RAB5A is sufficient to promote intratumoral cell motility and distant dissemination. (A) GFP-LifeAct control (Ctr) or GFP-LifeAct-RAB5A-(RAB5A) HeLa cells were injected into the mammary fat pads of NSG mice. Metastases were analyzed 4 wk after removal of primary tumor. (A, left) Ipsilateral metastasis (arrowheads) of control or RAB5A-HeLa tumors. (A, right) Quantitation of number and volume (mean ± SEM [error bars]; n = 10 mice/group) of disseminated tumors nodules. **, P < 0.005. (B, left) H&E and anti-GFP staining of FFPE lung tissue sections. Magnified boxed regions show metastasis. (B, right) The size of metastatic nodules is the mean tumor area/total lung area ± SEM (error bars; n = 9 mice/group repeated in two independent experiments). **, P < 0.005. (C) Tumors from GFP-LifeAct control (Ctr) or GFP-LifeAct-RAB5A-(RAB5A)-HeLa cells injected into the mammary fat pads of NSG mice were analyzed by two-photon microscopy. Green, GFP-LifeAct; gray, collagen structure (SHG). (D) Tumor invasive front visualized: in the top panels by projecting ∼40 serial z sections (green, GFP-LifeAct; gray, collagen structure [SHG]; or, in bottom panels, by IHC. (E) Intratumoral motion analysis of control and RAB5A-HeLa cells was obtained by overlaying 10 differentially colored, consecutive frames of time-lapse recording (Videos 1 and 2; left). Coloring indicates motile cells. The percentage of motility events/field of view/tumor is the mean ± SEM (error bars; n = 45). Bars: (B, left) 2 mm; (B, right) 0.2 mm; (C) 400 µm; (D, top) 200 µm; (D, bottom) 10 µm; (E) 80 µm.