8.0 m pore size inserts

Migration assay was performed using 24-transwell plates (Corning) with 8.0 µm pore size inserts (Corning). A total of 200,000 cancer cells were seeded in the lower chamber with complete medium. Four hours later, the complete medium was discarded and washed twice with DPBS and replaced with serum free medium. A total of 100,000 MSCs were seeded in the upper chamber with serum free medium. After 48 h, MSCs were fixed in 4% formaldehyde. Unmigrated MSCs were removed using a Q-tip and migrated MSCs that penetrated the porous membrane were stained with Hoechst 33342 (5 µg/mL), documented with a fluorescence microscope (EVOS M7000 Imaging System, Thermo Fisher Scientific) and quantified using EVOS Analysis software (Version 1.5.1479.304, Thermo Fisher Scientific).

hMel (4 x 10⁴ cells/cm²) were seeded in 8.0 µm pore size inserts (Corning) in non‐supplemented HEM medium, while complete medium was added to the bottom well. After 48 hours of culture under normoxia or physoxia, cells that migrated from the insert to the bottom well were fixed with 10% formalin (15 minutes, RT). Images were acquired with an AxioVert.A1 (Zeiss) for quantification (12 per triplicate) or with an Axio Observer (Zeiss) microscope after staining the cells' nuclei with DAPI.