Fig. 2.

Enhanced matrix plasticity promotes spreading and motility of cancer cells independent of proteases. a Schematic of experimental setup. b After 1 day in 3D culture, cells were imaged using bright field microscopy and cell outlines were traced. Example MDA-MB-231 cells and cell outlines shown. Top scale bar is 10 μm, and bottom scale bar is 50 μm. c Circularity was calculated for traces of cells from four different cell lines. Data shown are from one representative biological replicate experiment. d Cell circularity was also quantified for MDA-MB-231 cells in the presence of broad-spectrum protease inhibitor (10 μM GM6001) or vehicle-alone control. Data shown are from two pooled experiments each. The validity of comparing medians of pooled data sets was verified (Supplementary Tables 2 and 3). For c and d, bars indicate median circularity of number of cells indicated, error bars indicate interquartile range, and statistical tests compared medians (****P < 0.0001, Mann–Whitney). e Time-lapse microscopy was used to image RFP-LifeAct-transfected MDA-MB-231 cells stimulated with 50 ng mL⁻¹ EGF. Maximum intensity projections of RFP-actin signal are shown merged with bright field images. Scale bar is 10 μm. f Representative 3D cell track reconstructions for cells in LP (n = 143) and HP (n = 114) IPNs. Grid size is 10 μm. g Probability of cell motility shown for LP, MP, and HP IPNs, with vehicle alone (DMSO) or protease inhibitor (10 μM GM6001 or 100 μM marimastat) added to the medium. Differences in mean probability of motility indicated are statistically significant (****P < 0.0001, Fisher’s exact). Probability of motility trends with plasticity for protease inhibitor studies (^####P < 0.0001, χ² test for trend). h Maximum speeds for motile cells in HP IPNs, with vehicle-alone and protease inhibitor conditions (t tests; ns not significant). For both g and h, graph displays the number of cells analyzed per condition, taken from R = 3–5 biological replicate experiments, bars indicate mean probabilities, and error bars indicate 95% confidence intervals. i A representative cell in HP IPN exhibiting protrusion and retraction cycles prior to migrating. Scale bar is 10 μm