Neuregulin

Patient-derived organoids used in the current study were derived from post-surgery specimens of three female patients who underwent surgery at the Department of breast, Fudan University Shanghai Cancer Center. The organoids were cultured based on previously described methods^{61 (link),62 (link)}. The organoids were suspended in Basement Membrane Extract (BME) Type 2 (Trevigen, 3533-010-02) and cultured in breast cancer organoid medium (Advanced DMEM/F12 supplemented with R-spondin-1 [Peprotech], noggin [Peprotech], neuregulin [Peprotech], estradiol [Sigma], HEPES [Gibco], GlutaMAX [Gibco], nicotinamide [Sigma], N-acetylcysteine [Sigma], B-27 [Gibco], A83-01 [Tocris], primocin [InvivoGen], SB-202190 [Selleck], Y27632 [Selleck], FGF10 [Peprotech], FGF7 [Peprotech] and EGF [Peprotech]). After 3-5 passages, the organoids were added to each well of a 384-well plate, and different concentrations of Stattic (0, 5, and 15 μmol) were added to each well in duplicate and incubated for 5 days. Photos were taken on the last day to observe the changes in organoids under drug treatments. Acquisition of all clinical samples was approved by the Ethics Committee of FUSCC (Protocol number: 050432-4-1911D) and agreed to by each patient via signed informed consent.

Medium composed of DMEM- supplemented with 10% FBS, 10 nM neuregulin (recombinant heregulin-β1_177–244 from PreproTech) and 2 μM forskolin (Sigma) was used for the growth and expansion of both adult and postnatal SCs unless otherwise noted. Medium was typically replaced every 2–3 days. Washes with L-15 or DMEM prior to feeding were carried out when abundant myelin debris was observed in suspension. Determination of cell number and viability was carried out to assess the temporal course of cell expansion. Briefly, cells were collected from their respective dishes by mild trypsinization. Trypan blue exclusion assay was then performed to discriminate live versus dead cells. Estimation of the total number of live/dead cells was done using a Bio-Rad TC20™ automated cell counter (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. In turn, quantitative data was used to construct growth curves and calculate the doubling time using the algorithm provided by http://www.doubling-time.com/compute.php33 .

Explant medium (ExM): DMEM/F12 (Thermo, 11330032) containing B27 supplement (no vitamin A; Invitrogen, Paisley, UK, 12587010), 2 mM L-glutamine (SIGMA, G7513) and 100 U/mL Penicillin/100 µg/mL Streptomycin (SIGMA, P0781).
Clevers’ medium (CM[28 (link)]): DMEM/F12 containing 5% R-spondin conditioned medium, 5 nM neuregulin (Peprotech, 100–03), 5 ng/mL epidermal growth factor (Peprotech, AF-100–15), 100 ng/mL noggin (Peprotech, 120-10C), 500 nM A83-01 (Tocris, 2939), 5 µM Y27632 (Abmole, S1049), 500 nM SB202190 (Sigma, S7067), 1 × B27 (with vitamin A, Gibco, 1750444), 1.25 nM N-acetylcysteine (Sigma, A9165), 5 mM nicotinamide (Sigma, N0636), 1 × Glutamax (Invitrogen, 12634–034), 10 mM HEPES (Invitrogen, 15,630–056), 100 U/mL Penicillin/100 µg/mL Streptomycin and 50 ng/mL FGF2 (Thermo, 100-18B).
FCS medium: DMEM/F12 and 10% foetal calf serum (FCS, Thermo, 10270106), 100 U/mL Penicillin/100 µg/mL Streptomycin.