The mechanisms that cause tumors such as melanomas to metastasize into peripheral lymphatic capillaries are poorly defined. Non-mutually-exclusive mechanisms are lymphatic endothelial cell (LEC) chemotaxis and proliferation in response to tumor cells (chemotaxis-lymphangiogenesis hypothesis) or LECs may secrete chemotactic agents that attract cancer cells (chemotactic metastasis hypothesis). Using migration assays, we found evidence supporting both hypotheses. Conditioned medium (CM) from metastatic malignant melanoma (MMM) cell lines attracted LEC migration, consistent with the lymphangiogenesis hypothesis. Conversely, CM from mixed endothelial cells or LECs, but not blood endothelial cells, attracted MMM cells but not non-metastatic melanoma cells, consistent with the chemotactic metastasis hypothesis. MMM cell lines expressed CCR7 receptors for the lymphatic chemokine CCL21 and CCL21 neutralizing antibodies prevented MMM chemotaxis in vitro. To test for chemotactic metastasis in vivo tumor cells were xenotransplanted into nude mice B1 cm from an injected LEC depot. Two different MMM grew directionally towards the LECs, whereas non-metastatic melanomas did not. These observations support the hypothesis that MMM cells grow towards regions of high LEC density owing to chemotactic LEC secretions, including CCL21. This chemotactic metastasis may contribute to the close association between metastasizing tumor cells and peritumor lymphatic density and promote lymphatic invasion.

Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis -the growth of new vessels from preexisting vasculature -is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF xxx b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF xxx b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF xxx b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) Po0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF xxx b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

Objective-To determine whether chemotactic-metastasis, the preferential growth of melanomas towards areas of high lymphatic density, is CCL21/CCR7 dependent in vivo. Lymphatic endothelial cells (LECs) produce the chemokine CCL21. Metastatic melanoma cells express CCR7, its receptor, and exhibit chemotactic-metastasis, whereby metastatic cells recognise and grow towards areas of higher lymphatic density.Methods-We used two in vivo models of directional growth towards depots of LECs of melanoma cells over-expressing CCR7. Injected LEC were tracked by intravital fluorescence microscopy, and melanoma growth by bioluminescence.Results-Over-expression of the chemokine receptor CCR7 enables non-metastatic tumour cells to recognise and grow towards LECs (3.9 fold compared with control), but not blood endothelial cells (0.9 fold) in vitro and in vivo, in the absence of increased lymphatic clearance. Chemotactic metastasis was inhibited by a CCL21 neutralising antibody (4-17% of control). Furthermore, CCR7 expression in mouse B16 melanomas resulted in in-transit metastasis (50-100% of mice) that was less often seen with control tumours (0-50%) in vivo.Conclusion-These results suggest that recognition of LEC by tumours expressing receptors for lymphatic specific ligands contributes towards the identification and invasion of lymphatics by melanoma cells, and provides further evidence for a chemotactic metastasis model of tumour spread.

Cancer and dendritic cells recognize and migrate toward chemokines secreted from lymphatics and use this mechanism to invade the lymphatic system, and cancer cells metastasize through it. The lymphatic-secreted chemokine ligand CCL21 has been identified as a key regulatory molecule in the switch to a metastatic phenotype in melanoma and breast cancer cells. However, it is not known whether CCL21 inhibition is a potential therapeutic strategy for inhibition of metastasis. Here, we describe an engineered CCL21-soluble inhibitor, Chemotrap-1, which inhibits migration of metastatic melanoma cells in vivo. Two-hybrid, pull-down, and coimmunoprecipitation assays allowed us to identify a naturally occurring human zinc finger protein with CCL21 chemokine-binding properties. Further analyses revealed a short peptide (∼70 amino acids), with a predicted coiled-coil structure, which is sufficient for association with CCL21. This CCL21 chemokine-binding peptide was then fused to the Fc region of human IgG1 to generate Chemotrap-1, a human chemokine-binding Fc fusion protein. Surface plasmon resonance and chemotaxis assays showed that Chemotrap-1 binds CCL21 and inhibits CCL21-induced migration of melanoma cells in vitro with subnanomolar affinity. In addition, Chemotrap-1 blocked migration of melanoma cells toward lymphatic endothelial cells in vitro and in vivo. Finally, Chemotrap-1 strongly reduced lymphatic invasion, tracking, and metastasis of CCR7-expressing melanoma cells in vivo. Together, these results show that CCL21 chemokine inhibition by Chemotrap-1 is a potential therapeutic strategy for metastasis and provide further support for the hypothesis that lymphaticmediated metastasis is a chemokine-dependent process. Cancer Res; 70(20); 8138-48. ©2010 AACR.