Mda mb 231 breast cancer

Human epithelial cancer cell lines (U87 and LN229 glioma cells and MDA-MB-231 breast cancer cells) were obtained from American Type Culture Collection (ATCC). U87-MG cells stably transfected with EGFRvIII (U87 MG EGFRvIII, U87vIII) were provided by Prof. Huan Ren of the Department of Immunology at Harbin Medical University [28 (link)]. The cells were cultured at 37 °C in a 5% CO₂ humidified incubator and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin. Cells in the logarithmic growth phase or at 80% confluence were used for subsequent experiments.

HEK 293T cells, human A549-VIM-RFP lung adenocarcinoma cells, MDA-MB-231 breast cancer cells and human U2OS osteosarcoma cells were originally obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (DMEM, 11965092, Thermo) supplemented with 10% fetal bovine serum (FBS, S1860-500, BioWest) and 100 U/mL penicillin‒streptomycin (15140148, Thermo). The MCF10A-Ras breast epithelial cell line was derived from MCF10A cells that were transformed with Ha-Ras (kindly provided by Dr. Fred Miller) (Barbara Ann Karmanos Cancer Institute, Detroit, MI), and cultured in DMEM/F12 (11039047, Thermo) supplemented with l-glutamine with 5% horse serum (26050088, Thermo), 20 ng/mL epidermal growth factor (EGF 01–107, Merck Millipore), 10 mg/mL insulin (91077C, Sigma), 100 ng/mL cholera enterotoxin (C8052, Sigma), 0.5 mg/mL hydrocortisone (H0135, Sigma), and 100 U/mL penicillin‒streptomycin. All the cell lines were tested to confirm the absence of mycoplasma contamination and were authenticated by short tandem repeat (STR) profiling.

Human MDA-MB231 breast cancer cells from the American Type Culture Collection (ATCC, Rockville, MD, United States) were grown in MEM medium supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PS) and 1% non-essential amino acids (Gibco, Invitrogen, Karlsruhe, Germany). Telomerase immortalized human lymph endothelial cells (LECs) were grown in EGM2 MV (Clonetics CC-4147, Allendale, NJ, United States). The cells were kept at 37°C in a humidified atmosphere containing 5% CO₂. For CCID formation assays, LECs were stained with CellTracker^TM green purchased from Invitrogen (Karlsruhe, Germany).

MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, USA) at 37°C in an atmosphere of 5% CO₂. The stem-like ALDH^hiCD44⁺ cells and non-stem-like ALDH^lowCD44⁺ cells were separated from the MDA-MB-231 cell culture using fluorescence-activated cell sorting with an ALDEFLUOR™ assay kit (Stemcell Technologies, Inc., Vancouver, BC, Canada), and monoclonal fluorescence-conjugated rat anti-mouse antibodies against CD44-phycoerythrin (PE; 1:100) and immunoglobulin (Ig) G1-PE (1:100; Abcam, Cambridge, UK), according to the manufacturer’s instructions. Briefly, the cells were incubated in ALDEFLUOR assay buffer containing ALDH substrate. In each experiment, a sample of cells was incubated with 50 mmol/l diethylaminobenzaldehyde (a specific ALDH inhibitor) at 37°C for 45 min, for use as the negative control. Subsequently, the CD44 and IgG1 antibodies were added with ALDH into negative control Eppendorf tubes at a temperature of 4°C for 30 min. Finally, all the cells were resuspended with buffer for detection by flow cytometry.