Potent induction of invadopodia by HDFC. (A) Topography and orthogonal confocal views of HDFC, thin 3D collagen, globular collagen, and gelatin matrices labeled with Alexa Fluor 568F. Confocal z stacks of the matrices were deconvolved using blind deconvolution algorithm set to 10 iterations (AutoDeblur software; Media Cybernetics). (B) Confocal and orthogonal views of MDA-MB-231 carcinoma cell invading HDFC matrix. Invadopodia appear as yellow dots with colocalized actin and cortactin that penetrate into fluorescently labeled HDFC matrix. Inset (red rectangle) shows a magnified orthogonal view of the invadopodia. (C) Endogenous MT1-MMP accumulation at invadopodia of MDA-MB-231 cells on HDFC. Insets (red outlines) show magnified views of the invadopodia. (D) Quantification of total and degradative invadopodia in MDA-MB-231 cells invading HDFC and gelatin matrices. Total invadopodia were identified as aggregates of colocalized actin and cortactin at the cell membrane adherent to the matrix, and degradative invadopodia were actin/cortactin aggregates with colocalized MT1-MMP. Mean number of invadopodia per cell with 19–20 cells analyzed per condition. (E) Proteolytic degradation as indicated by black holes in a layer of fibronectin (FN) bound from serum to HDFC by MDA-MB-231 cells invading the matrix. Degradation is present at invadopodia (actin aggregate). Magnified views of the invadopodia and corresponding area of matrix degradation are shown in the red-framed insets. (F) Immunofluorescence labeling of 1/4 collagen type I fragment. Proteolytic cleavage of fibrillar collagen by MDA-MB-231 cells invading HDFC was readily detected in serum-free medium (gray–white localization). Insets (red boxes) show enlarged views of the invadopodia and localized collagen degradation. (G) Effect of MT1-MMP silencing in MDA-MB-231 cells on HDFC degradation identified by immunofluorescence labeling of cleaved 1/4 collagen type I fragment under cells. Cells were transfected with two different single duplex siRNAs specific to MT1-MMP (MT1-MMP si#1 and si#2). Controls were mock-transfected (not transfected [N/T]) or transfected with nonspecific control siRNA (N/S). Mean ± SEM from each of 100–106 cells from each of three independent experiments. ***, P < 0.0001. (H) Confocal images of MDA-MB-231 cells invading HDFC and 2D gelatin matrices. (I) Quantification of invadopodia in parental and c-Src–expressing MDA-MB-231 cells invading HDFC or gelatin matrices. Means ± SEM from each of 12–15 cells. wt, wild type. (J) Immunostaining of MDA-MB-231 carcinoma cells invading HDFC matrix in medium with or without serum. (K) Quantification of invadopodia formation in MDA-MB-231 cells invading HDFC or gelatin in media with or without serum. Means ± SEM of 80–130 cells from each of three independent experiments. **, P < 0.001. (L) Confocal images of primary HFFs invading HDFC matrix in the presence or absence of serum. (M) Quantification of results from L showing the number of invadopodia-forming HFFs plated on HDFC matrix in the presence or absence of serum in culture medium. Means ± SEM from each of 90–100 cells from each of three independent experiments. (N) Confocal images of the HFF invadopodia and associated HDFC degradation as detected by immunofluorescence of 1/4 collagen type I fragments. Bars: (A–C [main images], E and F [main images], H, I, and N) 10 µm; (C and F, insets) 3 µm; (E, insets) 2 µm; (L) 20 µm.