Vascular guidance tunnels are physical spaces in 3D collagen matrices that are generated by MT1-MMP-mediated proteolysis during EC tube morphogenesis, which support EC motility and vascular tube remodeling. (A) ECs were seeded within a 3D collagen matrix and allowed to form vascular tube networks for 48 hours. (B) Vascular networks were rapidly regressed for 10 minutes with the phosphatase inhibitor calyculin A. Arrows indicate borders of vascular guidance tunnels; arrowheads indicate collapsed EC aggregates. Bar equals 25 μm. (C) Silicone oil was injected into vascular guidance tunnels from a single injection site using a 2-μm diameter micropipette (arrows). Cultures were allowed to form (A), were collapsed (B) with calyculin A, and were microinjected. A montage of photographs are shown from this extensive vascular guidance tunnel network present within the 3D collagen matrices (arrowheads) as a result of EC lumen and tube morphogenesis. Bar equals 20 μm. (D) Photographs from time-lapse images showing EC tube remodeling (denoted by white arrows) whereby ECs and tube structures are observed moving through vascular guidance tunnels (outlined by white arrowheads). Black arrows indicate process extension and tube movement into a vacated vascular guidance tunnel space (white arrowheads). Bar equals 25 μm.