The mechanisms of tumor metastasis to the sentinel lymph nodes are poorly understood. Vascular endothelial growth factor (VEGF)-A plays a principle role in tumor progression and angiogenesis; however, its role in tumor-associated lymphangiogenesis and lymphatic metastasis has remained unclear. We created transgenic mice that overexpress VEGF-A and green fluorescent protein specifically in the skin, and subjected them to a standard chemically-induced skin carcinogenesis regimen. We found that VEGF-A not only strongly promotes multistep skin carcinogenesis, but also induces active proliferation of VEGF receptor-2–expressing tumor-associated lymphatic vessels as well as tumor metastasis to the sentinel and distant lymph nodes. The lymphangiogenic activity of VEGF-A–expressing tumor cells was maintained within metastasis-containing lymph nodes. The most surprising finding of our study was that even before metastasizing, VEGF-A–overexpressing primary tumors induced sentinel lymph node lymphangiogenesis. This suggests that primary tumors might begin preparing their future metastatic site by producing lymphangiogenic factors that mediate their efficient transport to sentinel lymph nodes. This newly identified mechanism of inducing lymph node lymphangiogenesis likely contributes to tumor metastasis, and therefore, represents a new therapeutic target for advanced cancer and/or for the prevention of metastasis.

V.Schacht and M.I.Ramirez contributed equally to this workWithin the vascular system, the mucin-type transmembrane glycoprotein T1a/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic-speci®c homeobox gene Prox1. In this study, we examined the role of T1a/podoplanin in vascular development and the effects of gene disruption in mice. T1a/podoplanin is ®rst expressed at around E11.0 in Prox1-positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1a/podoplanin ±/± mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1a/podoplanin is also expressed in the basal epidermis of newborn wild-type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1a/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA-mediated inhibition of T1a/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1a/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

Early during development, one of the first indications that lymphangiogenesis has begun is the polarized expression of the homeobox gene Prox1 in a subpopulation of venous endothelial cells. It has been shown previously that Prox1 expression in the cardinal vein promotes and maintains the budding of endothelial cells that will form the lymphatic vascular system. Prox1-deficient mice are devoid of lymphatic vasculature, and in these animals endothelial cells fail to acquire the lymphatic phenotype; instead, they remain as blood vascular endothelium. To investigate whether Prox1 is sufficient to induce a lymphatic fate in blood vascular endothelium, Prox1 cDNA was ectopically expressed by adenoviral gene transfer in primary human blood vascular endothelial cells and by transient plasmid cDNA transfection in immortalized microvascular endothelial cells. Transcriptional profiling combined with quantitative real-time reverse transcriptionpolymerase chain reaction and Western blotting analyses revealed that Prox1 expression up-regulated the lymphatic endothelial cell markers podoplanin and vascular endothelial growth factor receptor-3. Conversely, genes such as laminin, vascular endothelial growth factor-C, neuropilin-1, and intercellular adhesion molecule-1, whose expression has been associated with the blood vascular endothelial cell phenotype, were down-regulated. These results were confirmed by the use of specific antibodies against some of these markers in sections of embryonic and adult tissues. These findings validate our previous proposal that Prox1 is a key player in the molecular pathway leading to the formation of lymphatic vasculature and identify Prox1 as a master switch in the program specifying lymphatic endothelial cell fate. That a single gene product was sufficient to re-program the blood vascular endothelium toward a lymphatic phenotype corroborates the close relationship between these two vascular systems and also suggests that during evolution, the lymphatic vasculature originated from the blood vasculature by the additional expression of only a few gene products such as Prox1.