Meningeal lymphatic vasculature mediates cervical lymph node metastasis of brain tumors through VEGF-C/VEGFR3/eNOS signaling pathway

Purpose: The rediscovery of meningeal lymphatic vessels (MLVs) suggests a potential linkage between brain tumors and cervical lymph nodes (LNs). In this study, we investigated whether and how meningeal lymphatics regulate the metastasis of brain tumor cells. Experimental Design: We first examined the pattern of MLVs and LNs after tumor cells were injected into the cisterna magna (i.c.m.) or subdural space of mice. The meninges were isolated and analyzed by whole-mount immunofluorescence staining. LN metastasis was analyzed by detecting GFP+ tumor cells. Then we used inducible knock-out mice and AAV-VEGF-C delivery system to study the function of MLVs in mediating the metastasis of brain tumor cells. Finally, RNA-seq was used to determine the expression profiles of meningeal lymphatic endothelial cells (mLECs) in mice with or without tumors. The roles of the endothelial nitric oxide synthetase (eNOS) were evaluated both genetically and pharmacologically in vivo. Results: Meningeal lymphangiogenesis induced by GL261 and B16 tumors was correlated with dCLN swelling and metastasis. dCLN metastasis was suppressed in MLV-defective mice, while promoted in AAV-VEGF-C-treated mice. In addition, we found that VEGF-C/VEGFR3 signaling regulated both meningeal and tumor lymphangiogenesis. Tumor-associated mLECs have a distinct transcriptomic signature, which highly express eNOS. Furthermore, genetic or pharmacological inhibition of eNOS significantly suppressed meningeal lymphangiogenesis and dCLN metastasis. Conclusions: The meningeal lymphatic vasculature mediates the metastasis of brain tumors to cervical LNs, mainly through a VEGF-C/VEGFR3-eNOS signaling pathway, suggesting that MLV may be a potential therapeutic target for the extracranial metastasis of brain tumors.研究背景：脑膜淋巴管（meningeal lymphatic vessels, MLVs）的重新发现，提示脑肿瘤与颈淋巴结（cervical lymph nodes, LNs）之间存在潜在关联。本研究旨在探究脑膜淋巴管是否以及如何调控脑肿瘤细胞的转移进程。 实验设计：本研究首先向小鼠的小脑延髓池（cisterna magna, i.c.m.）或硬膜下腔注射肿瘤细胞，随后观察脑膜淋巴管与颈淋巴结的分布模式。分离脑膜组织并通过全铺片免疫荧光染色进行分析；通过检测GFP阳性肿瘤细胞以分析淋巴结转移情况。随后，我们利用诱导性基因敲除小鼠与AAV-VEGF-C递送系统，探究脑膜淋巴管在介导脑肿瘤细胞转移中的功能。最后，通过RNA测序（RNA-seq）分析荷瘤与未荷瘤小鼠的脑膜淋巴管内皮细胞（meningeal lymphatic endothelial cells, mLECs）的表达谱；并在体内通过遗传学与药理学手段，评估内皮型一氧化氮合酶（endothelial nitric oxide synthetase, eNOS）的作用。 研究结果：GL261与B16肿瘤诱导的脑膜淋巴管生成，与颈深淋巴结（deep cervical lymph nodes, dCLN）肿胀及转移呈正相关。颈深淋巴结转移在MLV缺陷型小鼠中受到抑制，而经AAV-VEGF-C处理的小鼠则出现转移增强。此外，我们发现VEGF-C/VEGFR3信号通路同时调控脑膜淋巴管生成与肿瘤淋巴管生成。肿瘤相关脑膜淋巴管内皮细胞具有独特的转录组特征，其eNOS表达水平显著升高。进一步研究表明，对eNOS进行遗传学或药理学抑制，可显著抑制脑膜淋巴管生成与颈深淋巴结转移。 研究结论：脑膜淋巴管系统介导脑肿瘤向颈淋巴结的颅外转移，其主要通过VEGF-C/VEGFR3-eNOS信号通路实现，提示MLVs可作为脑肿瘤颅外转移的潜在治疗靶点。