Visualization of cell cycle-dependent cancer cell mobilization and invasion.

<p>(A) Establishment and analyses of HCT116 colon cancer cells stably expressing Fucci. (<i>Upper</i>) The Fucci system enables monitoring of the cell cycle in live cells in real time. The nuclei of cells in the G1/G0, early S, and S/G2/M phases are labeled red, yellow, and green, respectively. (<i>Lower</i>) Snapshots of Fucci-expressing HCT116 cells. Scale bars represent 20 μm. (B) Intravital multiphoton imaging of Fucci-positive HCT116 cells inoculated into NOD/SCID mice. (<i>Left</i>) A representative image of Fucci-expressing HCT116 cells implanted in the cecum (green: Fucci-green (mAG), S/G2/M; red: Fucci-red (mKO2), G1; blue: collagen fibers (second harmonic generation (SHG) imaging)). Scale bars represent 75 μm. (<i>Right</i>) Quantification of the numbers of Fucci-green and -red HCT116 cells in different areas of inoculated tumors. Central and marginal zones were defined as areas further or closer than 75 μm from the border between the tumor and normal tissues, respectively. (C) Representative image at the edge of a Fucci-expressing HCT116 tumor mass. The entire area (<i>left</i>) and a time series (<i>right</i>) of magnified images (one per 400 s) of cancer cells invading the interstitium (green: Fucci-green (mAG), S/G2/M; red: Fucci-red (mKO2), G1; blue: collagen fibers (SHG imaging) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083629#pone.0083629.s015" target="_blank">Movie S1</a>). Actual images (<i>upper</i> panels) and cell trajectories (<i>lower</i> panels) are shown. Scale bars represent 100 μm (<i>left</i>) and 10 μm (<i>right</i>). (D) Representative image of extravasating Fucci-expressing HeLa cells. The entire area (<i>left</i>) and a time series (<i>right</i>) of magnified images of cancer cells extravasating from blood vessels (one per 12 min) (green: Fucci-green (mAG), S/G2/M; red: Fucci-red (mKO2), G1; blue: collagen fibers (SHG imaging) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083629#pone.0083629.s016" target="_blank">Movie S2</a>). Actual images (<i>upper</i> panels) and cell trajectories (<i>lower</i> panels) are shown. Scale bars represent 100 μm (<i>left</i>) and 10 μm (<i>right</i>). (E) Cellular motility in Fucci-green- and -red-positive cells was measured for 4 h (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083629#pone.0083629.s017" target="_blank">Movie S3</a>). (<i>Left</i>) Green and red spheres represent Fucci-green- and -red-positive cells, respectively, and yellow lines show the associated trajectories. Scale bars represent 100 μm. (<i>Right</i>) Mean tracking velocities of Fucci-green- and -red-positive cells. Data (n = 379 for Fucci green and n = 259 for Fucci red) were obtained from individual cells in three independent experiments. The velocities of the two groups were compared by Mann-Whitney <i>U</i>-test (p = 0.0191). The median and interquartile ranges for each group are overlaid on the dot plots.