Neuregulin 1

Human astroglioma cells (U87MG) were obtained from the Health Protection Agency (Culture Collections, Salisbury, UK). Human epidermoid cells (A431) were obtained from the RIKEN Cell Bank (Ibaraki, Japan). Human breast cancer cells (MDA-MB-453) was a kind gift from Dr. Higashiyama^{35 (link),36 (link)}. Cells were grown in DMEM or RPMI-1640 containing 10% fetal bovine serum and kanamycin (50 μg/mL). Cancer cells were plated at a cell density of 10³ onto the 96-well culture plates and grown for 24 h. Cell growth/survival for the following 48 h was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). For phosphorylation assays, cells were starved in serum-free DMEM for 10–12 h and then treated with 0–100 µM of the antipsychotic compounds for 20–30 min followed by the stimulation of EGF or neuregulin-1 (30 ng/mL, Peprotech Inc, Ehovot, Israel) for 5 min. The used neuregulin-1 was a core EGF domain peptide whose amino acid sequence is shared by all splice variants of neuregulin-1^{30 (link)}Whole cerebral neocortices of fetal rats (Sprague-Dawley, embryonic day 18–19, male and female) were enzymatically dissociated, plated on 35 mm-diameter dishes at a density of 1.5 × 10⁶ cells/dish, and grown in the serum-free condition as described previously^{37 (link)}. Cortical cultures were maintained for 5 days and subjected to treatment with EGF or neuregulin-1 as described above.

Tumor samples will be enzymatically and mechanically dissociated to obtain isolated cells or small cell clusters. Cells will be embedded in an extracellular matrix and cultured in a medium supplemented with growth factors and signal pathway inhibitors [Advanced DMEM (Gibco) supplemented with 100 UI/mL of penicillin and streptomycin (Gibco), 1% GlutaMAX (Gibco), 1X B27 (Gibco), 5 mM Nicotinamide (Sigma-Aldrich), 1.25 mM N-Acetyl-L-Cysteine (Sigma-Aldrich), 50 μg/mL Primocin (InvivoGen), 100 ng/mL Noggin (Peprotech), 5 nM Neuregulin 1 (Peprotech), 5 μM Y27632 (Interchim), 20 ng/mL FGF-10 (PeproTech), 500 nM A-83–01 (PeproTech), 50 ng/mL EGF (PeproTech), 5 ng/ml FGF-7 (PeproTech), 1 µM SB202190 (PeproTech) and 10% RSPO1- conditioned media (Cultrex HA-R-Spondin-1-Fc 293 T, Amsbio)]. Culture medium will be changed twice a week. Once formed, PDTO will be dissociated and reseeded to amplify them for experimental purposes. Cryovials will be prepared at regular intervals by dissociating and resuspending PDTO in Recovery Cell Culture Freezing Medium (Gibco) prior to be biobanked in liquid nitrogen. PDTO lines will be considered as established when it will be maintained for more than 3 passages. For each established PDTO line, samples will be kept frozen for DNA/RNA/protein analysis and others will be embedded in paraffin for histopathological analysis.