Humec medium

Myoepithelial and luminal cells isolated from human breast tissue were previously shown to reform ductal structures with internal luminal cell layer and external myoepithelial cell layer, and with luminal compartment when grown in collagen gels (Carter et al, 2017). Furthermore, in the same model, inducible expression of HER2 in luminal compartment was shown to induce luminal filling, recapitulating ductal carcinoma in situ. Thus, this 3D model appears to recreate physiologically reflective duct (Carter et al, 2017).
Pure populations of myoepithelial and luminal cells (n = 2) were obtained from the Breast Cancer Now Tissue Bank at the Barts Cancer Institute (REC:15/EE/0192). All patients donated tissues from which cells were derived following fully informed consent, and all experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Luminal cells were cultured in DMEM/F12 medium supplemented with 10% FBS, 0.5 μg/ml hydrocortisone, 10 μg/ml apo‐transferrin, 5 μg/ml insulin and 10 ng/ml EGF. Myoepithelial cells were cultured in HuMEC medium (Thermo Fisher Scientific) supplemented with 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 ng/ml EGF and 50 μg/ml bovine pituitary extract (Thermo Fisher Scientific; Carter et al, 2017).

Human HEK293T [American Type Culture Collection (ATCC), CRL-11268], MCF-7 (ATCC, HTB-22), and MDA-MB-231 (ATCC, HTB-26) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, 11885-084) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, 26140-079), 1% Penicillin-Streptomycin (Thermo Fisher Scientific, 15070-063) and 1% L-glutamine (Thermo Fisher Scientific, 25030-081). Human MCF-12F (ATCC, CRL-10783) cells were maintained in HuMEC medium (Thermo Fisher Scientific, 12753018) with HuMEC supplement kit (Thermo Fisher Scientific, 12755013). These cells were transfected using GenJet in vitro DNA transfection reagent (SignaGen Laboratories, SL100488) following the manufacturer’s instructions. Viability of cells was measured after each transfection. Only cells with viabilities of 90% and above were considered for further experiments.

European Cell Culture Collection cell lines MCF-7, T47D, MDA-MB-231 and HS578T were obtained from Sigma-Aldrich. CAL51 cells were obtained from the German Resource Centre for Biological Material (DSMZ). These cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1 g/L glucose, 10% fetal calf serum and 4 mM l-glutamine (Thermo Fisher Scientific). Medium for HS578T cells contained, in addition, 10 µg/mL recombinant human insulin (Sigma-Aldrich). MCF-10A cells were a gift from L. Buluwela (Imperial College London) and were routinely cultivated on serum-free HuMEC medium (Thermo Fisher Scientific). Where indicated, cells were treated with 5-azacytidine (azaC; Sigma-Aldrich) daily for 3 days.

All cell lines were obtained from ATCC except SUM149 and SUM159 cells were obtained from the lab of Dr. Charles Perou, and SUM229 parental and EpCAM-sorted cells were obtained from the lab of Dr. Gary Johnson. SUM149, SUM159, SUM229, and EpCAM-sorted SUM149 and SUM229 cells were cultured in HuMEC medium (Gibco, Waltham, MA, USA) with supplements added and 5% FBS. MDA-MB-231 cells were cultured in DMEM (Gibco) with 10% FBS. All other cell lines were cultured in RPMI-1640 (Gibco) with 10% FBS. Penicillin/Streptomycin (Gibco) was added to all culture media. Cells were occasionally tested for mycoplasma with the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland). DRD2 antibody was obtained from Millipore (AB5084P) and used at 1:1000. Actin antibody was obtained from Cell Signaling Technologies. Thioridazine and quinpirole were obtained from Sigma-Aldrich, and SKF83959 was obtained from Cayman Chemical.

A total of 1 × 10³ BrM cells were plated in low-attachment plates in Humec medium (12753018, Gibco) supplemented with 10 ng ml⁻¹ basic human fibroblast growth factor (13256-029, Gibco), 20 ng ml⁻¹ epidermal growth factor (EGF; E9644, Sigma-Aldrich), 5 µg ml⁻¹ insulin solution from bovine pancreas (IGF1; I0516, Sigma-Aldrich), and B27 supplement (17500-044, Gibco). H2030-BrM cells were grown for 7 days and E0771-BrM cells for 4 days, so formed oncospheres were approximately the same size or number per well. After this period, oncospheres were irradiated with a single dose of 10 Gy; 72 h later, oncosphere size was evaluated using ImageJ.

All cell lines (MCF7, T47D, MDA-MB-231, MDA-MB-468, BT-549, MCF10A and MCF12F) used in this manuscript were purchased from ATCC which utilizes STR technology for Cell Authentication, and they were used in a low passage (<20) within 6 months or less after receipt or resuscitation. MB231 cells were cultured in DMEM (Gibco) supplemented with Na Pyruvate (Sigma), MCF7 cells were cultured in MEM (Gibco) supplemented with 0.1% insulin (Sigma), MCF10A and MCF12F cells were cultured in HuMEC Medium (Gibco) supplemented with HuMEC supplement kit (Gibco); while T47D, BT549 and MB468 cells were cultured in RPMI 1640 (Gibco) supplemented with 0.1% insulin. All culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen).