metastases to lung (,4-fold), small intestine (,6-fold) and in a single tissue sample of brain metastasis (,4.5-fold). In contrast, notable MT1-MMP mRNA levels were detected in several of the normal skin samples, and the slightly increased expression in both lymph node metastases and benign nevi were not statistically significant (Fig. 1B). Furthermore, four different metastatic melanoma cell lines tested (WM852, WM164, WM165 and WM239) as well as the Bowes, G361 and WM793 cells derived from non-metastatic primary melanoma all expressed MT1-MMP, whereas MT3- MMP was expressed in three cell lines originally isolated from metastatic melanomas (WM852, WM165 and WM239) and WM793 cell line isolated from advanced primary melanoma (Fig. 1C). To investigate the growth and invasion of these cells, they were implanted as single cells in cross-linked collagen matrix that typifies the surrounding ECM environment in collagen-rich skin. Under these conditions, MT1-MMP expression remained high or was even enhanced as compared to cells in monolayer culture or inside a 3D fibrin matrix (Fig. S1). Significant MT3- MMP expression remained unaltered and restricted to the same cells in the monolayer and 3D cultures (Fig. S1). During a 4-d culture within collagen, Bowes, G361 and WM164 cells all devoid of significant MT3-MMP expression grew and invaded efficiently in elongated morphology (Fig. 1D, upper panel). In contrast, the MT3-MMP expressing cells grew as either non-invasive sphere- shaped colonies or groups of rounded cells within collagen (Fig. 1D, lower panel), suggesting that MT3-MMP could have an effect on melanoma cell invasion. MT3-MMP expression is associated with rapid fibrin invasion and poor collagen invasion WM852 cells, isolated from the metastatic lesions of nodular melanoma, and Bowes cells, derived from early stage superficially spreading melanoma [29–31], were used to begin characterizing the functions of MT1-MMP and MT3-MMP in melanoma. These cells expressed MT1-MMP at comparable levels (Fig. 1C and 2A). By contrast, only WM852 cells expressed significant levels of MT3-MMP (Fig. 1C and 2A), while neither Bowes nor WM852 cells expressed MT2-MMP (Fig. 1C). To asses the relative invasive activities of the cells, they were cultured atop 3D gels of type I collagen, or fibrin, which deposition is enhanced at perivascular sites as well as tumor-host interfaces of melanoma [32]. During a 7-d culture period, WM852 cells efficiently invaded fibrin gels, but displayed only minor collagen-invasive activity (Fig. 2B and C). By contrast, Bowes cells readily invaded both collagen and fibrin matrices (Fig. 2B and C). In accordance with previously described functions for MT-MMPs in cell invasion [15,18,33], the ability of either Bowes or WM852 cells to infiltrate collagen or fibrin gels was blocked completely by the synthetic MMP-inhibitor, GM6001 (Fig. 2B and C). Taken together, these results suggest that MT3- MMP expression is compatible with rapid tumor cell invasion into fibrin, whereas in WM852 cells it was associated with the limited ability of MT1-MMP to support collagen invasion. MT3-MMP knockdown enhances WM852 melanoma cell invasion into collagen To define the effects of MT3-MMP on the melanoma cell invasion, we assessed the impact of silencing endogenous MT1- MMP or MT3-MMP expression by specific siRNAs in WM852 cells (over 80% silencing efficiencies by qPCR; Fig. 3A). The down-regulation of MT1-MMP or MT3-MMP protein was confirmed by immunoblotting (Fig. 3B and C). MT3-MMP, which was detected as the major 65 kDa protein in both control MT3-MMP in Matrix-Dependent Melanoma Invasion Figure 2. MT3-MMP expressing WM852 melanoma cells invade readily fibrin but have limited ability to invade collagen type I. (A) MT1- and MT3-MMP mRNA expression in WM852 cells from nodular melanoma and Bowes cells from superficially spreading melanoma were analyzed by Northern blotting. GAPDH mRNA levels served as an internal standard. (B) The cells were allowed to invade 3D fibrin and type I collagen for 7 d. Quantitative results are expressed as the number of invasive cells per microscopic field (n = 3). The number of invasive cells in fibrin was set to 100%. (C) Light micrographs of fibrin and collagen cross-sections visualize the invasion of WM852 and Bowes cells. GM6001 (10 mM) was added where indicated. White dotted lines mark the surface of the matrix. doi:10.1371/journal.pone.0028325.g002 cells expressing endogenous protease and cells transiently transfected with MT3-MMP cDNA, was markedly suppressed by MT3-MMP siRNAs (Fig. 3C). Likewise, endogenous MT3- MMP detected by surface biotinylation followed by MT3-MMP immunoprecipitation in control WM852 cells was barely detect- able after transfection with the most efficient MT3-MMP siRNA (Fig. 3D). When plated atop the collagen matrices, the control siRNA-transfected cells displayed only minor invasive potential during a 5-d assay, which was completely abrogated by MT1- MMP silencing as expected (Fig. 3E). By contrast, MT3-MMP knockdown unexpectedly enhanced collagen invasion (1.760.3 fold, n = 3, p = 0.05), whereas fibrin invasion was inhibited by silencing either MT1-MMP or MT3-MMP (Fig. 3E). PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28325