Multimodal Cell Migration Assays

For 2D migration, we employed a wound closure assay using Ibidi wound closure inserts. Cells were seeded at 7 × 10⁵ per mL and allowed to adhere overnight e.g., ~16 h. Following insert removal, cells were supplemented with various treatments, and wound closure was imaged. For 3D invasion, cells were seeded at 3 × 10⁴ in Corning ultra-low attachment spheroid round bottom 96-well plates and allowed to form spheroids for 3 days. Following spheroid formation, cells were embedded in an invasion matrix (Trevigen, CT, USA) and invasion was monitored over 6 days. For donut migration, technical details can be found here (22 (link)). For transwell migration assays, cells were seeded at 1 × 10⁵ in the apical chamber of Corning BioCoat Matrigel Invasion Chambers (Corning #354480, USA) and allowed to invade for 24 h. Invaded cells were counted manually. All images were acquired on the BioTek Cytation 5 imaging station, in tandem with the BioTek BioSpa automated incubator and robotics system (BioTek, VT, USA). For each of the cell treatments, C.M. was supplemented at a final 1X concentration; 20 ng/mL VEGF, CCL-18, or IL-4; 1 μM Y-27632 or GSK429286A (Tocris, R&D, USA).