αv integrins are critical for cancer cell migration on CAF CDMs. (A and B) Quantifications of migration directionality (A) and speed (B) of DU145 cells on CAF CDMs when treated with control IgG, α5 integrin function-blocking antibody JBS5, or αv function-blocking antibody 17E6. Greater than 80 cells per condition were analyzed in four independent experiments. ***, P < 0.001 as determined by one-way ANOVA and Tukey’s post hoc test. (A and B) The box plots range from the 25th to the 75th percentile, the middle line indicates the median, and the whiskers range from the 5th to the 95th percentile. (C) Rose plots showing migration trajectories of 14 representative cells treated with control IgG, JBS5, or 17E6 antibodies. (D) Proposed model of CAF-mediated, directional cancer cell migration. The healthy (normal) fibroblastic matrix resembles a random meshwork of fibers that do not induce migration directionality. However, CAFs organize the Fn matrix into aligned fibers through increased MyoII, α5β1 integrin, and PDGFRα-mediated contractility and traction forces. Anisotropic organization of matrix fibers by CAFs promotes directional migration of cancer cells.