Human recombinant epidermal growth factor

The immortalised non-tumourigenic human epithelial cell line MCF12A (ATCC, CRL-10782)^{89 (link)} was cultured in DMEM/F12 (Gibco) supplemented with 5% horse serum (Sigma), 10 ng ml⁻¹ cholera toxin (Sigma), 10 μg ml⁻¹ insulin (Sigma), 0.5 μg ml⁻¹ hydrocortisone (Sigma), 20 ng ml⁻¹ human recombinant epidermal growth factor (StemCell Technologies), 1% Penicillin/Streptomycin (Gibco) and 1% L-Glutamine (Lonza).

Gliomaspheres were generated in analogy to well established standard protocols.37, 39, 40, 41 Briefly, glioblastoma cells were transferred from T75 to T25 flasks (to facilitate cell‐cell contacts) with serum‐free media supplemented with growth factors – as typically used for a spheric phenotype15, 36, 42: DMEM/Nutrient Mixture F‐12 medium (DMEM, Gibco, Life Technologies, Paisley, UK) supplemented with 20% BIT‐serum free supplement (bovine serum albumin, insulin, transferrin), human recombinant epidermal growth factor and human basic fibroblast growth factor at 20 ng/mL each (all STEMCELL Technologies, Vancouver, BC, Canada). For passaging and plating, spheres were transferred into conical tubes, centrifuged (200 g, 5 minutes), resuspended in serum‐free medium, dissociated into single‐cell suspensions using trituration (60‐70 times using a Pasteur pipette) and re‐plated. BIT as well as growth factors were added freshly to maintain the concentration after each passage. Tumor spheres were observed under a light microscope every day.

Human GBM samples were obtained from consenting patients, as approved by the Hamilton Health Sciences/McMaster Health Sciences Research Ethics Board. Brain tumor samples were dissociated as previously described [17 (link)] and cultured as neurospheres in Neurocult complete (NCC) media, a chemically defined serum-free neural stem cell medium (STEMCELL Technologies, Vancouver, BC, Canada), supplemented with human recombinant epidermal growth factor (20 ng/mL: STEMCELL Technologies, Vancouver, Canada), basic fibroblast growth factor (20 ng/mL; STEMCELL Technologies, Vancouver, Canada), heparin (2 μg/mL 0.2% Heparin Sodium Salt in PBS; STEMCELL Technologies, Vancouver, Canada), antibiotic-antimycotic (10 mg/mL; Wisent Bioproducts, Saint Bruno, QC, Canada) in ultra-low attachment plates (Corning, New York, NY, USA). Primary GBM cells (BT 428, BT 458 and BT 624) were cultured in NSC complete media and flow-sorted for CD133+ and CD133− populations as described previously [18 (link),19 (link)]. Transfections were carried out by Lipofectamine 2000 as per the manufacturer’s instructions.