Figure 1.
Protease inhibitors reduce tumor cell transendothelial migration.
(A) Model of extravasation stages. Three-dimensional confocal rotations or maximum projections of DiI labeled WM238 (red) at stages of extravasation from the top (B, D, F and H) and side (C, E, G, and I). BODIPY FL phallacidin (green) shows actin, and arrows indicate processes. Bars=10 μm. Note that confocal rotations or maximum projections allow for the fluorescence in all the focal planes to be seen in one image. Therefore, the ‘top view’ of the ‘attached’ stage allows for the visualization of actin in both the tumor cells and the endothelial cells in the same image, even if the latter cells are well below the top focal plane of the tumor cell. For reference please also compare with the ‘side view’ panel. Percent attached (●), seeking (■), migrating () and spreading (◆) cells at 1, 3, 5 and 24 hours for MDA-MB231 (J) and WM239 (K). (L) Percent spreading MDA-MB231 (grey bars) and WM239 (white bars) relative to the no inhibitor control (black bar). Proteinase inhibitors MPi (20 μM) and aprotinin (100 U) were used. A * Indicates a significant p-value compared to controls and ** indicates significant p-value relative to both controls and MPi or aprotinin alone. (M) The reduction in spreading cells with recombinant TIMP-1 or TIMP-2 (each at 2 μg/mL).
Figure 2.
The involvement of trimolecular complex components in the process of tumor cell transendothelial migration.
(A) Zymogram of serum-free, 24 hour conditioned media samples from EC, WM239 (WM) and MDA-MB231 (MDA). (B) Western blot of total cell lysates from MDA-MB231 (MDA), WM239 (WM) and EC (HMVEC) cultured in EC media stimulated with TNFα (10 ng/mL) show the expression of MT1-MMP. The arrowhead indicates the location of full length MT1-MMP. The dotted line represents an excised lane. (C) Tumor cells, with blue cell tracker (lighting up the entire cell cytoplasm and contours) and Hoechst (lighting up the nuclei) were incubated on EC (no blue cell tracker, but blue nuclei, which are not seen in this image) for 3 hours. Both tumor and EC cells were stained for actin (blue in both endothelial and tumor cells). Note that there are three ‘blue’ signals – the nuclei and actin of both tumor and endothelial cells, as well as the tumor cell cytoplasm. The upper panel shows 1 μm sections of a non-permeabilized seeking MDA-MB231 cell, the lower panel shows a non-permeabilized migrating WM239 cell labeled with MT1-MMP (green) and TIMP-2 (red). Arrows in the merged image indicate co-localization (yellow). Bars=10 μm. (D) Three reagents were tested to study trimolecular complex involvement during MDA-MB231 (gray bar) and WM239 (white bars) transendothelial migration. HxCD (1.6 mM) and the MT1-MMP mAb (MT1 Ab; 15 μg/mL) inhibited a 5-hour transendothelial migration assay. CBD (12.5 μM) stimulated melanoma extravasation at 3 hours. The relative control is the black bar. (E) Recombinant furin convertase (rFurin; 71 U/mL) stimulated transendothelial migration of MDA-MB231 (gray bar) and WM239 (white bars) cells at 3 hours. Increasing amounts of a furin inhibiting peptide decreased WM239 transendothelial migration. Increasing concentrations were 50, 100 and 200 mM. Error bars=mean±S.E.M. (F) Lamellipodia from MDA-MB231 (upper panels) and WM239 (lower panels) express furin in 1 μm confocal sections when cultured alone on Matrigel. Furin (green) localized to leading lamellipodial edges (arrowhead) with actin (red) (i and iv). Seeking Hoechst and blue cell tracker labeled (blue) MDA-MB231 (ii and iii)) and WM239 (v and vi) in 1 μm confocal sections. These images, taken on non-permeabilized transendothelial migration samples, show furin (red) at the cell surface and on blebs. Arrows described in the Results. Bars=10 μm.
Figure 3.
The association of cell adhesion molecules and MMPs during tumor cell transendothelial migration.
