Effect of different fibroblast-conditioned media on cell migration in two-dimensional cultures. PMC42-LA cells were cultured on glass in either normal medium (control), NMF-conditioned medium, or CAF-conditioned medium. Using scratch tests, over a period of 48 hours, PMC42-LA cells were analyzed to determine the rate and extent of wound closure, and vimentin expression. Four scratches averaging between 367 μm (minimum) and 392 μm (maximum) in width were made at time 0. Scratches were measured and averaged, and the extent of wound closure calculated and averaged. (a-c) Cells in normal medium (control) exhibited some wound closure at 24 hours (179 ± 21.4 μm), and this had progressed by 48 hours (106 ± 29.0 μm) with small, sparing vimentin expression appearing at 48 hours. (d-f) Cells in NMF-conditioned medium exhibited similar rates of wound closure at 24 hours (171 ± 32.9 μm) and 48 hours (160 ± 15.6 μm), with vimentin expression appearing at wound edges at 24 hours. (g-i) Cells in CAF-conditioned medium exhibited an accelerated wound closure rate, with the width of scratches reduced by 24 hours (333 ± 52.0 μm) and almost full wound closure by 48 hours (panel i part i: 41.63 ± 28.8 μm). Vimentin expression in these cells was abundant around wound area. (Panel i part ii) Closed wounds were identified at 48 hours by visualizing areas of elongated, vimentin-profuse cells in apparently normal areas of lesser vimentin expression. (j) Graphical representation of wound closure by these cultures. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.