title: Vascular endothelial growth factor C disrupts the endothelial lymphatic barrier to promote colorectal cancer invasion.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Tacconi
authors.affiliation: Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Correale
authors.affiliation: Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Gandelli
authors.affiliation: Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Spinelli
authors.affiliation: FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy; Department of Biosciences, School of Sciences, University of Milan, Milan, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Dejana
authors.affiliation: Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy; Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy. Electronic address: silvia.dalessio@humanitasresearch.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: D'Alessio
authors.affiliation: Humanitas Clinical and Research Center, IBD Center, Rozzano, Italy. Electronic address: silvio.danese@humanitas.it.
authors.forename: Silvio
authors.initials: S
authors.lastname: Danese
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D004729:Endothelium, Lymphatic / Q000378:metabolism* / Q000473:pathology; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D042583:Lymphangiogenesis; D008207:Lymphatic Metastasis; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D015398:Signal Transduction; D013997:Time Factors; D057705:Transendothelial and Transepithelial Migration; D014162:Transfection; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics / Q000378:metabolism*; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D042582:Vascular Endothelial Growth Factor C; D040321:Vascular Endothelial Growth Factor Receptor-3; D000099152:Cadherin 5; C467486:VEGFC protein, human; C581309:FLT4 protein, human
keywords: Colon Cancer; Mouse Model; Tumor Progression; VEGFC