Epidermal growth factor (egf)

Fresh tumor tissue was cut into pieces with a maximum diameter of 2 mm and implanted subcutaneously in anaesthetized NMRI-nu mice (RjOrl:NMRI-Foxn1^nu/Foxn1^nu, Janvier Labs SAS, Saint-Berthevin, France). After harvest from euthanized animals, tumor tissue was dissociated into a single cell suspension by the GentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) applying a standardized protocol. In brief, tissue was minced manually and subjected to repeated cycles of mechanical stirring and enzymatic digestion. The resulting suspension was filtered and centrifugalized. The pellet was gently resuspended in a medium (Airway Epithelial Cell Growth Medium with SupplementMix, PromoCell GmbH, Heidelberg, Germany) with the addition of 50 μg/ml ampicillin (Ratiopharm GmbH, Ulm, Germany). Cells were incubated at 37°C in 5% CO₂ for 4–7 days. Following that, the initial medium was gradually replaced by DMEM (Biowest, Nuaillé, France) with the addition of 10% fetal bovine serum, 14 μg/ml phosphoethanolamine, 11 μg/ml ethanolamine, 0.5 ng/ml EGF (all Biochrom AG, Berlin, Germany), 5.0 μg/ml hydrocortisone, 5.0 μg/ml insulin (Sigma-Aldrich, St. Louis, USA), 0.5 ng/ml FGF (R&D Systems Inc., Minneapolis, USA), and 50 μg/ml ampicillin.

Tumor samples were collected from patients diagnosed with newly diagnosed or recurrent GB and undergoing treatment at the Johannes-Gutenberg University Medical Center Mainz (UMM). Freshly resected tumor tissue was used for GSCs isolation in accordance with the approval of ethics committee (No. 837.178.17(11012) and patient’s informed consent. Human GSCs were isolated as previously described [22 (link),23 (link),24 (link)]. GSCs were maintained in NeuroBasal complete medium (Neurobasal^TM-A + B27 supplement, Gibco, Life Technologies, Darmstadt, Germany) containing the self-renewal promoting factors bFGF (basic fibroblast growth factor, 10 ng/mL) and EGF (epidermanl growth factor, 20 ng/mL) (Biochrom GmbH, Berlin, Germany). Serum-dependent glioma cell lines U87 and LN229 were purchased from the American Type Cell Culture Collection (ATCC) and propagated in DMEM medium supplemented with 5% fetal calf serum.