肿瘤相关巨噬细胞分泌VEGF/TGF-β1促进结直肠癌乏周细胞血管形成的机制及意义

There are lack of effective biomarkers for predicting response of bevacizumab in metastatic colorectal cancer. Our study found that patients with high tumor associated macrophage had much more survival benefit from bevacizumab, but the potential mechanism is unknown. It was reported tumor associated macrophages can induce angiogenesis in tumor acidic microenvironment, but whether it can promote forming of bevacizumab-sensitive vessels is unknown. We previously found that vessels without pericyte coverage were increased in tissue sample from patients with high tumor associated macrophage. Vessel without pericyte coverage is considered sensitive to bevacizumab. Preliminary experiment showed that the tumor associated macrophage conditioned medium can promote endothelial cell migration and lumen formation, and suppress pericyte migration. Tumor associated macrophage also had high expression of VEGF and TGF-β1. This research project aims to investigate that lactate promote VEGF and TGF-β1 expression and secretion through stabilization of HIF-1α after entering tumor associated macrophage by monocarboxylate transporter 1. Then, VEGF promotes endothelial cell migration and lumen formation, and TGF-β1 suppresses pericyte migration, thus forming a large number of new vessels without pericyte coverage. This vessel is the most sensitive type of blood vessels to bevacizumab. This project also aims to reveal the value of tumor associated macrophage as a predictive biomarker to bevacizumab, which will provide a new basis for precision treatment of metastatic colorectal cancer.

贝伐珠单抗缺乏有效的疗效预测指标，本课题组发现肿瘤相关巨噬细胞增多者贝伐珠单抗获益，机制不明。既往报道酸性微环境中的肿瘤相关巨噬细胞可以诱导肿瘤血管形成，但是否可以促进对贝伐珠单抗敏感的血管生成目前尚无报道。我们前期工作发现肿瘤相关巨噬细胞增多的晚期结直肠癌患者肿瘤组织中乏周细胞血管增多，后者对贝伐珠单抗敏感。预实验显示肿瘤相关巨噬细胞可以促进内皮细胞迁移以及管腔形成，并抑制周细胞迁移；肿瘤相关巨噬细胞VEGF以及TGF-β1表达上调。本课题拟探讨肿瘤酸性微环境下，乳酸经单羧酸转运体1进入肿瘤相关巨噬细胞后以依赖于HIF-1α方式促进VEGF以及TGF-β1表达并分泌，VEGF促进内皮细胞迁移以及管腔形成，TGF-β1抑制周细胞迁移，从而形成大量乏周细胞血管，这正是贝伐珠单抗最敏感的血管类型。研究还将揭示肿瘤相关巨噬细胞作为贝伐珠单抗疗效预测指标的价值，为晚期结直肠癌的精准治疗提供新依据。

贝伐珠单抗联合化疗是晚期转移性结直肠癌的一线治疗方案，但贝伐珠单抗缺乏有效的疗效预测指标，课题组发现肿瘤相关巨噬细胞增多者贝伐珠单抗获益，故拟探讨肿瘤相关巨噬细胞对贝伐珠单抗预测的作用机制，以期获得其作为疗效预测指标的数据支持。通过实验发现肿瘤相关巨噬细胞对内皮细胞与周细胞增殖、凋亡无明显影响，但抑制周细胞迁移并促进内皮细胞迁徙以及管腔形成能力。利用qPCR以及ELISA法检测发现，上述功能表型系由于肿瘤相关巨噬细胞所分泌的VEGF以及TGF-β1所致。通过对比单纯巨噬细胞以及肿瘤相关巨噬细胞在有或无HIF-1α特异性抑制剂的情况下VEGF/TGF-β1蛋白水平，提示TAM分泌VEGF/TGF-β1需要HIF-1α参与。我们还探讨了Wnt通路在结肠癌免疫逃逸中的作用及其机制。实验发现Wnt通路与IL-23的转录与蛋白表达有关。抑制Wnt通路后IL-23 mRNA以及蛋白表达水平升高，相反，激活Wnt通路后IL-23 mRNA以及蛋白表达水平下降。利用流式细胞术发现IL-23可上调树突细胞表面成熟分子标记HLA-DR、CD86的表达，而该结果在肿瘤细胞与树突细胞共培养模型中得到验证。利用免疫组化对比APC突变与野生型结肠癌组织中β-catenin、IL-23A表达以及CD83阳性树突细胞数目，APC突变的结肠癌细胞中β-catenin核内表达增加，IL-23A表达降低；同时CD83阳性的树突细胞显著减少。上述结果提示结肠癌细胞Wnt通路激活后下调IL-23表达从而抑制树突细胞的成熟，可能是结肠癌免疫耐受的机制之一。在临床研究方面，发现全身炎症指标Glasgow评分为0的患者可以从一线贝伐珠单抗联合化疗显著获益 (PFS 11.83 vs. 8.67 月, p=0.03; OS 30.80 vs. 24.87 月, p=0.04)，而Glasgow评分为1或2的患者未能从贝伐珠单抗治疗中获益。总体来讲，课题按原设计进行，并获得了相关数据。另外还探讨了Wnt通路抑制结直肠癌组织中浸润性树突细胞成熟的机制。所有研究均关注结直肠癌的药物敏感性问题，且可能阐明结直肠癌免疫耐受的机制，为提高结直肠癌疗效提供一个有效的策略发展方向。