Fig. 9

MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a (top) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies (white arrow), disseminations around colonies (green arrow), or protrusions emanating from colonies (red arrows). Scale bars = 100 μm. (bottom) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal (green), DAPI (blue) and Alexa633 phalloidin (red) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony (c) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin (red arrow) and zsGreen (blue arrow) protrusions emerging from a colony. Scale bars = 100 μm