MT3-MMP in Matrix-Dependent Melanoma Invasion Figure 6. MT3-MMP promotes invasion in 3D fibrin and adhesive, nodular-type cell growth in 3D collagen. (A) WM852 cells transfected with the siRNAs against MT1-MMP (siMT1-MMP) and MT3-MMP (siMT3-MMP) were embedded in 3D collagen type I or fibrin gels as single cell suspension. Light micrographs show representative cell colonies that were formed after 5-d assay. Chart on the right shows the relative sizes of the cell colonies (n = 3). (B) Stable WM852 and WM165 cell pools expressing control scrambled shRNA (scr) or MT3-MMP targeting shRNAs (shMT3-1, shMT3-2 and shMT3-3) were allowed to grow inside 3D collagen or fibrin gels for 14 d. The fixed 3D cultures were stained with TRITC-phalloidin and photographed (see Fig. S4D for representative micrographs of WM165 cell colonies). Chart shows relative areas of invasive sprouts. (C) Cell surface MT1-MMP and MT3-MMP were detected by surface biotinylation of stable WM852 cells expressing the most potent shRNA against MT3-MMP (shMT3- 2) or scrambled shRNA (Scr), followed by immunoprecipitation using MT1-MMP and MT3-MMP antibodies. WM852 cells after transient transfection with MT3-MMP cDNA (MT3-MMP) were used as a control for the immunoprecipitation of endogenous MT3-MMP. (D) Epifluorescence micrographs visualize the increased MT1-MMP (green) on the surface of shMT3-2 expressing cells. The cells were counterstained with DAPI for nuclei (blue) and phalloidin for filamentous actin (red). doi:10.1371/journal.pone.0028325.g006 or as an expansive, adhesive mass, which may in part depend on MT1-MMP surface levels and activity [38]. In contrast, recombinant MT3-MMP is a poor type I collagenase [16] and unable to drive cell invasion through native polymerized collagen type I matrix [17,33], although MT3-MMP itself has also been implicated in the remodeling of 3D collagen [40,41]. In our experiments, Bowes cells derived from superficially spreading melanoma invaded rapidly in 3D collagen as single cells as well as multicellular sprouts in an MT1-MMP dependent manner. As such, MT1-MMP activity could support both local superficial spreading in epidermal and upper dermal tissues as well as invasion into deeper portion of the dermis. However, the collagen- invasive phenotype of Bowes cells was reduced following co- expression of catalytically-active MT3-MMP. These observations are not confined to melanoma cells as COS-1 cells expressing MT1-MMP alone were also highly invasive in 3D collagen, while cells expressing both MT1- and MT3-MMP exhibited only limited collagen invasive ability. By restricting single cell invasion and sprouting, MT3-MMP co-expression led to the MT1-MMP- dependent expansion of nodular cells colonies in 3D collagen. Given the inability of MT3-MMP to degrade native collagen type I directly and the importance of MT1-MMP homodimeriza- tion for its activity in collagen degradation [42], we considered the possibility that MT1-MMP/MT3-MMP heterodimers might form a complex that fails to support collagen adhesion or unwinding. However, although both catalytically active and inactive MT3- MMPs co-immunoprecipitated with MT1-MMP, the catalytically- inactive MT3-MMP mutant did not suppress MT1-MMP- mediated collagen invasion. Alternatively, MT3-MMP could suppress collagen invasion by cleaving MT1-MMP to inactive fragments, or indirectly by catalyzing the processing of other co- operative or invasion regulating proteins such as cell surface PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28325