Mda mb 468

Four HER2 overexpressing cell lines were used in this study. The ovarian adenocarcinoma cell line SKOV-3 (ATCC, HTB-77™), purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), the human breast adenocarcinoma cell line SK-BR-3, kindly provided by the Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway, the human ovarian carcinoma cell line HOC-7, kindly provided by Dr. Yvonne Anderson, Department of Tumor Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway, and the rat ovarian cancer cell line NuTu-19, originally a gift from Dr. A.L Major, University of Geneva, Switzerland [22 ]. The HER2 low expressing human breast adenocarcinoma cell line MDA-MB-468 (ATCC, HTB-132^TM) was obtained from ATCC. SKOV-3 and SK-BR-3 cells were cultured in McCoy's 5A medium while HOC-7 and NuTu-19 cells were cultured in RPMI 1640 medium. Both media were obtained from Sigma-Aldrich (St. Louis MO) and modified as previously described [23 ]. The MDA-MB-468 cells were cultured in Leibovitz`s L-15 medium (Lonza, Basel, Switzerland) modified as previously described [23 ] with free gas exchange with atmospheric air.

The breast cancer cell lines MCF7, T47D, MDA-MB-231, SK-BR-3, MDA-MB-453, MDA-MB-468, Hs578T and MCF10A were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA), where they are regularly authenticated. The cell lines were grown at 37°C in 5% CO₂. Hs578T, MDA-MB-231, MDA-MB-453, MDA-MB-468, and SK-BR-3 cells were maintained in DMEM (Lonza, Basel, Switzerland) containing 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA), penicillin (100 units/ml; Gibco) and streptomycin (100 units/ml, Gibco). MCF7 and T47D cells were cultured in RPMI 1640 (Lonza), 10% FBS, penicillin (100 units/ml) and streptomycin (100 units/ml). MCF10A cells were cultured in DMEM/F12 media (1:1) (Invitrogen, Grand Island, NY, USA) supplemented with 5% horse serum (Gibco), 10 μg/ml bovine insulin (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/ml epidermal growth factor (EGF; Sigma), 0.5 μg/ml hydrocortisone (Sigma), 0.1 μg/ml cholera toxin (Sigma), penicillin (100 units/ml), and streptomycin (100 units/ml). Human umbilical vein endothelial cells (HUVECs; Lonza) were maintained in Lonza EGM-MV (normal growth medium) at 37°C in 5% CO₂. The cells were maintained in culture plates and used in assays between cell passages 3 and 8.

The human breast cancer cell lines MDA-MB-231, BT-549, MDA-MB-157, HCC1937, MDA-MB-468 (American Type Culture Collection), SUM149, and SUM159 (Asterand Bioscience, Detroit, MI) were authenticated using a panel of microsatellite markers. Cell lines were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air as follows: MDA-MB-231 and BT-549 in RPMI 1640 (Sigma-Aldrich); MDA-MB-468 in Dulbecco's modified Eagle's medium (DMEM) (Lonza); MDA-MB-157 in Leibovitz (Lonza); SUM149 and SUM159 in DMEM F12 (Lonza) that was supplemented with insulin (5 μg/ml); and HCC1937 in RPMI 1640 medium that was supplemented with 1 mM sodium pyruvate, 1% (v/v) nonessential amino acids, and 10 mM Hepes. Each medium was also supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine (both from Sigma-Aldrich). For the stimulation with WHF, cells were starved in serum-free medium for 24 h and then treated for 48 h with a pool of 5 WHFs at a final concentration of 5% as described [24 (link)].

MDA-MB-468 and Balb/c 3T3 cells were obtained from ATCC^® (Manassas, VA, USA). MDA-MB-468 and 3T3HAS3 cells were authenticated based on short tandem repeat profiles (IDEXX, Columbia, MO, USA) and were negative for mycoplasma (MycoAlert, Lonza, Allendale, NJ, USA). MDA-MB-468 cells were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum, and 3T3 lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal calf serum. Co-cultures of MDA-MB-468 and 3T3HAS3 cells were maintained in RPMI supplemented with 10% fetal bovine serum.

Breast carcinoma cell lines (MCF7, T47D, HCC1806, HCC1937, BT549, MDA-MB-453, SUM149, MDA-MB-468, SKBR3, BT20, and BT549) were kindly provided by Dr. M. Götte (Department of Gynecology and Obstetrics, Münster, Germany) and Dr. B. Greve (Department of Radiation Oncology, Münster, Germany). Cells were maintained in DMEM/high glucose medium (Lonza, Basel, Switzerland) (SKBr3, MDA-MB-453, and MDA-MB-468) or in RPMI medium (MCF7, T47D, HCC1806, HCC1937, BT20, and BT549). Both media were supplemented with 10% fetal calf serum (FCS; Gibco, Waltham, MA, USA) and 100 U/mL penicillin–streptomycin (PS; Gibco). SUM149 cells were cultured in Dulbecco’s modified Eagle’s medium-F12 (1:1) with 5% FCS, insulin (5 μg/mL) (Merk, Darmstadt, Germany), and hydrocortisone (1 μg/mL; Qiagen, Hilden, Germany). MDA-MB-231 cell line was obtained from DSMZ (Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures) and was grown in DMEM/high glucose medium containing 10% FCS and 100 U/mL PS. HUVECs were kindly provided by Dr. D. Vestweber (Max Planck Institute of Molecular Biomedicine, Münster, Germany) or purchased from Promocell (Heidelberg, Germany) and cultured up to passage five in ECGM-2 medium supplemented with SupplementPack (PromoCell).