RAB5A is necessary and sufficient to promote matrix-metalloprotease–dependent invasion into a 3D matrix. (A) RAB5A promotes organotypic cell invasion and remodels stromal collagen. (A, left) H&E-stained cross-sections of GFP-LifeAct control (Ctr) or GFP-LifeAct-RAB5A-(RAB5A) HeLa cells grown for 14 d on organotypic collagen matrix preconditioned with U937 macrophages, in the absence or presence of GM6001. Bar, 400 µm. (B) Quantification of the number of invading cells/condition, with respect to the number of invading RAB5A-HeLa cells, is the mean invasion index ± SEM (error bars) of four independent experiments (n = 45). *, P < 0.01. (C and D) Doxycycline-induced control- and RAB5AS34N-MDA-MB-231 (C) or RAB5AS34N-MCF10.DCIS.com (D) cells were assessed for their invasiveness by placing them on one side of a chamber slide in which 2.3 mg/ml acid extracted–only polymerized type I collagen gel and 100 ng/ml HGF were added. Examples of cell migration tracks are shown on the left (Videos 5 and 6). (C and D, right) Cell invasion is expressed as the mean forward invasion index ± SEM (error bars; n = 75 single cells/experiment repeated in four independent assays). *, P < 0.01; **, P < 0.005. (E) Doxycycline-inducible control (empty) or RAB5AS34N-MF10.DCIS.com cells were grown on a thick 1:1 Matrigel/type I collagen mixture and overlaid with diluted Matrigel (2 mg/ml). After 1 wk, cells were treated with doxycycline and/or HGF (20 ng/ml), or were mock treated. Bars, 400 µm. (E, right) The percentage of structures with invasive outgrowths was expressed as mean ± SEM (error bars) of four independent experiments; n = 35. **, P < 0.005.