Supplementary Figures S1-S9. MMP14 which is over-expressed in melanoma cell lines was not associated with patient survival (S1); Vasculature and morphology of human melanoma tumors (S2); MMP16 expression in WM852 cells and its correlation with tumor weight in melanoma xenografts (S3); No significant difference in the blood and lymphatic vessel densities was observed between shScr and shMMP16 tumors (S4); MMP16-silencing does not affect mouse collagen type I in xenografts (S5); Endogenous or exogenous MMP16 regulates collagen invasion and cell-cell contacts in WM852 and Bowes melanoma cells (S6); Endogenous or recombinant MMP16 regulates LEC spheroid intravasation of WM852 and Bowes melanoma cells (S7); Silencing of L1CAM cleaved by MMP16 rescued the transmigration across BECs but not the cell junction disassembly of shMMP16 cells (S8); L1CAM immunohistochemistry of xenografts and human samples (S9).
ARTICLE ABSTRACT
Lymphatic invasion and accumulation of continuous collagen bundles around tumor cells are associated with poor melanoma prognosis, but the underlying mechanisms and molecular determinants have remained unclear. We show here that a copy-number gain or overexpression of the membrane-type matrix metalloproteinase MMP16 (MT3-MMP) is associated with poor clinical outcome, collagen bundle assembly around tumor cell nests, and lymphatic invasion. In cultured WM852 melanoma cells derived from human melanoma metastasis, silencing of MMP16 resulted in cell-surface accumulation of the MMP16 substrate MMP14 (MT1-MMP) as well as L1CAM cell adhesion molecule, identified here as a novel MMP16 substrate. When limiting the activities of these trans-membrane protein substrates toward pericellular collagen degradation, cell junction disassembly, and blood endothelial transmigration, MMP16 supported nodular-type growth of adhesive collagen-surrounded melanoma cell nests, coincidentally steering cell collectives into lymphatic vessels. These results uncover a novel mechanism in melanoma pathogenesis, whereby restricted collagen infiltration and limited mesenchymal invasion are unexpectedly associated with the properties of the most aggressive tumors, revealing MMP16 as a putative indicator of adverse melanoma prognosis. Cancer Res; 75(10); 2083–94. ©2015 AACR.
