Hcc1428

ASCs were grown in M199 with 10% FBS. Two TNBC cell lines were utilized, MDA-MB-231 and HCC1428. Both MDA-MB-231 and HCC1428 (ATCC, Manassas, VA) were grown in RPMI (Gibco, Gaithersburg, MD) with 10% FBS. Media was changed every 3–4 days and cells were passaged when they reached 80–90% confluence. ASCs were expanded until passage 4–5, when they were used for the experiments. All TNBC cells were at passage 8–10 when used for experiments.

Human mammary epithelial cells (HMEC) were obtained from Invitrogen and cultured with the proprietary HuMEC Ready Medium. The immortalised human mammary epithelial cell line MCF10A (ATCC) was cultured in HuMEC Ready Medium supplemented with 100 ng/ml cholera toxin. Breast cancer cell lines MCF-7, HCC1954, HCC1428 (all from ATCC), MDA-MB-468, MDA-MB-231, BT549 and Hs578T (all from the NCI-60 cell collection, CRN cell bank, University of Nottingham) were grown in RPMI medium supplemented with 10% foetal calf serum (FCS), 1% Penicillin/Streptomycin (Pen/Strep), 1% L-Glutamine, 1% sodium pyruvate, 1% non-essential amino acids (NEAA). HEK 293 T (ATCC) cells were grown in DMEM medium containing 10% FCS supplemented, 1% Pen/Strep, 1% L-Glutamine, 1% sodium pyruvate, 1% NEAA. Cell lines obtained from ATCC were used within few passages from the original stocks, whereas other cell lines were authenticated by STR profiling (Eurofins Genomics, Germany) using the Promega PowerPlex 21 PCR kit and matched against the ATCC STR database. All cell lines were tested form mycoplasma contamination using the EZ-PCR Mycoplasma Test Kit (Geneflow). All cell culture materials were from Invitrogen and chemicals from Sigma-Aldrich, unless otherwise stated.

All wild type (wt) cell lines (MCF7, HCC1428 and SUM44) were purchased from ATCC or Asterand. Upon receipt cells were aliquoted to prevent phenotypic drift and authenticated by STR. All cell lines were routinely screened for mycoplasma infection. The wt cell lines were cultured in phenol red-free RPMI 1640 (Gibco, Thermo Fisher Scientific, UK) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, UK) and 1 nM 17-β-estradiol (E2) (Sigma-Aldrich, UK). Long-term oestrogen deprived (LTED) derivatives of the cell lines were cultured in phenol red-free RPMI 1640 in the absence of exogenous E2 and supplemented with 10% dextran charcoal-stripped bovine serum (referred to as 10% DCC medium).^{24 (link)} Two MCF7-LTED derivatives were used within the study one harbouring wt-ESR1 (MCF7-LTED^wt), the other harbouring a naturally occurring ESR1^Y537C mutation (MCF7-LTED^Y537C). Additionally SUM44-LTED express an ESR1^Y537S mutation (SUM44-LTED^Y537S) whilst HCC1428-LTED contain wt-ESR1.^{25 (link)}

MDA-MB-231 and Htb126 cells were cultured in basal medium supplemented with 10% fetal calf serum and were originally purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and kindly provided by M White, Department of Cell Biology, University of Texas Southwestern Medical School, Dallas, TX. SUM149PT was purchased from Asterand (Detroit, MI, USA) and grown as per supplier’s instructions. MCF7, Htb126, and HCC1428 were purchased from ATCC and grown per supplier’s instructions. PP2 was obtained from Tocris Bioscience (Bristol, UK) and used at the indicated concentrations.

Human BC cell lines MCF7, T47D, HCC1428, ZR75.1 and SUM44 were purchased from ATCC and Asterand. Cells were cultured in phenol red free RPMI containing 10% foetal bovine serum (FBS) and 1 nM estradiol (E2). LTED derivatives were cultured in phenol red free RPMI supplemented with 10% dextran charcoal stripped (DCC) FBS, as previously described [14 (link)]. Palbociclib-resistant cell lines were generated by long-term culture of parental cell lines in the continuous presence of 1 µM palbociclib until resistance developed (in average 5–6 months for all the cell lines). Resistance was authenticated by lack of response to escalating concentrations of palbociclib in comparison with their wild-type progenitor cell line and routine passage in the presence of the drug. From that point, palbociclib-resistant cell lines were routinely cultured in the presence of 1 µM palbociclib. Palbociclib was removed from the media 48 h prior to each experiment unless otherwise stated. All cell lines were authenticated by short tandem repeats (STR) profiling and routinely screened for mycoplasma contamination.

Human cancer cell lines MA-11, MDA-MB-231 (ATCC #HTB-26™), Hs578T (ATCC #HTB-126™), AU565 (ATCC #CRL-2351™), HCC1428 (ATCC #CRL-2327™), MCF7 (ATCC #HTB-22™), HCC1187 (ATCC #CRL-2322™), DU4475 (ATCC #HTB-123™), HCC1569 (ATCC #CRL-2330™), DLD1 (ATCC #CCL-221™), HPAF-II (ATCC #CRL-1997™) and LNCaP (ATCC #CRL-1740™), were maintained at 37°C in 5% CO₂ and cultured in either Dulbecco´s Modified Eagle's Medium (DMEM) or Roswell Park Memorial Institute Medium (RPMI) supplemented with 10% FBS.
All cancer cell lines, except MA-11, were obtained from the American Type Culture Collection (ATCC; www.atcc.org). The human MA-11 breast carcinoma cell line, established from bone marrow micrometastases of a patient with breast cancer [52 (link)], was obtained by Øystein Fodstad (Norwegian Radium Hospital, Oslo, Norway). Cell pellets and conditioned media from the Melmet 1 (MM1) and Melmet 5 (MM5) melanoma cell lines were a gift from Dr. Siri Tveito at the Norwegian Radium Hospital, Oslo, Norway, and were established from the biopsies of metastatic melanoma patients at the department of Tumor Biology, The Norwegian Radium Hospital Oslo, Norway [53 (link)].

The following breast cancer cell lines, namely triple negative breast cancer (TNBC) HCC1806 (CRL-2335^™), ER-positive HCC1428 (CRL-2327^™), and HER2-positive AU565 (CRL-2351^™) (ATCC, Manassas, VA, USA), were grown in RPMI-1640 medium (Corning, Tewksbury, MA, USA) supplemented with 10% FBS and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 g/mL amphotericin). MCF10F non-tumorigenic epithelial cells from mammary gland (CRL-10318^™, ATCC, Manassas, VA, USA) were cultivated in DMEM/F12 medium (Merck KGaA, Darmstadt, Germany) containing 5% horse serum, 10 ng/mL epidermal growth factor EGF, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin, and mix of antibiotics. Cells were cultured at 37 °C in a controlled humidified atmosphere containing 5% CO₂ and passaged with trypsin solution (0.05% trypsin for MCF10F cells and 0.25% trypsin for breast cancer cells, respectively, Corning, Tewksbury, MA, USA).