P4333

Human neuroblastoma SH-SY5Y cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM, #11995073, GIBCO) with 10% fetal bovine serum (FBS, vol/vol, #26140079, GIBCO) and a penicillin–streptomycin antibiotic solution (100 U/mL, #P4333, Sigma-Aldrich). PC12 cells, noradrenergic clonal cells obtained from the adrenal medulla of Rattus norvegicus (ATCC), were grown in DMEM (#11995073, GIBCO) containing 10% FBS (vol/vol, #26140079, GIBCO), 5% Horse serum (vol/vol, #26050-088, GIBCO), 2 mM Glutamine (#G7513, Sigma-Aldrich), and penicillin–streptomycin antibiotic solution (100 U/mL, #P4333, Sigma-Aldrich). Both types of cells were grown at 37°C in a humidified atmosphere consisting of 5% CO₂/95% air. X-tremeGENE HP transfection reagents (#6366546001, Roche) were used for transient transfection, following the manufacturer’s instructions.

For cell culture experiments, we used mesenchymal stem cells (hMSC): bone marrow-derived (female, 26 years) and adipose-derived (female, 18-30 years) and THP-1 cells, purchased from RoosterBio and American Type Culture Collection (ATCC), respectively. The hMSC cells were maintained in DMEM high glucose medium with L-glutamine (D5796, Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F6765, Sigma-Aldrich), 1% penicillin–streptomycin (P4333, Sigma-Aldrich), and 1% NEAA (M7145, Sigma Aldrich). The THP-1 cells were maintained in RPMI-1640 medium (R7388, Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F6765, Sigma-Aldrich), 1% penicillin–streptomycin (P4333, Sigma-Aldrich), and 0.05 mM 2-mercaptoethanol. All cells were cultured at 37 °C in 5% CO₂ and were mycoplasma-free. The hMSC passages were performed at 75-80% confluence using 0.25% trypsin (59418C, Sigma-Aldrich). The hMSCs were used below seven passages, and the THP-1 cells were used below 40 passages. Viable cells were counted using a hemocytometer and the trypan blue exclusion method (T8154, Sigma–Aldrich). The differentiation of THP-1 monocytes to macrophages occurred after treatment with 18 nM of phorbol 12-myristate 13-acetate (1585, Sigma-Aldrich) for 12 hrs. Macrophage M2 polarization was performed using 20 ng/mL of IL-13 and 20 ng/mL of IL-4 for 72 hrs.

30 samples derived from 24 patients with neuroblastoma were included in this study. All tumor samples were collected from patients at the Great Ormond Street Hospital following written informed consent. In the case of minors, consent was obtained from adults with parental responsibility, with written assent from the child being optional for older participants. Ethical approval for the study was obtained through the national United Kingdom research ethics application system of the UK Health Research Authority; reference 14/WM/1253 “Establishing primary cultures and cell lines from pediatric cancers”. Tumor tissue was disaggregated manually into 1–2 mm fragments in X-VIVO 15 media (BE02-060F, Lonza) supplemented with 5% heat-inactivated human AB serum (H3667, Sigma-Aldrich), 100IU/mL penicillin (P4333, Sigma-Aldrich) and 100 μg/mL streptomycin (P4333, Sigma-Aldrich).

BEAS-2B cells were purchased from American Type Culture Collection (ATCC^® CRL-9609^TM, In Vitro Technologies, Melbourne, Victoria, Australia) were cultured in BEGM^TM bronchial epithelial cell growth medium BulletKit^TM (CC3170, Lonza, Mount Waverley, Victoria, Australia) containing BEGM^TM basal medium (CC-3171, Lonza, Mount Waverley, Victoria, Australia) and BEGM^TM SingleQuots^TM supplements (CC-4175, Lonza, Mount Waverley, Victoria, Australia) supplemented with 1% penicillin/streptomycin (P4333, Sigma, North Ryde BC, New South Wales, Australia) and 1% amphotericin B (A2942, Sigma, North Ryde BC, New South Wales, Australia). Human small airway epithelial cells (SAECs) (CC-2547, purchased from Lonza, Mount Waverley, Victoria, Australia) were cultured in SAGM^TM small airway epithelial cell growth medium BulletKit (CC-3118, Lonza, Mount Waverley, Victoria, Australia) containing SABM^TM basal medium (CC-3119, Lonza, Mount Waverley, Victoria, Australia) and SAGM SingleQuots^TM supplements (CC-4124, Lonza, Mount Waverley, Victoria, Australia) supplemented with 1% penicillin/streptomycin (P4333, Sigma, North Ryde BC, New South Wales, Australia) and 1% amphotericin B (A2942, Sigma, North Ryde BC, Australia), grown in flasks coated in bovine collagen I (A10644-01, Life Technologies, Scoresby, Victoria, Australia). Cells were maintained in a humidified incubator (37 °C, 5% CO₂).