化疗诱变巨噬细胞极化与乳腺肿瘤干细胞富集及其细胞互作机制探索

Research has shown that cancer stem cells (CSCs) play important roles in chemo-resistance, and the maintenance and the progression of CSCs are tightly regulated by tumor microenvironment. Recently, some studies have shown that macrophages (mø) in tumor microenvironment play an essential role in the regulation network of CSCs, but the regulation mechanisms are still not clear. Some key questions in this field need to be answered: Can chemotherapies reform the macrophages in breast tumor microenvironment? Is this reformation related to the CSC chemo-resistance? In this proposed study, we will study the effects of chemotherapy-induced cytokines IL-6, MIP-3 alpha and MCP-1 and more on macrophage polarization or novel functional macrophage formation, and the mechanisms of these macrophages and related cytokines on the regulation of breast cancer stem cells (BCSCs); furthermore, we will define the effects and mechanisms of chemotherapy-related macrophages on regulation of BCSCs and breast cancer progression, and elucidate the clinical implications of antagonizing chemotherapy-induced and macrophage-related cytokines utilizing breast cancer PDX models and clinical samples. The most important object of this proposal is to clarify how the reformation of macrophages by chemotherapies affect the BCSCs as well as breast cancer formation and progression at the levels from the in-vitro cells to the clinic. Successful carry-out of these studies will provide novel and strong theoretical support for how to solve the chemotherapy resistance of cancer by antagonizing chemotherapy-induced macrophage reformation.

研究表明肿瘤干细胞（CSC）是导致肿瘤化疗耐药的根源；CSC的维持和发展受到肿瘤微环境的调控。近来研究提示肿瘤微环境中的巨噬细胞（mø）是CSC调节网络的核心部分，但mø对CSC调控的机理研究还处于起步阶段。化疗是否改造了乳腺肿瘤微环境里的mø，以及CSC的化疗耐药是否与这种改造相关等关键科学问题有待解决。本项目将研究化疗诱导的细胞因子IL-6, MIP-3a和MCP-1等对mø极化或形成新功能亚群的影响，以及极化的mø及其相关细胞因子对乳腺肿瘤干细胞（BCSC）的调控机制；进而阐明化疗相关mø对BCSC和乳腺肿瘤发展的影响及机制研究，最终利用乳腺移植瘤和临床样品，探明拮抗化疗诱导的mø相关细胞因子的临床意义。真正从细胞到临床阐明化疗对mø的改变如何影响CSC及肿瘤的发生发展，是本项目最重要的目标。这些研究的顺利实施，将会为用干预化疗诱导的mø的改变从而逆转肿瘤的化疗抗性提供新的理论基础。

乳腺肿瘤干细胞是三阴性乳腺癌化疗耐受或治疗后复发的根源。我们的前期研究中已经发现乳腺肿瘤干细胞可以通过多种内源性信号调控对化疗耐受，而在本项目中我们进一步探究了化疗药物诱导肿瘤细胞和巨噬细胞分泌的细胞因子对乳腺肿瘤微环境特别是巨噬细胞的影响及其对三阴性乳腺癌和乳腺肿瘤干细胞化疗耐药的影响。按照计划，我们利用RNA-seq、Antibody Array等技术手段从三阴性乳腺癌耐药患者样品与耐药细胞中筛选出一系列与化疗密切相关的细胞因子（如CCL20、CCL3等）以及在三阴性乳腺癌细胞特别是乳腺肿瘤干细胞中特征性表达的差异基因（如Notch3、IL1R2等），并进行了一系列功能与机制的研究。我们的主要发现包括：1）化疗耐受三阴性乳腺癌患者治疗早期血清中CCL20表达增高，CCL20可以通过与CCR6受体结合激活p38MAPK/PKC-NFkB-ABCB1信号轴，导致肿瘤干细胞自我更新和三阴性乳腺癌化疗耐药；2）化疗诱导的ROS堆积可以导致巨噬细胞中CCL3表达增加，CCL3促进了巨噬细胞M1极化并增强了三阴性乳腺癌的化疗敏感性；3）Notch3被靶向抑制后释放了下游Hey2对IL6的转录抑制，导致了广谱Notch抑制剂（GSI）治疗后三阴性乳腺癌中肿瘤干细胞的富集以及最终的治疗失败；4）乳腺肿瘤干细胞中IL1R2可以增强去泛素化酶USP15对BMI1蛋白稳定性的调控，IL1R2中和性抗体可以有效靶向肿瘤干细胞并增强三阴性乳腺癌化疗敏感性；5）三阴性乳腺癌细胞中MSN可以与NONO和pPKCζ形成复合体并激活CREB信号通路，联合CREB抑制剂治疗可以增强三阴性乳腺癌的化疗敏感性。综上所述，我们确认了CCL20、CCL3、IL6、IL1R2、CREB等一系列潜在的三阴性乳腺癌临床治疗新靶标，并探索了利用靶基因特异性抑制剂、中和性抗体等联合治疗增强三阴性乳腺癌化疗敏感性的新思路，项目产出的研究成果具有较大的潜在的社会和经济价值。