SDF1 chemotaxis differentially regulates protrusion dynamics and polarization in epithelial-like and mesenchymal NC clusters. (A) Representative images of mesenchymal and epithelial-like NC clusters without chemotactic cues. Cells were labeled with mbmCherry. Optical sections were acquired at the substrate level (pseudocolored in cyan, showing protrusions) and above the substrate (pseudocolored in magenta to highlight cell bodies) and overlaid to distinguish both planes. (B and C) Quantification of protrusion area at the cluster edge (mesenchymal, n = 10; epithelial-like, n = 9) and (C) internally cryptic protrusion (n = 10 per condition) in the absence of external chemotactic cues. (D) Orientation of internal protrusions measured as angles in mesenchymal and epithelial-like clusters (n = 10 per condition) without chemotactic cues. (E) Representative images of mesenchymal and epithelial-like NC clusters during chemotaxis to SDF1. (F and G) Quantification of protrusion area at the cluster edge (mesenchymal, n = 10; epithelial-like, n = 9) and (G) internally cryptic protrusion (mesenchymal, n = 10; epithelial-like, n = 9) during chemotaxis. (H) Orientation of internal protrusions during chemotaxis, showing polarization toward the SDF1 in epithelial-like clusters (n = 10 mesenchymal; n = 9 epithelial-like). Scale bar: 30 µm. Each dot represents one cluster. Data are from at least three independent experiments. Error bars show mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test. ****P ≤ 0.0001; *P ≤ 0.05.