应，本项目拟在以往研究的基础上，观察DNMTi在分别处理疫苗和小鼠后，能否增强疫苗的免疫应答，是否能有效地抑制结肠癌发展并延长荷瘤小鼠的生存；并观察DNMTi治疗荷瘤小鼠后肿瘤微环境中效应和记忆T细胞的变化，以及它们对疫苗抗肿瘤效应的影响；再在结肠癌患者活检和手术标本中分析DNMTi治疗前后肿瘤浸润淋巴细胞亚群和相关基因表达的变化，阐明甲基化调控对GVAX抗肿瘤免疫应答和肿瘤免疫微环境影响的机制，为通过甲基化调控来提高肿瘤疫苗治疗的疗效提供理论依据。

我们前期研究显示结肠癌 GVAX 疫苗安全有效，为进一提高结肠癌 GVAX 疫苗的抗肿瘤效应，我们建立了小鼠半脾切除结肠癌肝转移动物模型，发现去甲基化药物预先使用后，再应用GVAX疫苗治疗可以提高鼠结肠癌肝转移模型的生存，并能减少肿瘤的肝脏转移；进一步研究去甲基化药物预先使用后，GVAX 疫苗可以使免疫细胞产生的 IFN-γ增加， 还发现去甲基化药物可能会增加结肠癌的免疫原性，增强肿瘤特异抗原（TSA）的免疫应答。我们进一步研究发现在人升结肠癌组织的TILs中， 肿瘤组织CD4+ T细胞明显少于正常组织，而CD8+ T细胞比正常组织无减少；RAS基因突变不影响癌组织中 T细胞的浸润数目；微卫星不稳定（MSI）明显影响癌组织中的T细胞数目。我们再通过FACS流式分选联合NGS检测升结肠正常黏膜和癌组织中CD3+CD4+和CD3+CD8+T细胞转录组的差异和甲基化水平的差异。分析发现在CD4+T细胞中，通过甲基化调节，TNXB可下调表达而导致MMP9的上调表达；RNF144A的下调表达可导致S100A8的上调表达，而增强T细胞的迁移和抗肿瘤效应。说明癌细胞可以影响TILs的甲基化水平而影响相关基因的表达，最后影响肿瘤浸润淋巴细胞（TILs）的抗肿瘤效应。