Trypsin

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.

Mammary tumors were dissected from mice, rinsed three times in PBS, and sequentially digested with collagenase/hyaluronidase (37°C, 2 h), 0.05% Trypsin, DNAse I, and Dispase (Stem Cell Technology). The ensuing cell suspensions were treated with red blood cell lysis buffer, rinsed with PBS, resuspended in Opti-MEM medium (Gibco) and passed through a 40 µm mesh to remove cell chunks. Cells were plated on gelatin-coated plates and grown in CnT-BM1 medium (Cell-N-Tec). Unless indicated otherwise, lapatinib-resistant 125R cells derived from a lapatinib-resistant mammary tumor were routinely maintained in the presence of 300 nM of lapatinib. Heterozygous mutant p53 R172H/+ status was verified and confirmed by using genotyping primers^{27 (link)} in all established mouse cell lines.

Human neuroblastoma SH-SY5Y cells obtained from the American Type Culture Collection (ATCC^® No. CRL-2266TM) were routinely cultured following the ATCC’s instructions. A mixture of ATCC-formulated Eagle’s minimum essential medium (EMEM) (ATCC, USA) and F12 medium (Gibco, Gaithersburg, MD) was prepared at a 1:1 ratio (hereafter called EMEM-F12). To prepare a complete medium for human SH-SY5Y cells, EMEM-F12 medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone, USA), herein called complete EMEM-F12. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. At a cell density of 60–80% confluence, cells were subcultured using 0.25% trypsin in 0.5 mM EDTA solution (STEMCELL Technologies, Vancouver, Canada). The number of viable cells was counted using Trypan blue and a hemocytometer.

Number 4 (inguinal) mammary glands from virgin female C57Bl/6J mice (Harlan Laboratories) were digested overnight in DMEM/F-12 supplemented with 15 mM Hepes (Invitrogen), 1 mg ml⁻¹ collagenase A (Roche), 100 U ml⁻¹ hyaluronidase (Sigma) and 50 μg ml⁻¹ gentamycin (Gibco). No growth factors or serum were included in the digestion mixture, to avoid induction of cell proliferation. After dissociation, the red blood cells were lysed with NH₄Cl followed by sequential digestion with 0.25% trypsin (StemCell Technologies), 5 mg ml⁻¹ dispase (StemCell) and 0.1 mg ml⁻¹ DNase (Sigma). The cell preparation was then filtered through a 40-μm mesh (BD Biosciences) to obtain a single cell suspension. Ice-cold Hank's balanced salt solution (Gibco) supplemented with 2% fetal bovine serum (Gibco) and 10 mM Hepes (Sigma; collectively termed HF) was used for all the washing steps. In some experiments, the mice were oestrus staged as outlined above.

MCF10A and MCF10A-BMPR1B⁺ cells were maintained in phenol-free DMEM/F12 medium supplemented with 5% horse serum, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin, 20 ng/ml epidermal growth factor (EGF) (all from Sigma), and 1% penicillin/streptomycin (Life Technologies, Waltham, MA, USA). Rapamycin was obtained from Sigma.
Normal reduction mammoplasty tissue was obtained from premenopausal women with informed consent, according to procedures approved by the University of British Columbia Research Ethics Board. This tissue was then treated to obtain organoid-rich pellets, that were then viably cryopreserved^{28 (link)}. Thawed organoids were rinsed with Hank’s Balanced Salt Solution supplemented with 2% fetal bovine serum (FBS) (HF), and the cells then dissociated in 2.5 mg/ml trypsin with 1 mM EDTA and 5 mg/ml dispase (STEMCELL Technologies, Vancouver, Canada) with 100 μg/ml DNaseI (Sigma, St Louis, MO, USA) and washing with HF between each step. The resulting cell suspension was filtered through a 40 μm mesh and EpCAM^loCD49f.⁺ BCs, EpCAM^hiCD49f.⁺ LPs, EpCAM^hiCD49f.⁻ LCs and EpCAM⁻CD49f.⁻ SCs isolated from within the CD45⁻CD31⁻ (blood and endothelial) cells by FACS, as described^{28 (link)}.