Preclinical and clinical studies positively correlate the expression of vascular endothelial growth factor (VEGF)-C in tumors and the incidence of lymph node metastases. However, how VEGF-C regulates individual steps in the transport of tumor cells from the primary tumor to the draining lymph nodes is poorly understood. Here, we image and quantify these steps in tumors growing in the tip of the mouse ear using intravital microscopy of the draining lymphatic vessels and lymph node, which receives spontaneously shed tumor cells. We show that VEGF-C overexpression in cancer cells induces hyperplasia in peritumor lymphatic vessels and increases the volumetric flow rate in lymphatics at the base of the ear by 40%. The increases in lymph flow rate and peritumor lymphatic surface area enhance the rate of tumor cell delivery to lymph nodes, leading to a 200-fold increase in cancer cell accumulation in the lymph node and a 4-fold increase in lymph node metastasis. In our model, VEGF-C overexpression does not confer any survival or growth advantage on cancer cells. We also show that an anti-VEGF receptor (VEGFR)-3 antibody reduces both lymphatic hyperplasia and the delivery of tumor cells to the draining lymph node, leading to a reduction in lymph node metastasis. However, this treatment is unable to prevent the growth of tumor cells already seeded in lymph nodes. Collectively, our results indicate that VEGF-C facilitates lymphatic metastasis by increasing the delivery of cancer cells to lymph nodes and therapies directed against VEGF-C/VEGFR-3 signaling target the initial steps of lymphatic metastasis. (Cancer Res 2006; 66(16): 8065-75)

Lymphatic metastasis is a critical determinant of cancer prognosis. Recently, several lymphangiogenic molecules such as vascular endothelial growth factor (VEGF)-C and VEGF-D were identified. However, the mechanistic understanding of lymphatic metastasis is still in infancy. Nitric oxide (NO) plays a crucial role in regulating blood vessel growth and function as well as lymphatic vessel function. NO synthase (NOS) expression correlates with lymphatic metastasis. However, causal relationship between NOS and lymphatic metastasis has not been documented. To this end, we first show that both VEGF receptor-2 and VEGF receptor-3 stimulation activate eNOS in lymphatic endothelial cells and that NO donors induce proliferation and/or survival of cultured lymphatic endothelial cells in a dose-dependent manner. We find that an NOS inhibitor, L-NMMA, blocked regeneration of lymphatic vessels. Using intravital microscopy that allows us to visualize the steps of lymphatic metastasis, we show that genetic deletion of eNOS as well as NOS blockade attenuates peritumor lymphatic hyperplasia of VEGF-C-overexpressing T241 fibrosarcomas and decreases the delivery of metastatic tumor cells to the draining lymph nodes. Genetic deletion of eNOS in the host also leads to a decrease in T241 tumor cell dissemination to the lymph nodes and macroscopic lymph node metastasis of B16F10 melanoma. These findings indicate that eNOS mediates VEGF-C-induced lymphangiogenesis and, consequently, plays a critical role in lymphatic metastasis. Our findings explain the correlation between NOS and lymphatic metastasis seen in a number of human tumors and open the door for potential therapies exploiting NO signaling to treat diseases of the lymphatic system.

Blood vessels are required for a tumor to grow and functional lymphatic vessels are required for it to disseminate to lymph nodes. In an attempt to eradicate both the primary tumor and its lymphatic metastasis, we targeted both blood and lymphatic vessels using two different tyrosine kinase inhibitors (TKIs): cediranib and vandetanib, which block vascular endothelial growth factor receptor (VEGFR)-2 and -3 in enzymatic assays. We found that although both cediranib and vandetanib slowed the growth rate of primary tumors and reduced blood vessel density, neither agent was able to prevent lymphatic metastasis when given after tumor cells had seeded the lymph node. However, when given during tumor growth, cediranib reduced the diameters of the draining lymphatic vessels, the number of tumor cells arriving in the draining lymph node, and the incidence of lymphatic metastasis. On the other hand, vandetanib had minimal effect on any of these variables, suggesting that vandetanib did not effectively block VEGFR-3 on lymphatic endothelial cells in our animal model. Collectively, these data indicate that the response of lymphatic vessels to a TKI can determine the incidence of lymphatic metastasis, independent of the effect of the TKI on blood vessels. [Mol Cancer Ther 2008;7(8):2272 -9]