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Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor
Yulong He1, Ken-Ichi Kozaki, Terhi Karpanen
1Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Finland.
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.
Insights
Inhibiting vascular endothelial growth factor receptor 3 (VEGFR-3) signaling suppressed tumor lymphangiogenesis and lymph node metastasis. However, lung metastasis was not affected, suggesting other factors regulate cancer spread.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Metastasis Research
Background:
- Vascular endothelial growth factor C (VEGF-C) drives tumor lymphangiogenesis and metastasis.
- VEGF-C interacts with VEGF receptor 3 (VEGFR-3) to promote lymphatic vessel formation.
- Targeting VEGFR-3 signaling is a potential strategy to inhibit tumor spread.
Purpose of the Study:
- To investigate if inhibiting VEGFR-3 signaling reduces tumor lymphangiogenesis and metastasis.
- To determine the role of VEGFR-3 in lung cancer metastasis to lymph nodes and lungs.
Main Methods:
- Used human lung cancer cell lines (NCI-H460-LNM35 and NCI-H460-N15) with varying metastatic capacities.
- Engineered cell lines to secrete VEGF-C or VEGFR-3-Ig (VEGFR-3 inhibitor).
- Utilized xenograft mouse models and adenovirus-mediated gene delivery to assess tumor growth, lymphangiogenesis, and metastasis.
Main Results:
- LNM35 cells showed higher VEGF-C expression and metastatic potential than N15 cells.
- VEGFR-3 inhibition significantly reduced intratumoral lymphatic vessels and lymph node metastases.
- Lung metastasis occurred regardless of VEGFR-3 signaling inhibition, indicating alternative spread mechanisms.
Conclusions:
- VEGFR-3 signaling inhibition effectively suppresses tumor lymphangiogenesis and regional lymph node metastasis.
- Factors beyond VEGF-C regulate lymph node metastasis.
- VEGFR-3 inhibition does not prevent lung metastasis, highlighting the complexity of cancer spread.