Consistent with the interaction in Bowes cells, both MT3-MMP and MT3E/A were co-precipitated with MT1-MMP (Fig. 5C). Interestingly, when 3-fold higher amount of cDNA for MT3- MMP than for MT1-MMP was transfected, MT1-MMP process- ing to 37–43 kDa fragments was increased also in COS-1 cells (Fig. 5C) suggesting that a dose-dependent competition may at least partially dictate the order of the cleavages between these MT- MMPs. Furthermore, even after equal transfection of the MT1- MMP and MT3-MMP cDNAs, MT3-MMP notably enhanced the processing of the fraction of MT1-MMP and MT1E/A exposed to cell surface biotinylation (Fig. 5D). Accordingly, MT3-MMP, but not the catalytically-inactive MT3E/A, decreased cell surface MT1-MMP in tandem with intracellular MT1-MMP accumula- tion as detected by immunofluorescence (Fig. 5E and S3). Since MT1-MMP surface levels can affect 3D growth and invasion pattern of the cells [37–39], we assessed next the impact of MT1-MMP and MT3-MMP on 3D cell growth and mode of invasion. As such, COS-1 cells transiently transfected to express MT1-MMP, MT3-MMP, catalytically-inactive MT3E/A, or MT1-MMP and MT3-MMP in combination, were embedded as single cells in either type I collagen or fibrin gels. During a 5-d culture period, mock-transfected cells formed small colonies which were not invasive in either matrix (Fig. 5F and G). As expected, MT3-MMP-expressing cells invaded only in fibrin (21.666.0 -fold increase in invasive area compared to mock cells, with a negligible increase in collagen invasion), whereas MT1-MMP-expressing cells generated both invasive colonies characterized by multicel- lular sprouts as well as clusters of cells invading in a single-cell fashion in either collagen or fibrin gels (in collagen, 9.960.4 –fold increase in invasive area as compared to mock-transfected cells, and in fibrin, 17.064.6 –fold increase compared to mock- transfected cells; Fig. 5F and G). Interestingly, the co-expression of MT3-MMP with MT1-MMP resulted in weakly sprouting or sphere-shaped colonies in collagen (Fig. 5F and G), while increasing fibrin-invasive activity of the cells (28.565.9 –fold increase compared to mock-transfected cells; Fig. 5F and G). By contrast, the expression of MT3E/A did not inhibit collagen invasion or induce fibrin invasion (Fig. 5F and G). These results indicate that MT3-MMP-dependent proteolytic activity promoted fibrin invasion, but resulted in decreased MT1-MMP surface levels and inhibition of collagen invasion. MT3-MMP enhances fibrin invasion and MT1-MMP- dependent nodular-type cell growth in 3D collagen To assess next the impact of the endogenous proteases on melanoma cell growth and pattern of invasion, WM852 cells were cultured within 3D collagen gel. The control cells grew as compact nodular cell colonies, while in 3D fibrin the cells formed invasive colonies with multicellular sprouts (Fig. 6A and B). As expected, 3D growth in collagen was blocked when MT1-MMP expression was silenced, a result consistent with previous reports (Fig. 6A; [37]). Remarkably, following MT3-MMP knockdown by siRNAs or by lentiviral shRNAs (Fig. S4A), the cell colonies in collagen became irregular in outline and displayed invasive activity, while the average size of the colonies was less affected (Fig. 6A and B, Fig. S4B). Concomitantly with increased collagen invasion, MT1- MMP levels on the cell surface were increased after MT3-MMP knockdown, as assessed by surface biotinylation and immunoflu- orescence (6C and D), while MT1-MMP mRNA remained unaffected (Fig. S4A). In contrast, efficient MT3-MMP silencing by shMT3-1 and shMT3-2 significantly reduced both colony size and invasive sprout formation in 3D fibrin (Fig. 6A and B). These changes correlated with the dose of MT3-MMP, as the modest MT3-MMP knockdown by shMT3-3 had only minor effects on MT3-MMP in Matrix-Dependent Melanoma Invasion the matrix composition dependent 3D growth and invasion (Fig. 6B and S4A). Likewise, stable MT3-MMP knockdown in the MT3-MMP expressing WM165 melanoma cells (Fig. S4C) led to increased cell elongation and invasive sprouting of cell colonies in collagen (Fig. 6B and S4D). Unexpectedly, MT3-MMP knockdown reduced the invasion of WM852 cells in fibrin even more efficiently than MT1-MMP knockdown (Fig. 6A; relative colony size 0.6060.04, and invasion 0.4160.1 for siMT1-MMP transfected cells; size 0.4660.04, and invasion 0.2060.02 for siMT3-MMP transfected cells, n = 3). MT3-MMP silencing also dramatically reduced the invasive growth of WM165 cell colonies in fibrin (Fig. 6B and S4D). In contrast, the invasion and growth of WM793 cells with higher MT1-MMP to MT3-MMP expression ratio was only slightly affected in 3D collagen by MT3-MMP knockdown (Fig. 1, S4E and S4F). The MT3-MMP knockdown also did not alter growth or invasion of Bowes cells in 3D collagen or fibrin, consistent with the low expression levels of endogenous MT3-MMP in these cells (Fig. S5A–D). To confirm the specific invasion regulating functions of MT3- MMP in collagen and fibrin matrices, we constructed two MT3- MMP rescue plasmids with silent mutations in the shMT3-2 targeting sequence (rescMT3-1 and rescMT3-2; Fig. 7A). Tran- sient transfection of these constructs in the MT3-MMP knock- down WM852 cells diminished collagen invasion and facilitated fibrin invasion (over 70% transfection efficiency in the shMT3-2 cells using TransIT-2020), indicating that the observed 3D cell phenotypes were indeed MT3-MMP-dependent (Fig. 7B and C). Likewise, the enhanced collagen invasion of the MT3-MMP knockdown cells was reversed to the level seen in scrambled shRNA expressing cells after transient expression of the rescue constructs in the knockdown cells (Fig. 7D). Taken together, these results suggest that MT3-MMP, essential for WM852 and WM165 melanoma cell invasion in fibrin, is a strong negative regulator of melanoma cell invasion within 3D collagen, where it induced adhesive nodular-type growth pattern. Discussion Unlike the strongly pro-invasive MT1-MMP that is widely overexpressed in the aggressive and metastatic tumors, MT3- MMP expression has been reported in only a few tumor types [11,19–21]. These include nodular melanoma, the most aggressive melanoma subtype characterized to date [24,25]. Consistent with the compact sphere-like growth pattern characteristic for nodular melanoma, we found that WM852 cells, originally derived from a nodular melanoma metastasis, and the other MT3-MMP expressing cell lines from advanced melanomas grew within 3D type I collagen gels as nodular-type adhesive cell colonies. These colonies displayed a limited ability for invasion of the surrounding collagen matrix as single cells or multicellular sprouts. Unexpect- edly, this cell phenotype was dependent on MT3-MMP as silencing its expression enhanced MT1-MMP-driven collagen invasion. Since the nodular-type growth phenotype was rescued when MT3-MMP was overexpressed in the knockdown cells, MT3-MMP is not only unable to support type I collagen-invasive activity [16,17], but also restricts the proinvasive activity of MT1- MMP in our 3D experimental model. By contrast, MT3-MMP – both alone and in combination with MT1-MMP – conferred cells with prominent fibrin-invasive activity. In addition to invasion, MT1-MMP is required for tumor growth within the confines of cross-linked matrices of type I collagen, a hydrogel whose structural and mechanical character- istics mimic those of most interstitial tissues in vivo [33,37]. The pattern of tumor cell growth in collagen can occur either invasively PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28325