Human epidermal growth factor

Bradford assay reagent was purchased from Bio-Rad Laboratories, Inc. (Mississauga, ON, Canada). Alamethicin and UDP-glucuronic acid (UDPGA) were obtained from Corning Life Sciences (Tewkbury, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), F12/DMEM, fetal bovine serum (FBS), L-glutamine, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), penicillin–streptomycin (Pen-strep) solution, TryPLE Express, and trypsin [0.25% with EDTA (1 ×)] were obtained from Life Technologies Inc. (Burlington, ON, Canada). Docosahexaenoic acid (DHA) was purchased from Nu-Chek Prep Inc (Elysian, MN, USA). Human epidermal growth factor (EGF) was purchased from PeproTech (Rocky Hill, NJ, USA). NADPH was obtained from Roche Diagnostics (Laval, QC, Canada). Bovine insulin, bovine serum albumin (BSA), hydrocortisone, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), phloridzin (PZ), quercetin, S-adenosylmethionine (SAM) were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Cell lines were grown in the media indicated in Table S5. All growth media were supplemented with antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM l-glutamine (Thermo Fisher Scientific, Waltham, MA, USA). Normal murine neural stem/progenitor cells were isolated from the sub-ventricular zone of adult C57BL/6J mice. Tumoral murine GL261 stem/progenitor cells were derived from C57BL/6J mice harboring GL261 glioma. Both murine normal and tumoral stem/progenitor cells were expanded as floating neurospheres and maintained in DMEM (Dulbecco’s Modified Eagle Medium)/ Ham’s F-12 medium (1:1) supplemented with B-27 (Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL human epidermal growth factor (EGF), 10 ng/mL human basic fibroblast growth factor (bFGF) (PeproTech, London, UK), and 0.0002% heparin (Merck KGaA, Darmstadt, Germany). The analysis of normal and tumor mouse neurospheres was performed on cells derived from the same mouse, which were collected at different passages. Cell lines and primary cultures were grown in a humidified 37 °C incubator with 5% CO2.

Enriched-cancer stem cells (CSCs) were obtained under specific culture conditions as reported in the literature (Brown et al., 2017 (link); Bassi et al., 2020 (link)) as a sarcosphere-forming method starting from a human MG63 osteosarcoma cell line. The MG63 cell line was seeded in Ultra-Low Attachment T25 flasks (Corning Inc., NY) with a density of 2.0 × 10³ cells/cm² in serum-free DMEM F12-GlutaMAX™ Modified Medium supplemented with a specific cocktail of factors: 10 μL/ml N2 (Gibco), 20 μL/ml B27 (Gibco), 0.1 μL/ml human Basic-Fibroblast Growth Factor (bFGF) (Invitrogen), and 0.01 μL/ml human Epidermal Growth Factor (EGF) (PeproTech). The cocktail was added to each flask every 2/3 days for a total of 10 days of culture. After their formation, the CSCs were collected and centrifugated for 10 min at 130 × g; the pellet was resuspended in the same medium conditions, well mixed, and directly seeded in Ultra-Low Attachment 96 well-plate and Ultra-Low Attachment 6 well-plate with 200 µL/well and 1.5 ml/well volume of cell culture medium, respectively. The factors’ cocktail was added every 2/3 days during the experiment following the above-reported manufacturer’s instructions.

Hippocampal neural stem and progenitor cells (HNSPCs) derived from the juvenile/adult rat (P43-55; Merck-Millipore, SCR022) were cultured in a mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s Nutrient Mixture F-12 (DMEM/F-12; Wako Pure Chemical) containing B-27 supplement (Invitrogen), human basic fibroblast growth factor (FGF-2; 20 ng/ml; PeproTech) and human epidermal growth factor (EGF; 20 ng/ml; PeproTech), in a CO₂ incubator at 37 °C. Cells were sub-cultured upon reaching 80% confluency using Accutase (Innovative Cell Technologies).

As representative of mesenchymal stem cells, the human immortalized stem cell line SCP-1 was used [37 (link)]. SCP-1 cells were cultured in Minimal Essential Medium Alpha (MEM-α, HiMedia Laboratories, Thane West, Maharashtra, India), supplemented with 5% fetal calf serum (FCS, Thermo Fisher, Waltham, MA, USA). Human umbilical cord vein cells (HUVEC) were used as a proxy for endothelial cells. HUVECs were cultured in Endothelial Cell Growth Basal Medium 2 (EBM-2, Peprotech, Hamburg, Germany), supplemented with 2% FCS, 1% Antibiotic/Antimycotic (A/A, PAA Laboratories, Toronto, Canada), 0.5 ng/mL human VEGFA165, 10 ng/mL human FGF-b, 5 ng/mL human epidermal growth factor (EGF), 20 ng/mL human IGF-R3 (all Peprotech, Hamburg, Germany), 22.5 µg/mL heparin (Leo Pharma, Bellerup, Denmark), 0.2 µg/mL hydrocortisone (Pfizer, New York, NY, USA), and 1 µg/mL L-ascorbic acid (Sigma-Aldrich/Merck, Darmstadt, Germany) in culture flasks coated with 0.1% gelatin (Sigma-Aldrich/Merck, Darmstadt, Germany). The culture medium was replaced every 3–4 days. The cells were subcultured upon reaching confluency. SCP-1 cells in passages between 10 and 20 and HUVECs in passages between 5 and 10 were used for the experiments.

To test the ability of cells with modified gene expression levels to form tumor spheres, we generated tumor spheres as previously described [18 (link)]. In detail, the adherent growing cell lines were dissociated into single cells using trypsin/EDTA and 2000 single cells per well seeded in ultra-low attachment 6-well plates (Corning, NY, USA) using serum-free MEBM (Lonza, Basel, Switzerland) medium (SFM) supplemented with 1xB27 supplement (Gibco), 20 ng/ml human epidermal growth factor EGF (Peprotech, Hamburg, Germany), 10 ng/ml human basic fibroblast growth factor FGF (Peprotech), 20 IU/ml heparin (Baxter, Vienna, Austria), and 1% antibiotic/antimycotic solution (Thermo Fisher Scientific, containing 10,000 units/mL of penicillin; 10,000 μg/mL of streptomycin; and 25 μg/mL of Gibco Amphotericin B). After 14 days, the number of spheres was counted using a bright-field microscope in three independent replicates.