4.9. Transwell and IncuCyte Migration Assays

MSCs were persistently treated by TNFα + IL-1β/vehicles, generally for 14–18 days. Serum- and cytokine-devoid CM were collected (prepared as described above) and were added to mCherry-expressing MCF-7 and T47D cultures for 48 or 72 h, respectively. Then, tumor cells were plated in transwells with 8 μm pore membranes (#3422, Corning) for 21.5–24 h in fresh cytokine-devoid CM. The bottom wells contained fresh cytokine-devoid CM supplemented with 10% FBS. The cells that migrated to the lower part of the transwells were fixed and stained with Hemacolor (#111661, Merck). The cells were photographed using light microscopy and counted at multiple fields.

In parallel, migration studies were performed using an IncuCyte^® Live-Cell Imaging System. Here, cytokine-devoid CM obtained from MSCs (that were persistently stimulated beforehand with TNFα + IL-1β/vehicles; prepared as described above) were added to MCF-7 cells that were plated in IncuCyte^® ImageLock 96-well plates (#4379, Essen BioSciense, Ann Arbor, MI, USA). After scratching the cells in the middle of the wells, kinetics analyses of tumor cell migration were performed for 24 h. When appropriate, the following inhibitors were added during the IncuCyte^® migration assay: for Ras, farnesylthiosalicyclic acid (FTS, Salirasib; 7 µM; #SML1166, Sigma-Aldrich); for Gαi, Pertussis Toxin (PTx; 0.1–0.2 µg/mL; #516560, Calbiochem, Merck); for CCR2, CAS 445479-97-0 (0.02–8 µM; #227016, Calbiochem, Merck); for CCR5, Maraviroc (30 µM; #14641, Cayman Chemical, Ann Arbor, MI, USA); and for CXCR1/2, Reparixin (30–40 µM; #A12383, ADOOQ Bioscience, Irvine, CA, USA). Inhibitor concentrations were determined in preliminary analyses that guaranteed that they did not affect tumor cell proliferation or viability (except for combined treatment with FTS and the three chemokine receptor inhibitors (15–25% reduction, in different experiments)). The final concentrations of the inhibitors that were selected for use were within the range used in other studies and within the range recommended by the manufacturers.