2.3. Cell Migration and Invasion Assays

Differences in cell migration and invasion between resistant and sensitive cell models were assessed using transwell migration assays. Falcon^® transwell inserts (8 µm pore size) were placed in 24-well dishes, coated with gelatin (migration) or Matrigel™ (invasion; 15% v/v; BD Science, San Jose, CA, USA) and exposed to culture media (10% FBS) in the bottom well. Cells (5 × 10⁴ cells/well) were seeded onto the top portion of each transwell chamber and treated with inhibitors (5 µM erlotinib or 5 µM cabozantinib), vehicle control (DMSO), or proliferation control (mitomycin C). Following a 24-h incubation at 37 °C and 5% CO₂ under the different treatment conditions, transwells were fixed with 1% glutaraldehyde and mounted and stained with Vectashield^® Antifade Mounting Medium with DAPI (Vector Laboratories). Five high-powered fields of view (HP-FOVs) were analyzed for each well, and the mean number of migrated or invaded cells per FOV was calculated using FIJI ImageJ software.