Rac3 impacts invasion, dissemination, and metastasis. (A) The percentage of invading cells in the in vitro invasion assay for cells with control, Rac3, CIB1, GIT1, Arf6, Vav2, or βPIX siRNA. n ≥ 900 cells per condition; three independent experiments. (B) Representative image showing MCF10A cells transfected with Rac3 G12V mutant (left cell) or untransfected (right cell) plated on 405 nm fluorescent gelatin for 16 h. Zooms show invadopodia (cortactin colocalizing with spots of matrix degradation). (B, bottom) Quantification of the percentage of cells transfected with the indicated constructs that have at least one degrading invadopod (identified by the colocalization of cortactin, Tks5, and spots of matrix degradation). The Western blot shows Rac3 expression levels in lysates from the indicated cell lines. (C) Representative images of lung metastases in MTLn3 nontargeting (NT) control (top) or Rac3-knockout (KO) cells (bottom) expressing EGFP. (C, top) Normalized lung metastases in mice spontaneous metastasis assay with nontargeting control or Rac3-knockout MTLn3 cells. (C, middle) Number of circulating tumor cells in the blood of the same mice. n = 9 control or 16 Rac3-knockout tumor-bearing mice. A Mann-Whitney U test was used to determine significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P < 0.001. (C, bottom) Representative H&E-stained sections of lungs in control mice versus Rac3-knockout mice. (D) Model of the signaling axes centering on Rac3 addressed in this study. All error bars are SEM.