Quantifying Carcinoma-Associated Fibroblast Migration

The migratory behavior of CAFs on a 2-dimensional substrate was assessed as previously described (44 (link)). Even though a 2-dimensional setting imposes nonphysiological constraints, it offers a reliable readout regarding the adhesive and migratory machinery of the cells (65 (link)). Briefly, CAFs originating from PDAC-bearing mice were seeded in 12.5 cm² flasks that were precoated with ECM components resembling the desmoplastic stroma (66 (link)), containing the following components: 40 μg/mL laminin (Sigma-Aldrich, Merck KGaA), 40 μg/mL fibronectin (Sigma-Aldrich, Merck KGaAy), 800 μg/mL collagen I (Corning), 12 μg/mL collagen III (Corning), and 5.4 μg/mL collagen IV (BD Biosciences). Following cell adhesion overnight at 37°C containing 5% CO₂, flasks were sealed air-tight and inserted into 37°C temperature-controlled chambers. In the chambers, cell motility was observed using an inverted microscope (Zeiss Axiovert 40, Zeiss) and recorded every other 15 minutes using the MicroCamLab 3.1 software (Bresser). From the recordings, cell contours were segmented using the Amira software (Thermo Fisher Scientific) and further processed to determine cell velocities (μm/min) from the displacement of the cell centroid over time (quantification detailed by Dieterich et al.; ref. 67 (link)). Cell translocation indicates the start-to-end net cellular displacement.