DNA去甲基化治疗对多发性骨髓瘤肿瘤微环境的调控机制研究

Multiple myeloma is still an encurable disease. One of the major reason is that tumor microenvironment of myeloma promotes myeloma cells’ proliferation, inhibit myeloma cells’ apoptosis, induce myeloma cells' resistance to chemotherapy and assist myeloma cells to escape from immune attack. As is well known, myeloid derived suppressor cells (MDSC) play a cental role in tumor microenvironment. Our team had confirmed previously that demethylating agent decitabine can clear MDSC in vitro and in vivo, followed by enhanced T cell immune response against tumor cells. Thus we hypothesized that DNA demethylation treatment can treat myeloma by clearing MDSC in the tumor microenvironment. In this project, we aim to: firstly, elucidate the internal molecular mechanism of the clearance of MDSC by decitabine through molecular biological techniques; secondly, emply transwell system to mimic tumor microenvironment in vitro, and investigate the change of biological behavior of murine myeloma cells or primary myeloma cells from clinical patients before and after decitabine treatment; finally, employ myeloma mice model to elucidate the relevance between MDSC clearancane in the tumor microenvironment and the treatment efficacy of decitaibne against myeloma in vivo. Our research will clarify the regulation mechanism of demethylation treatment against tumor microenvironment of myeloma. Our result may provide theoretical basis for combining DNA demethylation treatment with other treatment regimens currently available in clinical myeloma treatment, and improving the prognosis of multiple myeloma patients in future.

多发性骨髓瘤目前疗效欠佳的重要原因之一是肿瘤微环境对骨髓瘤细胞起到促进增殖、抑制凋亡、诱导耐药以及协助免疫逃逸等作用。髓系来源抑制细胞（MDSC）是构成骨髓瘤肿瘤微环境的核心细胞。本团队前期研究发现DNA去甲基化药物地西他滨能清除肿瘤微环境中MDSC，增强T细胞免疫应答。因此我们提出假说：地西他滨能通过清除肿瘤微环境MDSC对骨髓瘤发挥治疗作用。本项目计划利用分子生物学技术明确去甲基化治疗清除MDSC的机制；然后在体外环境下利用Transwell共培养体系模拟肿瘤微环境，从骨髓瘤细胞系和患者原代骨髓瘤细胞两个层次观察去甲基化治疗清除MDSC后对肿瘤微环境及骨髓瘤细胞本身的影响；最后通过小鼠模型，在体内条件下研究去甲基化治疗对骨髓瘤的疗效及与肿瘤微环境中MDSC的相关性。本项目有望发现骨髓瘤治疗的新机制，将DNA去甲基化治疗与现有骨髓瘤治疗手段结合，形成协同效应，进一步提高骨髓瘤的疗效。

研究背景：多发性骨髓瘤（MM）仍然无法治愈。MM微环境支持MM细胞的存活和免疫逃逸。因为髓源性抑制细胞（MDSCs）在MM微环境中非常重要，并且已知去甲基化药物地西他滨（DAC）可以在体外和体内清除MDSCs，所以我们假设DAC治疗可以通过在MM微环境中清除MDSC来抑制MM的增殖。.主要内容和方法：本研究以分泌IL6的小鼠骨髓瘤细胞系MPC11为模型。使用磁珠分选原代MDSCs并进行培养。使用Transwell共培养法在体外模拟肿瘤微环境，使用MPC11荷瘤小鼠模型来观察体内DAC治疗的疗效。.重要结果和关键数据：体外共培养实验表明，MPC11细胞与不含MDSCs（nonMDSCs）的骨髓细胞或DAC处理后的骨髓细胞（DAC BMs）共培养后，相比与MDSCs或PBS处理后的骨髓细胞共培养后，细胞增殖率显着降低，IL6产生减少，细胞凋亡增加。向DAC BMs中补充M-MDSC挽救了DAC BMs的抑制作用，而额外的NOHA抑制剂则进一步拮抗了M-MDSC的挽救作用。在携带MPC11的小鼠中，DAC与抗Gr1抗体的联合处理显示出对抑制肿瘤生长和促进肿瘤组织中T细胞浸润的协同作用。M-MDSC再输注也拮抗了DAC治疗的疗效。.结论及其科学意义：DAC治疗可以通过清除MM微环境中的M-MDSCs来抑制骨髓瘤细胞增殖，并诱导增强的自体T细胞免疫应答。我们相信DAC治疗有望在未来改善MM的预后。