唑来膦酸通过启动M1型巨噬细胞和Th17细胞间正反馈环路提高肝癌放疗疗效的机制研究

Radiotherapy is one of the effective treatments for hepatocelluar carcinoma, however, recurrence out of radiation field and radiotherapy-associated tumor progression are still the main causes of treatment failure. Our previous studies found that, the micro- environment change after irradiation is an important reason, which macrophages play an important role. The post-irradiated tumor has a greater recruitment and influx of macrophages which usually tend to M2 phenotype and these M2 populations are able to secrete of tumor growth factors, induce immune suppression, promote the invasion and metastasis of tumor cells, and stimulate tumor angiogenesis. Zoledronic acid (ZA) can reverse macrophage phenotype to M1, although the mechanism was still unknown. It has also been demonstrated that there is a positive loop between M1 macrophages and Th17 cells. We hypothesize that ZA is involved in this process. In this study, the first study on the changes of microenvironment after radiotherapy by establishment of hepatocellular carcinoma orthotopic implant model, we also investigate the synergistic antitumor effects after ZA intervention by reversed macrophages from M2 phenotype to M1. The mechanism of initiating the positive loop between M1 macrophages and Th17 cells by ZA and the involved pathways are also studied. Finally, we intend to further verify the results in clinical data. It is expected to establish a novel strategy targeting radiotherapy combined with micro-environmental intervention to improve the outcome, as well as provide the experimental and theoretical basis for clinical practice.

放疗是肝癌的有效治疗手段，但存在容易出现照射野外复发和放疗相关肿瘤进展的问题。我们前期研究发现，照射后微环境的改变是重要因素，其中巨噬细胞（macrophages）起关键作用。放疗后聚集的巨噬细胞通常表现为M2型，具有分泌肿瘤生长因子，促进肿瘤细胞侵袭转移，诱导免疫抑制，刺激肿瘤血管生成等作用，而唑来膦酸可将其逆转为“抗瘤”的M1型，具体机制尚未阐明。有研究发现M1型巨噬细胞和Th17细胞间存在正反馈环路，我们推测唑来膦酸参与了这一过程。因此，本项目首先建立肝癌原位种植模型，观察肝癌放疗后微环境的改变及其影响，明确唑来膦酸通过逆转巨噬细胞表型，与放疗产生协同抗瘤效应，继而阐明唑来膦酸通过启动M1型巨噬细胞和Th17细胞间正反馈环路的具体机制以及所涉及的通路，最后，通过临床资料加以验证。通过以上研究，有望建立肝癌放疗结合微环境干预的综合治疗新策略，为临床实践提供实验和理论基础。

放疗是肝癌的有效治疗手段，但存在容易出现照射野外复发和放疗相关肿瘤进展等问题。照射后免疫微环境的改变是重要因素，而肿瘤相关巨噬细胞在其中又起着关键的作用。本研究从临床和基础两个层面着手，围绕肝癌放疗后免疫功能的变化，以及如何改变肿瘤相关巨噬细胞极性以提高疗效等方面进行。通过肝癌放疗的病例研究，我们发现放疗后淋巴细胞数量较放疗前明显减少，其减少的程度与放疗疗效呈负相关，而淋巴细胞亚群（CD3+细胞、CD4+细胞、CD8+ T 细胞、NK细胞等）也表现出相应地变化，炎性指数PLR（血小板/淋巴细胞）的升高也影响放疗疗效，因此放疗后免疫炎性的变化是影响放疗疗效的重要因素。实验研究部分我们发现唑来磷酸可逆转肿瘤相关巨噬细胞极性。进一步研究探讨C-C趋化因子受体5（CCR5）与CCL5结合组成的CCR5-CCL5轴在改变巨噬细胞极性中的作用。研究发现，射线一方面可促进肝癌细胞自身分泌更多的CCL5，另一方面可导致癌细胞双链DNA断裂释放dsDNA促进巨噬细胞CCL5的分泌。CCL5具有趋化巨噬细胞迁移，并促使其向“促瘤”的M2型巨噬细胞极化的能力，从而减低放疗的效果。应用小干扰RNA技术下调CCL5相应受体CCR1、CCR3、CCR5的基因表达后，发现CCL5是通过与CCR5结合参与巨噬细胞趋化和极化。在小鼠肝癌原位模型中，给予X线照射和CCR5抑制剂（马拉维若，maraviroc，MV）处理后，可产生协同作用，进一步提高放疗效果。通过以上研究，有望建立肝癌放疗结合微环境干预的综合治疗新策略，为临床实践提供实验和理论基础。