Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy.
FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy; Department of Biosciences, School of Sciences, University of Milan, Milan, Italy.
Gastroenterology. 2015 Jun;148(7):1438-51.e8. doi: 10.1053/j.gastro.2015.03.005. Epub 2015 Mar 6.
BACKGROUND & AIMS: Colorectal cancer (CRC) is highly metastatic. Metastases spread directly into local tissue or invade distant organs via blood and lymphatic vessels, but the role of lymphangiogenesis in CRC progression has not been determined. Lymphangiogenesis is induced via vascular endothelial growth factor C (VEGFC) activation of its receptor, VEGFR3; high levels of VEGFC have been measured in colorectal tumors undergoing lymphangiogenesis and correlated with metastasis. We investigated VEGFC signaling and lymphatic barriers in human tumor tissues and mice with orthotopic colorectal tumors.
We performed immunohistochemical, immunoblot, and real-time polymerase chain reaction analyses of colorectal tumor specimens collected from patients; healthy intestinal tissues collected during operations of patients without CRC were used as controls. CT26 CRC cells were injected into the distal posterior rectum of BALB/c-nude mice. Mice were given injections of an antibody against VEGFR3 or an adenovirus encoding human VEGFC before orthotopic tumors and metastases formed. Lymph node, lung, and liver tissues were collected and evaluated by flow cytometry. We measured expression of vascular endothelial cadherin (CDH5) on lymphatic vessels in mice and in human intestinal lymphatic endothelial cells.
Levels of podoplanin (a marker of lymphatic vessels), VEGFC, and VEGFR3 were increased in colorectal tumor tissues, compared with controls. Mice that expressed VEGFC from the adenoviral vector had increased lymphatic vessel density and more metastases in lymph nodes, lungs, and livers, compared with control mice. Anti-VEGFR3 antibody reduced numbers of lymphatic vessels in colons and prevented metastasis. Expression of VEGFC compromised the lymphatic endothelial barrier in mice and endothelial cells, reducing expression of CDH5, increasing permeability, and increasing trans-endothelial migration by CRC cells. Opposite effects were observed in mice and cells when VEGFR3 was blocked.
VEGFC signaling via VEGFR3 promotes lymphangiogenesis and metastasis by orthotopic colorectal tumors in mice and reduces lymphatic endothelial barrier integrity. Levels of VEGFC and markers of lymphatic vessels are increased in CRC tissues from patients, compared with healthy intestine. Strategies to block VEGFR3 might be developed to prevent CRC metastasis in patients.
结直肠癌（CRC）具有高度转移性。转移可通过血液和淋巴管直接扩散到局部组织或侵犯远处器官，但淋巴管生成在 CRC 进展中的作用尚未确定。淋巴管生成是通过血管内皮生长因子 C（VEGFC）激活其受体 VEGFR3 诱导的；在经历淋巴管生成的结直肠肿瘤中测量到高水平的 VEGFC，并与转移相关。我们研究了人肿瘤组织和荷直肠肿瘤的小鼠中的 VEGFC 信号和淋巴管屏障。
我们对来自患者的结直肠肿瘤标本进行了免疫组织化学、免疫印迹和实时聚合酶链反应分析；来自无 CRC 患者手术期间的健康肠组织被用作对照。将 CT26 CRC 细胞注入 BALB/c-裸鼠的直肠远端后部。在形成原位肿瘤和转移之前，给小鼠注射抗 VEGFR3 抗体或编码人 VEGFC 的腺病毒。收集淋巴结、肺和肝组织并通过流式细胞术进行评估。我们测量了小鼠和人肠淋巴管内皮细胞中淋巴管上血管内皮钙黏蛋白（CDH5）的表达。
与对照组相比，结直肠肿瘤组织中 podoplanin（淋巴管标志物）、VEGFC 和 VEGFR3 的水平增加。与对照小鼠相比，表达来自腺病毒载体的 VEGFC 的小鼠淋巴结、肺和肝脏中的淋巴管密度增加，转移更多。抗 VEGFR3 抗体减少了结肠中的淋巴管数量并阻止了转移。VEGFC 的表达削弱了小鼠和内皮细胞的淋巴管内皮屏障，降低了 CDH5 的表达，增加了通透性，并增加了 CRC 细胞的跨内皮迁移。在阻断 VEGFR3 时，在小鼠和细胞中观察到相反的效果。
VEGFC 通过 VEGFR3 信号促进小鼠原位结直肠肿瘤的淋巴管生成和转移，并降低淋巴管内皮屏障的完整性。与健康肠道相比，患者的 CRC 组织中 VEGFC 水平和淋巴管标志物水平增加。阻断 VEGFR3 的策略可能被开发用于预防患者的 CRC 转移。