(A) Blue cell tracker and Hoechst labeled tumor cells (bright blue cells and nuclei) incubated on EC for 3 hours. Both tumor and EC were stained for actin (pale blue). 1 μm sections through a seeking MDA-MB231 cell (upper panel) and a seeking WM239 cell (lower panel) labeled for CD44 (green) and MMP-9 (red) are shown. Arrowhead and arrow point to CD44 in heterotypic tumor cell-endothelial cell contacts or on the cell surface, respectively. Arrowheads also indicate little cell surface, or abundant MMP-9 staining of blebs (merged images). Note that the blebs also show circumferential actin staining in blue. Bars=10 μm. (B) Inhibitory MMP-9 mAb (Ab; 15 μg/mL) used to block MDA-MB231 (gray bars) and WM239 (white bars) extravasation. (C) 1 μm confocal sections of a blue cell tracker and Hoechst labeled WM 239 cell (bright blue cells and nuclei) incubated on EC for 3 hours and undergoing the spreading stage of transendothelial migration. Experiments were subjected to labeling of actin in both tumor and EC cells (pale blue). Labeling of αvβ3 (green) and MMP-2 (red). Arrows indicate spectral co-localization (yellow) in merged image. Bars=10 μm. (D) An αvβ3-specific blocking cyclic peptide (RGD; 90 μM) reduced WM239 melanoma extravasation compared with a non-blocking cyclic RAD peptide (RAD; 90 μM) as shown in the white bars. The black bar is the relative control with no peptide. (E) Western blot of total cell lysates from MDA-MB231 (MDA), WM239 (WM) and EC cultured in EC media with TNFα (10 ng/mL) show the expression of the β3 integrin subunit. Tubulin is a protein loading control. The dotted line represents an excised lane.
Figure 4.
Matrix digestion occurs during transendothelial migration.
A three-dimensional optical rotation of a DiI labeled MDA-MB231 cell (red) from the top (A) and side (B) as it digested and internalized extracellular matrix (green). Substrate digestion observed during extravasation (C-F). An orange cell tracker labeled WM239 (red; D and F) imaged by epifluorescence. Digestion of fluorogenic substrate is seen around cell contours of migrating and spreading cells (green; C and E). (G) A blue cell tracker and Hoechst labeled WM 239 cell (bright blue cell and nucleus) incubated on EC for 3 hours and undergoing the spreading stage of transendothelial migration. (H) Digestion of FITC-labeled fluorogenic substrate is seen around cell contours (green). Bar=10 μm (I) MDA-MB231 breast cancer cells (gray bar; MDA) and WM 239 melanoma cells (white bar; WM) digest FITC gelatin as measured by a fluorescence plate reader in serum-free conditions relative to a media only control (black bar). Error bars=mean±S.D. (J) 1μm confocal section of a Hoechst and blue cell tracker (blue) tagged WM239 cell with surface MMP-2 (red) and corresponding matrix digestion as revealed by the fluorescence signal given off from the fluorogenic substrate incorporated in the Matrigel (green). Native substrate prior to enzymatic digestion, as well as without cells provides no detectable confocal fluorescence signature due to quenching of the FITC label on the substrate. Bar=10 μm.
Figure 5.
The migratory stage is most dependent on protease activity.
Patches of FITC-matrix digestion were scored for both MDA-MB231 (A) and WM239 (B). “-“ = no digestion around cell contours, “+” = single patch of digestion, “++” = patchy digestion and “+++” = digestion around the entire cell contour. The cartoon panel on the right represents the scoring scheme. Error bars=mean±S.E.M.. Percent WM239 cells at each stage of extravasation upon treatment with (C) TIMP-1 and (D) TIMP-2. Percent WM239 cells at each stage of extravasation upon treatment with (E) HxCD or (F) RDG peptide. Percent MDA-MB231 cells at each stage of extravasation upon treatment with the (G) MT1-MMP blocking antibody and (H) CBD. In graphs C-H, treated cells (black bars) are compared to untreated controls (white bars). Error bars=mean±S.E.M..
Figure 6.
Model depicting the redistribution, complex formation and activation of MMPs during tumor cell transendothelial migration.
This model indicates the sites of action of inhibitors, cell surface anchoring proteins and activators during the process of transendothelial migration in this study.