Breast Cancer Tumorsphere Formation Assay

To measure tumorsphere formation [26 (link)] BT474 (1 × 10⁴ cells/well), SKBR3 (5 × 10³ cells/well), and HCC-1954 (5 × 10³ cells/well) cells were plated in DMEM-Gluta MAX medium (Invitrogen) containing 2% B27 (Invitrogen), 4 ng/mL heparin (Sigma-Aldrich), 20 ng/mL epithelial growth factor (Sigma-Aldrich), and 20 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA) in 6-well ultra-low attachment surface plates (Corning, Corning, NY, USA). JIMT-1 (1 × 10⁴ cells/well) cells were similarly seeded in DMEM-Gluta MAX medium supplemented with 2% B27, 4 ng/mL heparin, 20 ng/mL EGF, 20 ng/mL bFGF, 5 mg/mL insulin, and 0.5 mg/mL hydrocortisone (Sigma-Aldrich), in ultra-low attachment surface plates. An inverted microscope was used to assess and quantify tumorsphere formation after 5 days.