Mda mb 231

MCF7, T-47D and MDA-MB-231 cell lines (ATCC, Manassas, VA, USA; HTB-22™, HTB-133™ and HTB-26™ respectively) were cultured in accordance with ATCC recommendations. MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, 1% L-glutamine, 1% Antibiotic-Antimycotic (all Thermo Fisher Scientific) and 1% MEM non-essential amino acids (Sigma-Aldrich); while T-47D and MDA-MB-231 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% streptomycin and penicillin, 1% L-glutamine, 1% Sodium Pyruvate (all Thermo Fisher Scientific) and 1% Antibiotic-Antimycotic. All cultures were performed in humidified chambers at 37 °C with 5% CO₂, and cell lines were confirmed as mycoplasma- free (Mycoplasma PCR Detection Kit, Applied Biological Materials Inc., Richmond, BC, Canada).
Patient-derived scaffold slices were cultured with MCF7, T-47D or MDA-MB-231. 3 × 10⁵ cells were added to each PDS slice in 48-well plates containing 0.5 mL cell line specific media. Patient-derived scaffolds were transferred to a new plate with fresh media after 24 h and every four to seven days, depending on the cell growth rate, and incubation was continued for 21 days. Regular 2D cultures in plastic dishes were performed in parallel to the PDS cultures and used as reference for gene expression adaptation analyses.

Human breast cancer cell lines MDA-MB-231 (derived from pleural effusion), MDA-MB-468 (derived from pleural effusion), MDA-MB-453 (derived from pericardial effusion), and T47D (derived from pleural effusion) were obtained from American Type Culture Collection (ATCC), and cultured in either α-MEM or RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Human normal mammary gland epithelial cells (MCF-10A) were obtained from Dr. Linda Penn (Princess Margaret Cancer Centre Research Institute) and cultured in DMEM/HAM F12 supplemented with 5% horse serum, insulin, hEGF, hydrocortisone (Clonetics), and cholera toxin (Sigma-Aldrich). All cell lines were maintained at 37°C and 5% CO2, authenticated using Short Tandem Repeat analyses, and tested to be free from mycoplasma contamination.
Anti-miR-449a or pre-miR-449a mimic (Ambion) were reverse transfected into cells using Lipofectamine 2000 (Invitrogen) at a final concentration of 40 nM (unless otherwise indicated), according to the manufacturer's instructions.
CRIP2-expressing vectors were purchased (Applied Biological Materials) and transfected into MDA-MB-231 cells using Lipofectamine 2000. Independent single clones were obtained after 2 weeks of drug selection. qRT-PCR and Western blot were used to screen for positive clones.