Cdc42 regulates PC3 cancer cell spreading on lung vascular ECs, retention in the lungs, and metastasis formation in vivo. CFP-PC3 cells transfected with Cdc42 siRNA and YFP-PC3 cells transfected with control siRNA were coinjected into mice. (A) 3D reconstructions of representative confocal 3D stacks of cells in the lung vasculature. Arrows indicate cancer cell protrusions along the vessels. Bar, 20 µm. (B) Quantification of spread cells in the lung vasculature. At least 50 single cells per condition were analyzed from at least three independent experiments. Data are expressed as the percentage of the total number of cells analyzed. (C) Number of cells in the lungs. 60 random independent fields were analyzed and the cell number was scored from three independent experiments. Data are expressed as the percentage of total cells analyzed. Values are means ± SEM (error bars); *, P < 0.05. (D) Effects of Cdc42 depletion on experimental metastasis formation. Mice were injected intravenously with PC3 cells transfected with control siRNA or Cdc42 siRNA. Quantification of metastatic foci on the lung surface (8 mice for control, 6 mice for Cdc42 siRNA). Values are means ± SEM (error bars); **, P < 0.01. (E) Representative images of lungs from mice injected with Cdc42-depleted cells (right) or control siRNA-transfected cells (left). Boxed regions are shown at higher magnification (bottom). Bars, 10 mm.