Epicult b

3D organotypic cultures were prepared as described elsewhere (30 (link), 82 (link)). Briefly, mammary glands from 8- to 12-week-old mice were dissociated in 10 mL DMEM/F12, 1× penicillin/streptomycin, 50 mg/mL gentamycin (Wisent, 450-135-XL), and 2 mg/mL collagenase B (Roche, 11088831001) for 1 hour at 37°C, and then washed in PBS with 5% FBS (Wisent, 080-150), centrifuged at 1,500g for 15 seconds, and resuspended in 0.25% trypsin (Wisent, 325-143-ES) for 20 minutes at 37°C. trypsin was quenched with FBS, and cells were resuspended in 3D medium consisting of Epicult-B (STEMCELL Technologies, 05610), with 1% (vol/vol) knockout serum replacement (Gibco, Thermo Fisher Scientific, 10828010), 50 μg/mL penicillin/streptomycin (Wisent, 450-200-EL), 10 ng/mL EGF, 25 μg/mL insulin, 1 μg/mL hydrocortisone, and 2% Geltrex (Thermo Fisher Scientific, A1413202) and then filtered through a 40 μm mesh. Single cells (10,000 cells/well) were plated on Geltrex-coated coverslips in a 24-well plate and grown in 3D medium until organoids formed (5–7 days), and then MIC transgene expression was induced by treatment with 2 μg/mL doxycycline for 12 days.

Cells (200 cells per well) were seeded into a 24-well plate with EpiCult-B serum-free (Stem Cell, USA) and 5 % FBS and 2 × 10⁴ irradiated NIH-3T3 cells. After incubation at 37 °C in 5 % CO₂ for 8–10 days, colonies of over 50 μm in diameter were counted [27 (link)]. The colonies were fixed with acetone: methanol (1:1), stained with Giemsa (Sigma-Aldrich, USA), and observed and photographed under an inverted microscope. Then clonogenic potential was calculated. Clonogenic potential (%) = colony numbers/seeding cell numbers × 100 %. The MCF-10A cells excluding MUC1⁻ESA⁺ were also plated as control.