Migration chamber

To study the cellular invasion towards chemo-attractant using a transwell migration assay, 100 μL of Matrigel (Corning, Corning, NY, USA) at 1 mg/mL was added to the migration chambers (8 microns, BD, San Jose, CA, USA) and allowed to stabilize at room temperature for 30 min. To analyze the invasion, 50,000 cells were seeded on the Matrigel in a 5% FBS medium and left to migrate toward a 10% FBS medium for 48 to 72 h depending on the cell line characteristics. To detect the migrated cells, the Matrigel was removed from the chambers, cells were fixed with 7% paraformaldehyde (Sigma), washed with PBS, and stained with cristal violet (Sigma). Migrated cells were counted from the high-power microscope fields (Leica DMI3000B microscope and Leica Application Suite camera software, Leica Application Suite X 1.1.0.12420, Wetzlar, Germany). Naïve counterpart fibroblasts isolated from the same patients were used as TAFs as controls.

Transwell assays were performed with migration chambers (BD Biosciences, USA). Briefly, cells (1 × 10⁴) were seeded and medium with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h, cells were fixed in methanol, stained with 0.1% crystal violet and counted.

Migration assay was conducted with migration chambers (BD Biosciences, UK). 5 × 10⁴ cells were plated per well in the upper chamber without serum, and the lower chamber was filled with DMEM medium with 10% FBS. 18–20 h later, the bottom cells were fixed and stained, while non-invading cells were removed. Finally, cells were extracted by 33% acetic acid and quantitatively detected (OD at 570 nm).