Establishing HER2+ Breast Cancer Cell Lines

Human HER2-positive breast cancer cell lines (Table 1) were obtained from the National Institute for Cellular Biotechnology (NICB), Dublin City University, and the Division of Haematology/Oncology, University of California, Los Angeles (UCLA). Resistant variants were developed as previously described [14 (link), 15 (link)] and BT474-RES (UCLA) was developed by twice weekly dosing of 100 ug/ml trastuzumab for 6 months. All cell lines (Table 1) were grown in RPMI-1640 medium (Sigma) supplemented with 10% FCS and 1% Penicillin/Streptomycin (P/S) and maintained at 37 °C with 5% CO₂. Cell line identities were confirmed by DNA fingerprinting, which was performed by Source Biosciences (Supplementary Table 2). Cell lines were Mycoplasma tested before and after the in vitro experiments. Trastuzumab (21 mg/ml) was obtained from St James University Hospital and prepared in bacteriostatic water. Lapatinib was purchased from Sequoia Chemicals and a stock solution (10.8mM) was prepared in dimethylsulfoxide (DMSO). BAY 80-6946 (copanlisib) (a PI3K inhibitor (PI3Ki)) and BAY86-9766 (refametinib) (a MEK1/2 inhibitor (MEKi)) were obtained under MTA from Bayer pharmaceuticals and stocks (5mM copanlisib; 10mM refametinib) were prepared in 100% DMSO with 10mM TFA, and 100% DMSO respectively. The MEKi GDC-0973 was obtained under MTA from Genentech and stocks (10mM) were prepared in 10% DMSO.