Poly 2 hydroxyethyl methacrylate

For RAR pathway modulation, cells were treated for 24 hours either with 0.5 µM RA or with 5 µM BMS493 (a pan‐retinoic acid receptor inverse agonist) or with a combination of RA and BMS493, directly diluted in the culture media. For EBs formation assay, cells were dissociated into single cells using StemPro Accutase (Thermo Fisher Scientific) and cultured for 7 days on poly (2‐hydroxyethyl methacrylate) (Sigma‐Aldrich) – coated dishes in mTeSR1 medium supplemented with 10 µM of the Rho‐kinase inhibitor Y‐27632 (Selleckchem) for the first three days. At day 7, floating EBs were transferred on 5 µg/mL Biolaminin 521LN (Biolamina)‐coated plates and cultured in adhesion for additional thirteen days in medium consisting of DMEM/F12 containing 20% knockout serum replacement (KSR, Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% non‐essential Amino Acids (Thermo Fisher Scientific), 100 µM 2‐mercaptoethanol and 0.5% penicillin and streptomycin.

MCF7 (and derivative lines LCC1 and LCC9), T47D, CAMA‐1 and ZR‐75‐1 (ER⁺/HER2⁻); SK‐BR‐3 (HER2⁺) and BT‐474 (ER⁺/HER2⁺); TNBC cell lines BT‐20, BT‐549, HCC1806, MDA‐MB‐157, MDA‐MB‐231, MDA‐MB‐453, MDA‐MB‐468 and PMC‐42; and non‐transformed MCF10A cells (basal phenotype) and FaDu (squamous cell carcinoma) were obtained from ATCC, ECACC or DSMZ. Cells were grown at 37 °C with 5% CO₂ in high glucose DMEM or McCoys 5A (for SKBR3), each with 10% fetal bovine serum (all from Invitrogen, Paisley, UK). MEBM with growth supplements (SingleQuots; Lonza, Slough, UK) was used for MCF10A. Cells were split regularly when approximately 80% confluent and all assays were performed when cells were subconfluent.
For mammosphere growth, cells were detached by trypsin and single suspensions prepared by passing through a 21G needle at least five times. Cells were counted and checked for doublets in a haemocytometer and 5000 cells/cm² were plated in dishes pre‐coated with 1% poly(2‐hydroxy‐ethyl‐methacrylate) (Sigma‐Aldrich, Dorset, UK) in 90% ethanol. Cells were grown in standard mammosphere culture conditions; serum‐free DMEM/F12 (Invitrogen) containing B27 (Invitrogen) and SingleQuots growth factor supplements (Lonza) 11.

Six-well plates were incubated overnight on a rocker with 1.2% poly(2-hydroxyethyl methacrylate) (Cat# P3932; Sigma) diluted in ethanol. mCherry-expressing MCF-7 cells were plated in the coated wells, in DMEM/F12 medium supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin (all from Biological Industries), 0.4% BSA (Cat# 0332-TAM; Amresco, Solon, OH, USA), B-27 serum-free supplement (Cat# 17504044; Gibco, Life technologies, Grand island, NY, USA), 20 ng/ml rh-basic FGF (Cat# 100-18B; PeproTech), 20 ng/ml rh-EGF (Cat# 236-EG; R&D systems, Minneapolis, MN, USA), and 5 µg/ml insulin (Cat# I9278; Sigma). After 72 h, tumor spheroids were collected, centrifuged (1,200 rpm for 7 min, + 4°C) and resuspended in the different MSC-derived CM or the respective control media (as described above). Tumor spheroids were photographed daily using fluorescent microscopy.

Briefly, 1 × 10⁶ CCSCs were cultured in DMEM/F-12 (MACGENE), containing 20 ng/mL EGF, 20 ng/mL bFGF, 2% B27, and 10 μg/mL heparin, in ultralow-attachment six-well plates (Corning, Corning, NY, USA). All the plates were covered with 95% poly (2-hydroxyethyl methacrylate) (Sigma-Aldrich, St. Louis, MO, USA). After two weeks of cultivation, spheroids of size > 80 μm were observed and counted using an inverted fluorescence microscope (CKX53, Olympus, Tokyo, Japan).