Phox2b

Samples taken from 17 NB tumors on relapse were kept in culture medium (RPMI-1640) for 24 h or stored in a freezing solution containing 90% of serum and 10% DMSO until use (histological immunophenotyping and DNA extraction). After paraffin embedding, immunohistochemistry phenotyping was carried out. Tumor tissue sections (3 µm) were de-paraffinized and incubated with the immune-cell marker CD45 (LCA) (Mouse Monoclonal Antibody (mAb)—Cell Marque/Roche), and with the NB markers CD56 (MRQ-42) (Rabbit mAb—Cell Marque/Roche), TH (F-11) (Mouse mAb—Santa Cruz Biotechnology), PHOX-2B (EPR14423) (rabbit mAb—Abcam) and S100 (4C4.9) (Mouse Monoclonal Antibody—Ventana/Roche). In some cases, primary Abs were diluted with Ventana/Roche Ab diluent (for TH mAb) or with BOND Primary Antibody Diluent—Leica (for PHOX-2B mAb). The ultraView Universal DAB detection kit from Ventana and the BOND Polymer Refine Detection kit from Leica (for PHOX-2B mAb) were used to detect the binding of primary antibodies. Sections were counterstained with HEMATOXYLIN—Ventana/Roche.

Gelatin from porcine skin (Type A, SLCC7838), methacrylic anhydride (MA), Irgacure (2‐Hydroxy‐4′‐(2‐hydroxye‐thoxy)‐2‐methylpropiophenone), and PBS were obtained from Sigma‐Aldrich (Wisconsin, U.S.A.). AlamarBlue and qPCR kits were purchased from Bio‐Rad (Hercules, U.S.A). Carbopol ETD 2020 polymer was purchased from Lubrizol (Wickliffe, U.S.A.). Calcein AM and propidium iodide (PI) were obtained from Biotium (Fremont, U.S.A.). HUVEC culture medium (VascuLife VEGF) was purchased from Lifeline Cell Technology (Oceanside, U.S.A.). Conjugated F‐actin and wheat germ agglutinin (WGA) were purchased from ThermoFisher, USA; CD31, Connexin 43, Phox2B and Synaptophysin antibodies were purchased from Abcam, USA.

Tumors were fixed in 4% formalin, embedded in paraffin, and cut in 4-μm sections for staining. H&E (Histolab Products) staining was performed for histopathological analysis. The slides were blinded for morphological differentiation assessment and evaluated by an independent viewer. TUNEL assay (Abcam, ab206386), using either methyl green or hematoxylin as counterstain, was performed to asses cell death. Quantification was performed using the software QuPath 0.2.3. 3,3’-Diaminobenzidine (DAB) staining was performed using the Autostainer Plus (Dako), and fluorescence staining was performed manually. Heat-induced antigen retrieval was done using sodium citrate buffer (pH 6.0). The antibodies used are PHOX2B (1:1000; Abcam), PHOX2B-AF647 conjugate (1:100; Santa Cruz Biotechnology), Ki67 (1:200; Dako), CD34 (1:200; Santa Cruz Biotechnology), MYCN (1:200; Proteintech), TH (1:1000; Abcam), SOX9 (1:500; Abcam), and LGR5 (1:150; Abcam). Processing and quantification of fluorescence images were performed with ImageJ.