Sphere Formation and Differentiation Assay

The sphere formation and differentiation (SFD) assay was performed as described previously [20 (link)]. Sorted cells were cultured in ultralow attachment 96-well plates (Corning, Midland, MI) with mouse mammosphere culture medium (EpiCult-B, StemCell Technologies) (150 μL per well) that was supplemented with 2% B27 (Invitrogen), 20 ng/mL bovine fibroblast growth factor, 20 ng/mL epidermal growth factor, 10 μg/mL heparin, 10 μg/mL insulin, 1 μg/mL hydrocortisone, and 50 μg/mL gentamicin. After suspension culture (7 days), mammospheres were counted and collected by centrifugation at 400 ×g. A total of 30 to 50 individual spheres were resuspended in 60 μL gel (Matrigel, BD Biosciences) for sphere differentiation. The sphere-gel drop was allowed to solidify inside a 37°C incubator for 15 min, covered with mammosphere medium supplemented with 5% fetal bovine serum, and incubated at 37°C for 9 days. The solid and hollow organoids (Supplemental Fig. S1c-d) were counted in ≥ 3 wells (approximately 120 spheres).