Fig. 1.

Dynamic reorganization of collectively invading breast cancer cells in vitro and ex vivo. (A) A representative MDA-MB-231 spheroid expressing the CycleTrak nuclear marker (green/yellow, different colors indicate different cell cycle phases) is invading upward into 4.5-mg/mL collagen with collective strands. (B) Cells dynamically reorganize their relative positions within a typical strand (boxed region from A) during invasion, resulting in leader cell turnovers (asterisks; frames under white lines are enlarged on the Right to highlight leader turnover). Note that the invasion is interrupted when the original leader cells (black arrowheads) pause or move backward before the emergence of the new leaders (red arrowheads). (C) Leader–follower switching during spheroid invasion is more frequent in high-density collagen than in lower-density collagen (n > 50 for each condition; P < 0.0001 from a logarithmic-rank test for trend; shades represent standard error (s.e.); complete leader cell lifetime data are available in Dataset S1). (D) A representative mouse mammary tumor virus–polyomavirus middle T-antigen (MMTV-PyMT) mouse tumor organoid invading into 4.5-mg/mL collagen matrix. (E) A representative strand (boxed region from D) exhibits intermittent short-period pauses or retractions during forward invasion, many of them correlate with leader cell turnover events (as indicated with asterisks; black arrowheads, old leader; red arrowheads, new leader; frames under the white lines are enlarged on the Right to highlight leader turnover). (F) Leader cell lifetime during organoid invasion decreases with increasing collagen density (n > 45 for each condition; P = 0.05 from a logarithmic-rank test for trend; shades represent s.e.). [Scale bar, 50 µm (A and B, Left and D and E, Left), 25 µm (B, Right and E, Right).]