Figure 2.

Aligned Fn organization by CAFs mediates directional cancer cell migration. (A) Representative images of NHS–ester-488 (green) and anti–Fn (red) staining in CDMs generated by NFs and CAFs. Bar, 50 µm. (B) Measurements of angles between Fn fibers in NF and CAF CDMs. More than 100 angles per condition were measured from at least 12 images from three independent experiments. ***, P < 0.001, Mann-Whitney U test. (C) FFT analysis of CDMs stained with the Fn antibody shown in A. (D) Time-lapse images showing DU145 cells (red) migrating on NF- and CAF-derived matrices (labeled with NHS–ester-488 dye, green). Bar, 50 µm (Videos 5 and 6). (E–H) Box plots showing DU145 cell migration directionality ratio on NF and CAF CDMs in 2D (E) or 3D (G), and migration speed in 2D (F) and 3D (H). Greater than 70 cells were analyzed per condition from three independent experiments. ***, P < 0.001, analyzed by Mann-Whitney U test. Box plots range from the 25th to the 75th percentiles; the central line indicates the median, and the whiskers range from the 5th to the 95th percentiles. (I) Fn staining of control or Fn-KD CAFs at 48 h. Nucleus, DAPI, blue; F-actin, phalloidin, cyan. Bar, 25 µm. (J) Representative images of CDMs generated by control or Fn-KD CAFs. Bar, 50 µm.