B27 and n2 additives

Before any therapy, glioblastoma samples were obtained after informed consent from patients admitted to the neurosurgery department at Toulouse University Hospital. Tumors were histologically diagnosed as glioblastoma according to WHO criteria. For patients 1 and 2, two tumor samples were removed from the cortical area (CT1, CT2) and from the periventricular zone (PVZ1 and PVZ2) by utilizing stereotactic image-guided sampling. These patients had a large tumor that was in contact with both CT and PVZ. After mechanical dissociation of tumor tissues, cells were seeded at 37° C in a humid atmosphere of 5% CO₂ in glioblastoma stem cell medium (GSM) composed of DMEM-F12 (Lonza) supplemented with B27 and N2 additives (Invitrogen), EGF (20 ng/mL) and basic FGF (20 ng/mL) (Peprotech). When neurospheres were formed, they were isolated, dissociated with trypsin and cultured as previously described [23 (link)]. The percentage of GSCs into the neurosphere was evaluated by flow cytometry [23 (link)] (Supplementary Table 10). For other patients (A1, G, I, K, SC1, SC3), only one tumor sample was removed from different brain zones.

The experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. All tumors were frozen after surgical resection. These tumors were either clinically or genetically characterized in the department of neurosurgery of the Pellegrin Hospital (Bordeaux, France), and informed consent was obtained in accordance with the French legislation or was obtained from the processing of biological samples through the Centre de Ressources Biologiques (CRB) Santé of Rennes BB‐0033‐00056. The research protocol was conducted under French legal guidelines and fulfilled the requirements of the local institutional ethics committee. GBM were classified according to (i) the presence of IDH1, OLIGO2 and TP53 expression and (ii) tumor phenotype (size and form of tumor cells, hyperplasia, necrosis, proliferation index). Primary GBM lines were generated as previously described (Avril et al, 2017). Briefly, fresh tumor tissues were mechanically dissociated using gentleMACS dissociator following the manufacturer's instructions (Miltenyi Biotec, Paris, France). RNS cells (neurospheres enriched in cancer stem cells) were directly cultured in DMEM/Ham's F12 (Lonza, Verviers, Belgium) supplemented with B27 and N2 additives (Invitrogen, Cergy Pontoise, France), EGF (20 ng/ml), and FGF2 (20 ng/ml) (Peprotech, Tebu‐Bio).