Fig 3. β1 integrins mediate invasion past the basement membrane.

(A) Representative cross sections of lumens in microvascular devices (scale bars = 10 μm) depicting the 3 distinct states in which a transmigrated tumor cell can be found relative to the endothelium after 6 hours (Position 1: directly adjacent to abluminal surface, Position 2: adjacent and elongated normal to vessel wall, Position 3: migrated away from the endothelium) (scale bars = 10 μm). Quantification of the number of fully transmigrated β1KD or control cells found in each “state” (50 cells analyzed per condition). (B) Quantification of the migration distance of extravasated tumor cells away from the endothelium. Distance is defined as the shortest length between the endothelium (at the point where transmigration occurred) and the nearest point on the transmigrated tumor cell body. Elongation length of the same transmigrated tumor cells is defined as the maximum length of the cell normal (perpendicular) to the vessel wall at the point of transmigration (n=50 transmigrated cells per condition, ***p<0.001, bars represent mean +/− standard deviation). (C) Representative immunofluorescence staining depicting the possible position of transmigrated tumor cells relative to the sub-endothelial laminin layer (LN: white, HUVEC LifeAct: green, MDA-MB 231: red, scale bars=10 μm). State 1: breached ECs but not laminin, State 2: fully transmigrated but no breaching of laminin, State 3: simultaneously breaching EC and laminin layer, State 4: fully breached EC and laminin layers. (D) Percentage of total fully transmigrated control or total β1-5 KD cells that are found in states 1 to 4 relative to the laminin layer (n=2 experiments, 4 devices per condition).