Mda mb 231

MCF-7 and MDA-MB-231 cells were purchased from ATCC- LGC Standards GmbH and tumor cell spheroids before and after immune cell infiltration were washed with PBS and blocked with 4% FcR Blocking Reagent (Miltenyi Biotec) prior to antibody staining using EpCAM (CD326, Brilliant Violet421, BD Biosciences) and CD45 (Alexa Fluor 700, BD Biosciences). For tumor cell proliferation, cells were stained with Ki-67 (Anti-human Ki-67 APC, Miltenyi Biotec). To discriminate viable from apoptotic and necrotic cells, spheroids were dissociated as described before and stained with FITC-labeled Annexin V (Immunotools, Friesoythe, Germany) and propidium iodide (PI; Fluka, Buchs, Switzerland). Cells were analyzed with a LSRII/Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo V10 (BD Biosciences).

The human non-IBC cell lines MCF-7 (luminal A) and MDA-MB-231 (triple-negative) were purchased from ATCC/LGC Promochem (Wesel, Germany). The IBC SUM149 (triple-negative) cell line was a gift from Prof. Dr. Robert J. Schenider (School of Medicine, New York University, New York, NY, USA). MCF-7 and MDA-MB-231 cells were cultured in RPMI and DMEM with 1% glutamine, 1% penicillin/streptomycin antibiotic mixture, and supplemented with 10% fetal calf serum (FCS) (Biochrom GmbH, Berlin, Germany), and maintained in a humidified atmosphere of 5% and 7.5% CO₂ at 37 °C, respectively. SUM149 cells were cultured in HAM’s-F12 containing 1% penicillin/streptomycin, 1% glutamine, and supplemented with 5% fetal calf serum (FCS), 1 µg/mL hydrocortisone, and 5 µg/mL insulin. SUM149 cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C. All reagents were purchased from Sigma-Aldrich Chemie (Taufkirchen, Germany). Total cell lysates of the aforementioned cell lines were used to extract miRNAs and perform Taqman probe-based real-time PCR as described above.

MCF7, MDA-MB-231, and Hs58T cell lines were from ATCC-LGC Promochem (Teddington, U.K.). All cell culture materials and classical chemotherapeutic drugs were from Sigma-Aldrich Ltd. (Gillingham, U.K.). TissueScan qPCR Breast Cancer Disease Panels I, II, and IV (BCRT101, BCRT102, and BCRT104) were purchased from OriGene Technologies. MIAT specific siRNAs were from Qiagen (Crawley, U.K.). Negative control siRNA, TRIzol, reverse transcription reagents, and TaqMan assays were from Life Technologies Ltd. (Paisley, U.K.). SensiFast Probe Hi-ROX kit was obtained from Bioline (London, U.K.). RQ1 RNase-free DNase was from Promega (Southampton, U.K.). Nucleofector solution V was from Lonza Biosciences (Verviers, Belgium).

The human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from ATCC/LGC Promochem (Wesel, Germany). MCF-7 cells were cultured in RPMI-1640 containing 1% glutamine (cat. no. R8758), 10% fetal calf serum (FCS) (Biochrom GmbH, cat. no. S0615, Berlin, Germany), and 1% penicillin/streptomycin (cat. no. P4333) in a humidified atmosphere of 5% CO₂ at 37 °C. MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (cat. no. D0819) containing 10% FCS, 1% glutamine, and 1% penicillin/streptomycin in a humidified atmosphere of 7.5% CO₂ at 37 °C. All reagents except for FCS were purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany.

Human acute T leukemia (Jurkat), human epidermoid carcinoma (A-431), estrogen (ER) and progesterone (PR) receptor-positive human breast cancer (MCF-7), and ER, PR, and epidermal growth factor receptor-2 (HER2)-negative human breast cancer (MDA-MB-231) cells were obtained from LGC Standard (LGC Group, Middlesex, UK).
Jurkat, A-431, and MDA-MB-231 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine 200 mM, and 1% penicillin (10,000 units)/streptomycin (10 mg/mL) solution (all provided by Euroclone, Pero, Italy). MCF-7 cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM, Euroclone) supplemented with 10% FBS, 1% L-glutamine 200 mM, 1% penicillin/streptomycin, and 0.1% insulin. All cells were maintained at 37 °C under 5% CO₂ in a humidified incubator.

Human breast cancer cell lines, such as MDA-MB-231 and MCF-7, and the human mammary epithelial cell line MCF-10A were purchased from ATCC-LGC-Promochem (Wesel, Germany). These cancer cell lines were cultured in 4.5 g/l DMEM with added 10% Fetal Calf Serum and 1% penicillin-streptomycin (Biochrom, Berlin, Germany) in an incubator at 37°C, 5% CO₂ and 95% humidity. Instead, the mammary epithelial cell line MCF-10A was cultured in a 1:1 mixture of DMEM (4.5 g/l glucose, L-glutamine) and Ham’s F12 medium with added 5% Horse Serum, 1 g/l Cholera toxin stock solution, 10 mg/ml Insulin (Sigma-Aldrich, I9278), 1 g/l Hydrocortison stock solution, 100 μg/ml epidermal growth factor (EGF) and 1% penicillin/streptomycin 100× (P/S) under the same conditions as mentioned above. For all experiments, cells with passage numbers of 5–25 at about 80% confluency were harvested.