Anti-VEGF-A antibody, 2C3, inhibits the formation of the lymphatic vessels in the metastatic MDA-MB-231 tumor model

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Neutralization of the classic angiogenic factor, VEGF-A, has been long reported to inhibit angiogenesis as well as metastatic spread. Anti-metastatic properties of anti-VEGF-A antibodies are presumably mediated through a decrease in density of tumor blood-transporting vessels. However, breast cancer metastasis preferentially occurs through the invasion of the lymphatic vascular bed. Formation of the lymphatic vessels is thought to be primarily induced by two lymphangiogenic factors, VEGF-C and VEGF-D. However, it has been recently established that over-expression of VEGF-A also leads to the formation of lymphatic vessels strongly resembling malignant lymphatic vasculature. This observation prompted us to examine whether neutralization of VEGF-A would inhibit tumor lymphangiogenesis in the metastatic model of human breast cancer MDA-MB-231. To enable a sensitive detection of the metastatic spread this line has been stably transfected with the luciferase gene. Mice bearing orthotopic, luciferase-expressing 231 tumors were treated with a monoclonal anti-VEGF-A antibody, 2C3, which has no cross-reactivity with the VEGF-C protein. Tumors, lymph nodes and lungs were harvested from control and 2C3-treated mice following 10 weeks of treatment. Blood and lymphatic vessels densities were determined on frozen tumor sections using MECA 32 and Lyve-1 antibodies, respectively. Lymphatic vessels were additionally identified by an antibody detecting Prox-1 protein. The presence of metastatic cells in normal organs was determined immunohistochemically by using 6w/32 antibody against human HLA antigens and by luciferase activity measurements in tissue extracts. Results showed that anti-VEGF-A therapy significantly suppressed the formation of both blood-transporting vessels and lymphatic vessels by 70% and 85%, respectively. Incidence of lymph nodes and pulmonary metastases in 2C3-treated mice was reduced by 3.2 fold and 2.4 fold, respectively, as compared to mice receiving the control antibody. These findings strongly suggest that VEGF-A is involved in the formation of the tumor lymphatics. This regulation may be direct, through up-regulation of the expression of VEGF-C and its cognate receptors VEGF-R2 and VEGF-R3, or indirect, through an increase in vascular permeability. Accumulation of excessive interstitial fluid is a potent inducer of the lymphangiogenesis at inflammatory sites and is likely to play a role in the formation of malignant lymphatics. These data also suggest that anti-metastatic properties of anti-VEGF-A antibodies might be equally related to their anti-angiogenic as well as anti-lymphangiogenic effects.