PDOs were established from 4 NB patients stage M (HR-NB with metastatic disease) (N691, N700, N711, N772) cells kindly provided by J. Molenaar (Academic Medical Centre, University of Amsterdam). Tumour biopsies were handled according to procedures reported by Bate-Eya et al.  to obtain single-cells and for their molecular and genomic characterization. The tumour sample was classified as NB Schwannian stroma-poor according to the International NB Pathology Committee  and contained more than 60% of neuroblasts in tumour cells. To initiate PDOs culture, we modified the procedure reported by Hubert CG et al., 2016 . Cells suspension was embedded in Matrigel (5,250,005, Sacco S.r.l., Como, Italy) and 20 μl droplets were placed on parafilm molds and incubated for 1 h at 37 °C. Then, droplets were transferred in 12-well plates and cultured in DMEM-F12 (Aurogene, Rome, Italy) supplemented with 1% Glutamine (Life Technologies, Carlsbad, CA, USA), 1% Penicillin/Streptomycin antibiotics (Life Technologies, Carlsbad, CA, USA), 40 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich, Missouri, USA), 1% B27 (Gibco, USA), 20 ng/ml epidermal growth factor (EGF) (Cell Guidance System Ltd., Cambridge, UK), 1% N2 (ThermoFisher Scientific, Massachusetts, USA), 10% BIT 9500 Serum Substitute (STEMCELL Technologies, Canada Inc.), 5% MEM non essential amino acids (Biowest, Nauillé, France), 55 μM β-mercaptoethanol (Sigma Aldrich, Missouri, USA) and 1000 U/ml leukaemia inhibitory factor (LIF) (Voden medical instruments, Monza-Brianza, Italy). PDOs were cultured for 40 days before characterisation. Images of growing organoids were acquired using DeltaPix DP 200 program (Exacta-Optech Labcenter, Modena, Italy). To quantify viable cells numbers, PDOs were collected after 30 and 60 days and dissociated by means of a solution containing PBS 1X (Carlo Erba reagents, Cornaredo, Italy), DNase (Roche, Basilea, Switzerland) and Collagenase/Dispase (Roche, Basilea, Switzerland) enzymes. Cell viability and number was evaluated with Trypan Blue (Invitrogen, California, USA) and Countess Automated Cell Counter (Invitrogen, California, USA). Two different tests of cryoconservation and expansion were performed on PDOs grown for 3 weeks. i. PDOs were dissociated to obtain single-cell suspension; cells were resuspended in cryoprotective medium (12-132A, Lonza, Walkersville, MD, USA), frozen and maintained in the vapor phase of liquid nitrogen. After 1 month, cells were thawed, resuspended in fresh organoids’ medium, tested for their viability with Trypan Blue (Invitrogen, California, USA) and directly embedded in Matrigel to re-establish the organoids, according to protocols previously described. ii. whole PDOs were frozen in 50% conditioned medium and 50% cryoprotective medium (12-132A, Lonza, Walkersville, MD, USA). Organoids were maintained for 1 month in the vapor phase of liquid nitrogen, then thawed and cultured in organoids’ complete medium. Graphs and statistical analyses were performed using GraphPad Prism software (GraphPad, La Jolla, CA, USA). All data in graphs represent the mean of at least three independent experiments ± SEM.
Immunohistochemical staining of PDOs was performed on 5 μm formalin fixed, paraffin-embedded tissue sections using a fully automated system (Bond-maX, Leica, Newcastle Upon Tyne, UK). Sections were de-waxed, rehydrated and incubated in retrieval buffer solution (Leica, Newcastle Upon Tyne, UK) for antigen recovery. Specimens were then washed with phosphate-buffered saline (pH 7.0) and incubated with the Bond Polymer Refine Detection Kit (Leica, Newcastle Upon Tyne, UK) according to the manufacturer’s protocols. Staining of proteins was performed with 3,3′-diaminobenzidine (DAB), and the slides were counterstained with Mayer’s haematoxylin (Diapath, Martinengo, Italy). Immunostains for NB84a (Leica; Newcastle, Clone NB84a, dilution 1:50), Synaptophysin (Dako, Clone DAK-SYNAP; dilution 1:100), Chromogranin A (Dako, Clone DAK-A3 dilution 1:200) and pHH3 (Thermo Scientific, dilution 1:100) were performed using an automated immunostainer. Images were acquired using Leica DM4000B microscope with Leica DFC295 camera. Measurement of the mitotic index was performed as follows: the percentage of positive tumour cell nuclei was counted in 5 × 100 cells for each case (magnification 400x, field size 0.18 mm2) in areas that are defined as “hot spots” (exactly where there are more positive nuclei).
