Fig. S1. ML7 and Y27632 alter MDAwt and MDAtwist cell sphericity. Average shape index (sphericity, round cell=1.0) for mock-treated or drug-treated MDAtwist and MDAwt cells (24 h.p.i.).

Movie 1. MDAwt tumor cell (horizontally migrating cell, blue, CFP) migrating within the ISV lumen (green, GFP). The tumor cell is migrating in the direction of the blood flow. Another MDAwt cell (vertically migrating cell, blue, CFP) can be briefly seen migrating in the connecting vessel lumen in the left part of the field in the direction of the blood flow as well. Note that both of the cells are rounded as they move inside the vessel lumen. Bright green areas within the vasculature are endothelial cell nuclei that show high expression levels of the EGFP. 60× objective, 18 frames, 1 frame/5 minutes.

Movie 2. Isosurface rendering of the MDAwt tumor cell (white) migrating within the vessel lumen from Movie 1 (horizontally migrated MDAwt cell was used for Isosurface rendering, frames 5-13 of Movie 1) using Imaris Software. Note cell shape change and small rounded protrusion formation at the leading cell edge (red arrows). 60× objective, 8 frames, 1 frame/5 minutes.

Movie 3. RFP-expressing MDAwt cell (red) migrating within the ISV lumen (green, GFP). During the imaging period the tumor cell is first seen migrating against blood flow (frames 1-23). It is then pushed backwards by normal blood flow (frame 24) and then again migrates against the blood flow (frames 24-29). Note the increased movement of endothelial cell nuclei around the vessel arrested tumor cell compared with endothelial nuclei in a normal ISV (right) without a arrested tumor cell. Another MDAwt cell can be seen being carried by blood flow in the lower part of the field. Red fluorescent objects in the lower part of the imaging field are auto-fluorescent zebrafish cells. 60× objective, 29 frames, 1 frame/5 minutes.

Movie 4. RFP expressing MDAtwist tumor cell (red) migrating within the ISV lumen (green, GFP) against the direction of blood flow. Note that MDAtwist cells display dramatically increased cellular membrane protrusion activity and rapid cell shape change when compared with control MDAwt cells (Movie 1). Light-red fluorescent objects in the right part of the imaging field are auto fluorescent Zebrafish cells. Another MDAtwist cell can be seen carried by the blood flow in the far right section of the imaging field. 60× objective, 20 frames, 1 frame/5 minutes.

Movie 5. Imaris isosurface rendering of the intravascular migrating MDAtwist tumor cell (red) and ISV (green) from Movie 4 (frames 6-14). Note that the migrating MDAtwist cell is extending several large rounded protrusions and makes dramatic shape changes as it navigates the narrow openings and branch points of the small ISV. The imaging field is rotated 90° clockwise in relation to Movie 4. 60× objective, 8 frames, 1 frame/5 minutes.

Movie 6. RFP-expressing MDAβ1KO tumor cell (red) arrested in an ISV (green, GFP). Note that the MDAβ1KO cell is completely immotile and does not change shape. Red fluorescent objects in the lower part of the imaging field are autofluorescent zebrafish cells. 60× objective, 30 frames, 1 frame/5 minutes.

Movie 7. 10 m Sepharose bead (red, Texas red) arrested in ISV (green, GFP). Note that the Sepharose bead is not in close contact with vessel wall, is completely immotile, and does not induce endothelial nuclei movement, or thickening of the vessel wall. 60× objective, 20 frames, 1 frame/5 minutes.

Movie 8. CFP transfected MDAwt (blue) and RFP transfected MDAtwist (red) tumor cells arrested in ISVs (green, GFP). Note that the MDAtwist cell extravasates approximately 2.5 hours after the start of time-lapse imaging (frame 10) whereas the MDAwt cell does not. Also, note that there is no apparent damage to the surrounding endothelium as the cell penetrates the vascular wall. 20× objective, 19 frames, 1 frame/15 minutes.

Movie 9. Imaris isosurface rendering of the MDAtwist tumor cell extravasating from Movie 8. Note that the ISV vessel wall is being severely distorted by the MDAtwist, cell but no vessel-wall fragmentation is observed as the tumor cell extravasates through the vessel wall. 20× objective, 19 frames, 1 frame/15 minutes.

Movie 10. Imaris isosurface rendering of the MDAwt tumor cell (blue) and ISV (green) from Movie 8. Note that the MDAwt cell is arrested in the small vessel, is significantly less protrusive than the MDAtwist cell, and does not penetrate the vessel wall. 20× objective, 19 frames, 1 frame/15 minutes.