缺氧促进肿瘤微环境中NRP-1+肿瘤相关巨噬细胞募集诱导宫颈癌淋巴管生成的作用及机制研究

As an essential feature of the tumor microenvironment (TME), hypoxia has been implicated as pivotal cause to promote lymph node metastasis in cervical cancer. Tumor-associated macrophages (TAMs) are main component of stromal cells in TME and related to cancer progression evidently. However, the underlying mechanism is still elusive. Our preliminary studies have found that CD163+ TAMs and lymphatic microvessels density marked by D2-40 significantly enriched in and around the anoxia zone in the TME of cervical cancer. In addition, cultured under hypoxia condition in vitro may induce the over-expression of NRP-1 in TAMs and cervical carcinoma cells. Therefore, we proposed that regional hypoxia can promote TAMs recruited into TME of cervical cancer and polarized, activate NRP-1 signal pathway to induce lymphangiogenesis, thereby promoting lymph node metastasis. This study was designed to utilize some key technologies, such as gene-targeted knockout, Luminex liquid chip, protein microarray and IPA, combined with macrophage-specific NRP-1 knockout C57BL/6 mice and visualization mice model of orthotopically growth and metastasis of cervical cancer model to verify this new hypothesis both in vitro and in vivo. Consequently, we can elucidate the effect and mechanism in promoting lymphangiogenesis induced by hypoxia in the TME of cervical cancer through changing the function of cervical cancer cells, TAMs, NRP-1 signal pathway, which will provide scientific foundation for searching early diagnostic markers and effective therapeutic targets of metastasis in cervical cancer.

肿瘤微环境(TME)中缺氧是促进宫颈癌淋巴结转移的重要原因，肿瘤相关巨噬细胞(TAMs)作为TME间质细胞的主要成分与肿瘤转移明显关联，其调控机制仍不明确。前期预实验显示宫颈癌组织的缺氧区域明显富集CD163+ TAMs和D2-40标记的微小淋巴管生成；而体外低氧培养诱导TAMs和宫颈癌细胞高表达NRP-1；文献报道NRP-1与特异配体结合促进肿瘤淋巴管生成。因此我们推测缺氧可能促进宫颈癌TME中TAMs募集极化，激活NRP-1通路诱导淋巴管新生而发生淋巴结转移。本项目拟利用定点基因敲除、Luminex液态芯片、蛋白芯片、IPA等关键技术，构建巨噬细胞特异性基因敲除小鼠和可视化宫颈癌原位及转移动物模型，从体内、外两方面验证这一新设想，阐明缺氧TME诱导的肿瘤细胞～TAMs～NRP-1信号通路变化在促进宫颈癌淋巴管生成中的作用和调控机制，为开发宫颈癌转移早期标志物和有效治疗靶点提供科学依据。

淋巴结转移是宫颈癌的主要转移途径和影响宫颈癌治疗及预后的独立指标。淋巴结转移是受多种因素影响的复杂过程，肿瘤微环境（TME）中局部缺氧、间质细胞活化、淋巴管新生、淋巴管免疫抑制状态等与宫颈癌淋巴结转移密切关联，其调控机制仍不甚明了。本项目围绕上述关键因素，通过体内、外功能研究及临床样本验证，探讨宫颈癌TME重塑促淋巴结转移具体作用机制。我们的研究内容和结果表明：1）宫颈癌TME缺氧可促进TAMs极化，NRP-1作为中枢环节参与其调控机制；2）缺氧诱导宫颈癌细胞高表达ZEB1，转录上调CCL8的表达，通过CCL8-CCR2信号轴促进TME中TAMs募集；3）宫颈癌组织缺氧区域存在一种新型的促淋巴管转移模式LVEM，该模式由缺氧TAMs介导及活化；4）TAMs上调宫颈癌细胞MMP-9的表达，通过诱导肿瘤细胞发生EMT改变促进宫颈癌侵袭转移；5）宫颈癌细胞外泌体、肿瘤相关成纤维细胞等均通过特有机制作用于TME中淋巴内皮细胞，促进淋巴结转移。以上研究结果阐明宫颈癌缺氧TME中肿瘤细胞~免疫细胞（因子）~淋巴内皮细胞~淋巴结转移这一级联过程中的信号分子、互动关系及调控机制，为开发宫颈癌转移的早期标志物和有效治疗靶点提供科学依据。在本项目资助下共发表 SCI论文15篇（总影响因子达100.739），中文核心期刊9篇，国际会议投稿交流8篇，申报国家发明专利3项，得到同行的广泛关注和认可。