Mammosphere Formation Assay Protocol

The ability of selected drugs to affect mammosphere-forming efficiency (MSF) and mammosphere viability (MSV) was studied in ultra-low attachment 96-well plates (Corning^®, One Riverfront Plaza Corning, Corning, NY, USA). Cells were plated at low density (1000 viable cells/well) in serum-free RPMI 1640 media supplemented with 2% antibiotic-antimycotic mixture and additional factors enhancing CSC maintenance [12 (link),20 (link)]. In detail, glucose (60 mg/mL), L-glutamine (10 µL/mL), heparin (4 µg/mL), BSA (2 mg/mL), EGF (0.02 µg/mL), FGFb (0.01 µg/mL), putrescin (10 µg/mL), apo-transferrin (0.1 mg/mL), insulin (25 µg/mL), 30 µM selenium and 20 µM progesterone (all from Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hours after seeding, the cells were treated with drugs for 7 days. The formed spheres were observed by the optical microscope (Olympus IMT-2 model) and quantified by presto blue (Invitrogen, Thermo Fisher Scientific, Madrid, Spain) to further calculate IC₅₀ as previously described [12 (link),25 (link)].