Mcf 7

The human breast adenocarcinoma cell lines MCF-7 (HTB-22) and MDA-MB-231 (HTB-26) were purchased from the American Type Culture Collection (ATCC). MCF-7 cells stably transfected with the full-length MT1-MMP vector (MCF-7 MT1-MMP) and MCF-7 cells transfected with the empty vector (MCF-7 VEC) were obtained as previously described (Maquoi et al., 2012 (link)). MCF-7 and MDA-MB-231 cell lines were cultured in DMEM (4,5 g/l glucose) with Glutamax I (PAN-Biotech, p04-04500) supplemented with 10% fetal bovine serum (Dominique Dutscher, S1810-500) and 1% penicillin-streptomycin (Invitrogen, 15140). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂ (v/v). Cells were routinely passaged at preconfluency using 0.05% trypsin, 0.53 mM EDTA (Invitrogen, 25300) and screened for the absence of mycoplasma using PCR methods.

The human cancer cell lines were purchased from ATCC (Manassas, VA, USA): SW620 (ATCC^® CCL-227™, metastatic-derived colon cancer), the MCF7 (ATCC^® HTB-22™, breast cancer,) and A375 (ATCC^® CRL-1619™, malignant melanoma). Cells were incubated at 37 °C in a humidified incubator with 5% CO₂ in air. For the SW620 and MCF7 cell lines, we generated a RPMI formulation with physiological glucose concentration (5 mM) supplemented with 10% dialyzed fetal bovine serum (FBS) (PAN BIOTECH, Aidenback, Germany, P30-2102) to reduce the interference with serum metabolites. For the A375, we generated a DMEM with 5 mM glucose supplemented with 10% dialyzed FBS. The new HPLM (Thermo Fisher Scientific, A4899101) was supplemented with 10% dialyzed FBS. Cells were counted in a Cellometer^® counter (Nexcellon Bioscience, Lawrence, MA, USA) following the manufacturer’s instructions. Briefly, a 100 µL aliquot of the cells was diluted in 0.1% trypan blue and measured in a Cellometer^® chamber.
For the sunitinib treatments, SW620 cells were cultured either in RPMI or HPLM supplemented with 10% dialyzed FBS. At the day of the experiment, cells were treated for 3 h with 10 µM sunitinib (MedChemExpress, Monmouth Junction, NJ, USA HY-10255A) dissolved in DMSO. After the incubation period, the cells were washed with PBS, detached with trypsin EDTA, counted, and subjected to respirometry.

Human breast cancer cell lines MCF-7 (HTB-22, ATCC), T-47D (HTB-133, ATCC), ZR-75-1 (CRL-1500, ATCC) and BT-474 (HTB-20, ATCC) were obtained from the American Type Culture Collection. The cells were grown in DMEM with L-Glutamine (MCF-7 and BT-474, PAN Biotech, Aidenbach, Germany) or RPMI-1640 with L-Glutamine (ZR-75-1 and T-47D, PAN Biotech) supplemented with 10 % FCS (Gibco, Life Technologies, Carlsbad, CA) and 1 % penicillin/streptomycin (Gibco). Culture media for T-47D, MCF-7 and BT-474 additionally contained 0,1 % bovine insulin (Sigma. St. Louis, MO). The tamoxifen-resistant cell lines (MCF-7 Tam1, T-47D Tam1 & Tam2, ZR-75-1 Tam1 & Tam2, BT-474 Tam1 & Tam2) were derived from the parental cell lines by continuous exposure to 4-OH-tamoxifen (Sigma, 1 μM in ethanol) for 8–12 months. Culture media was replaced every 2–3 days. All cells were incubated at 37 °C with 5 % CO₂ and passaged when ca 80 % confluent. The approximate doubling times of the cells were as follows: parental MCF-7, T-47D, ZR-75-1 and BT-474: 1–3 days. Resistant MCF-7 Tam1, T-47D Tam1 and Tam2: 1–2 weeks, ZR-75-1 Tam1 and Tam2: > 1 week, BT-474 Tam1 and Tam2: 2 weeks. The cells were free of mycoplasma and verified for their authenticity (Technology Centre, Institute for Molecular Medicine Finland, Helsinki, Finland).