Analyzing Fibronectin Degradation and Invadopodia Formation in Cancer Cells

All experiments in this study were performed using MTLn3 cells cultured on FN/gelatin matrix unless indicated. FN/gelatin matrix was prepared as described previously (Chen, 1989 (link)). In brief, MatTek dishes were treated with 2.5% gelatin/2.5% sucrose, cross-linked with 0.5% glutaraldehyde, treated with 10 μg/ml of fluorescently labeled fibronectin (FN) (Alexa 568 [Invitrogen]) or unlabeled FN (Sigma-Aldrich), and then with 1 mg/ml NaBH₄ in PBS. 100,000 MTLn3 cells were plated on FN/gelatin matrix 16 h before fixation. The cells were fixed and immunofluorescence was performed as described previously (Eddy et al., 2000 (link)). FN degradation was analyzed by quantifying the average area of degraded FN pixels per field. For live cell thin-matrix experiments, Oregon Green 488-gelatin (Invitrogen) was used and the thin-matrix coverslips were prepared as described previously (Artym et al., 2006 (link)). MTLn3 cells were stimulated with EGF and images were acquired every 1 min. Gelatin degradation was analyzed by measuring the change in 488-gelatin fluorescence over time in the cortactin-containing invadopodia region corrected for background.