Three-dimensional CDMs were generated as previously described13 (link). The thickness of the generated CDMs was measured using a Leica SP5 scanning confocal with a HCX PL APO 63×/1.40–0.60 OIL λ BL objective. The orientation of the fibronectin stained fibers was quantified using the OrientationJ plugin for ImageJ52 (link). The modal angle, as defined as the angle that had the highest percentage of fibers oriented, was set to 0 to allow for direct comparison between images.
For migration analysis, parental MDA-MB-231 human breast cancer cells (ATCC) were seeded into the generated CDMs for 4 hours in the presence of serum. After 4 hours, time lapse imaging was performed on a Nikon TE2000 microscope equipped with an environmental chamber and imaged using a HCX Plan Fluotar 10×/0.30 NA objective with images taken every 10 minutes for 16 hours. The percentage of cells exhibiting plasticity was assessed by scoring the number of cells undergoing at least one amoeboid to mesenchymal phenotypic switch. The Manual tracking plugin in ImageJ was used to track the cell centroid over the length of the movie. The Chemotaxis and Migration plugin in ImageJ was used to calculate directionality and migration velocity.
For migration analysis, parental MDA-MB-231 human breast cancer cells (ATCC) were seeded into the generated CDMs for 4 hours in the presence of serum. After 4 hours, time lapse imaging was performed on a Nikon TE2000 microscope equipped with an environmental chamber and imaged using a HCX Plan Fluotar 10×/0.30 NA objective with images taken every 10 minutes for 16 hours. The percentage of cells exhibiting plasticity was assessed by scoring the number of cells undergoing at least one amoeboid to mesenchymal phenotypic switch. The Manual tracking plugin in ImageJ was used to track the cell centroid over the length of the movie. The Chemotaxis and Migration plugin in ImageJ was used to calculate directionality and migration velocity.
