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Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.

The receptors for at least two hematopoietic growth factors, namely the stem cell factor and colony-stimulating factor 1, belong to class III receptor tyrosine kinases. Here we describe cloning of a partial complementary DNA for FLT4, an additional member of this gene family from human leukemia cells. The FLT4 tyrosine kinase domain is 79% homologous with the previously cloned FLT1 (M. Shibuya et al., Oncogene, 5: 519-524, 1990) tyrosine kinase and maps to the chromosomal region 5q33-qter. We have found FLT4 expression in human placenta, lung, heart, and kidney, whereas the pancreas and brain appeared to contain very little if any FLT4 RNA. The results suggest that FLT4 functions in multiple adult tissues.