Proteolytic-driven migration of HTERT-HDLECs cells into 3-D collagen gels. Spheroid and collagen fibers are visualized with a reflectance process (A-C,G-I). Dequenched (DQ) FITC collagen (green) becoming fluorescent after cleavage is used to visualize matrix degradation (D-F,J-L). (A-F) Insets show higher magnification of migrating cells. Under control conditions (A-F), a sprouting cell extends protrusion sensing the environment (A-B). Collagen degradation leads to a hole behind moving cell (C; see also supplemental Figure 2). Cleaved collagen appears in the cells (D-F) and at pseudopod–cell body interface (arrow; E). After MMP2 inhibition (G-L), no cell detached from the spheroid (G-I) and cleaved collagen is absent after MMP2 inhibition (J-L). On inhibitor treatment, cells extrude (white arrow) and retract (arrow head) pseudopods (K-L). Bars represent 50 μm in each panel. Live tissues were used in their culture medium and time-lapse imaging was performed using a Fluoview FV1000 confocal microscope (Olympus) at 37°C with Fluoview 3.0a acquisition software (Olympus) and a 60× NA 1.35 UPL SAPO lens (Olympus).