Membrane-Type-3 Matrix Metalloproteinase (MT3-MMP) Functions as a Matrix Composition-Dependent Effector of Melanoma Cell 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).