Array comparative genomic hybridisation (aCGH) analysis
aCGH was performed according to Agilent oligonucleotide array-based CGH for Genomic DNA Analysis Protocol (version 7.3). Samples were compared with commercial genomic DNA (Promega, Madison, Wisconsin, USA) representative for female and male. 1200 ng/μl of DNA were digested at high temperature and labelled by random priming with CY5-dCTP for the patients and CY3-dCTP for controls. Purification of the samples was performed by means of Micron filters YM-30 (Sigma-Aldrich, Missouri, USA) DNA was hybridised to a 244 K platform, at 65 °C for about 40 h. Chips were scanned on DNA microarray Agilent Scanner and digital analysis was carried out with Agilent Genomic Workbench 7.0 using the Aberration Algorithm ADM-1 with an Aberration Threshold of 5.0.
Protein extraction and western blot analysis
PDOs were collected and dissociated in cold lysis buffer (Biosource International, USA) supplemented with Phosphatase Inhibitor Cocktail 2 (P5726-Sigma Aldrich, Missouri, USA), Phosphatase Inhibitor Cocktail 3 (P044-Sigma Aldrich, Missouri, USA), Protease Inhibitor Cocktail (P8340-Sigma-Aldrich, Missouri, USA) and PMSF (Sigma-Aldrich, Missouri, USA). Protein concentration was determined using Pierce BCA Protein assay kit (ThermoFisher Scientific, USA) and Wallac Victor spectrophotometer (Perkin Elmer, Massachusetts, USA) and loaded in Criterion TGX Precast gel (BioRad, California, USA). Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-DBH (CSB-PA958833, Flarebio biotech LLC., MD, USA); anti-LGR-5 (CSB-PA267899, Flarebio biotech LLC., MD, USA); anti-MycN (CSB-PA965036, Flarebio biotech LLC., MD, USA); anti-p75 NGF receptor (ab93934, Clone 2F1C2, Abcam, Cambridge, UK); anti-Vimentin (NB100–74564, Clone J144, Novusbiologicals, Colorado, USA); anti-GAPDH (NB300–221, Clone 1D4, Novusbiologicals, Colorado, USA); anti-YAP (#4912, Cell Signaling, Massachusetts, USA). Images were acquired using Alliance 9.7 Western Blot Imaging system (Uvitec limited).
In vitro limiting dilution colony assay
PDOs were collected and dissociated to obtain single-cell suspension as described above. Cells were plated into 96-well plates with decreasing numbers of cells per well (1000, 750, 500, 250, 125, 100, 50, 25, 10, 5, 2 and 1). Every dilution was replicated for 24 wells for three independent experiments. Cultures were monitored at light microscope for colony formation and, after 15 days of culture, wells were scored positive or negative for the presence of at least one colony. Data were analysed using extreme limiting dilution assay. Extreme limiting dilution analysis was performed using available software (http://bioinf.wehi.edu.au/software/elda/).
PDOs were dissociated to obtain single-cells as described above. 200.000 cells/tube were stained for 30′ at room temperature in the dark with: anti-CD15 FITC (IM1423U, Clone 80H5, Beckman Coulter, California, USA), anti-CD44 PE (550,989, Clone 515, Becton Dickinson, California, USA), anti-CD133 PE (130-1,/1, Miltenyi Biotech, Cologne, Germany), anti-CD29 PE (556,049, Clone HUTS-21, Becton Dickinson, California, USA), anti-CD24 ECD (IM2645, Clone ALB9, Beckman Coulter, California, USA) and anti-CD56 PeCy7 (A21692, Clone N901, Beckman Coulter, California, USA). Samples were analyzed on FC500 flow cytometer (Beckman Coulter, USA): percentages of different populations were calculated based on live-gated cells relative to physical parameters, side scatter and forward scatter. At least 30.000 live gate events were acquired. Analyses were performed using Kaluza Flow Cytometry analysis software (Beckman Coulter, USA). Five independent experiments were performed.