Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling.
BACKGROUND
Vascular endothelial growth factor C (VEGF-C) stimulates tumor lymphangiogenesis (i.e., formation of lymphatic vessels) and metastasis to regional lymph nodes by interacting with VEGF receptor 3 (VEGFR-3). We sought to determine whether inhibiting VEGFR-3 signaling, and thus tumor lymphangiogenesis, would inhibit tumor metastasis.


METHODS
We used the highly metastatic human lung cancer cell line NCI-H460-LNM35 (LNM35) and its parental line NCI-H460-N15 (N15) with low metastatic capacity. We inserted genes by transfection and established a stable N15 cell line secreting VEGF-C and a LNM35 cell line secreting the soluble fusion protein VEGF receptor 3-immunoglobulin (VEGFR-3-Ig, which binds VEGF-C and inhibits VEGFR-3 signaling). Control lines were transfected with mock vectors. Tumor cells were implanted subcutaneously into severe combined immunodeficient mice (n = 6 in each group), and tumors and metastases were examined 6 weeks later. In another approach, recombinant adenoviruses expressing VEGFR-3-Ig (AdR3-Ig) or beta-galactosidase (AdLacZ) were injected intravenously into LNM35 tumor-bearing mice (n = 14 and 7, respectively).


RESULTS
LNM35 cells expressed higher levels of VEGF-C RNA and protein than did N15 cells. Xenograft mock vector-transfected LNM35 tumors showed more intratumoral lymphatic vessels (15.3 vessels per grid; 95% confidence interval [CI] = 13.3 to 17.4) and more metastases in draining lymph nodes (12 of 12) than VEGFR-3-Ig-transfected LNM35 tumors (4.1 vessels per grid; 95% CI = 3.4 to 4.7; P<.001, two-sided t test; and four lymph nodes with metastases of 12 lymph nodes examined). Lymph node metastasis was also inhibited in AdR3-Ig-treated mice (AdR3-Ig = 0 of 28 lymph nodes; AdLacZ = 11 of 14 lymph nodes). However, metastasis to the lungs occurred in all mice, suggesting that LNM35 cells can also spread via other mechanisms. N15 tumors overexpressing VEGF-C contained more lymphatic vessels than vector-transfected tumors but did not have increased metastatic ability.


CONCLUSIONS
Lymph node metastasis appears to be regulated by additional factors besides VEGF-C. Inhibition of VEGFR-3 signaling can suppress tumor lymphangiogenesis and metastasis to regional lymph nodes but not to lungs.
He
Yulong
Y
Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Finland.
Kozaki
Ken-Ichi
K
Karpanen
Terhi
T
Koshikawa
Katsumi
K
Yla-Herttuala
Seppo
S
Takahashi
Takashi
T
Alitalo
Kari
K
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002455:Cell Division; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000098:blood supply* / Q000473:pathology / Q000556:secondary*; D008207:Lymphatic Metastasis / Q000473:pathology / Q000517:prevention & control*; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D042582:Vascular Endothelial Growth Factor C; D042643:Vascular Endothelial Growth Factor D; D040321:Vascular Endothelial Growth Factor Receptor-3
D000906:Antibodies; D016228:Endothelial Growth Factors; D017978:Receptors, Growth Factor; D042582:Vascular Endothelial Growth Factor C; D042643:Vascular Endothelial Growth Factor D; D020794:Receptor Protein-Tyrosine Kinases; D040321:Vascular Endothelial Growth Factor Receptor-3