2.1. Cell and Tumorsphere Culture

MCF-7 and BT-549 breast cancer cell lines (ATCC, USA) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, USA) in a humidified incubator at 37°C and 5% CO₂. For BT-549 cells, the cells were maintained in the media supplemented with 0.023 IU/ml insulin (Sigma Aldrich).

For the generation of tumorspheres, 80-90% confluent cell plates were trypsinized, washed, and resuspended in Cancer Stem Premium™ medium (ProMab Biotechnologies, Richmond, CA, USA). 1x10⁴ cells/ml were cultured in Cancer Stem Premium™ media in ultra-low attachment Nunclon Sphera plate (Thermo Scientific, Nunclon Sphera, Roskilde, Denmark). The cells were incubated for 5 to 10 days for tumorsphere formation. Media change was done by collecting the tumorspheres in 15 ml falcon tubes and allowed to settle by gravity. The pellets were washed with 1X PBS. Tumorspheres older than seven days were used for the subsequent experiments.