4.1. Cellular Models and Functional Assays

BTICs were derived from resected, newly diagnosed human malignant gliomas as previously described [12]. The sampling of tumor specimens and enrichment of BTICs was approved by the Ethics Committee of the University of Regensburg (No° 09/101), and all patients gave written informed consent. We used the following core set of lines for our experiments: BTIC-8, BTIC-11, BTIC-13, and BTIC-18 (Supplementary Table S1). BTICs were kept in DMEM low glucose medium (DMEM with 1 g/L of glucose; Sigma-Aldrich, #D6046) containing Epidermal Growth Factor (Miltenyi Biotec, #130-097-751, Bergisch Gladbach, Germany) and Fibroblast Growth Factor (Miltenyi Biotec, #130-093-842, Bergisch Gladbach, Germany) supplemented with 50 U (v/v) Penicillin, 0.05% (v/v) Streptomycin (#P4333), 2 mM (v/v) L-Glutamine (#G7513), 1% (v/v) MEM Vitamin Solution (#M6895), and 1% (v/v) non-essential amino acids (#M7145) (all Sigma-Aldrich, St. Louis, USA). For differentiation, growth factors were withdrawn, and cells were exposed to fetal calf serum (FCS) for at least two weeks. Cells were incubated at 37 °C, 5% CO₂, 95% humidity in a standard cell culture incubator. All experiments were performed with mycoplasma-free cells.

Proliferation was assessed using the CyQUANT^® Direct Cell Proliferation Assay (Thermo Scientific, #{"type":"entrez-nucleotide","attrs":{"text":"C35012","term_id":"2371153","term_text":"C35012"}}C35012, Waltham, MA, USA) according to the manufacturer’s protocol. The spheroid migration assays were performed as previously described [11]. To distinguish proliferation from migration effects, we used early time points, i.e., after 16 and 24 h, which preceded the cell doubling time (average doubling time of BTICs and TCs = 45.8 h).

BTICs und TCs were lentivirally transduced using U57 pHR SFFV GFP (BTIC-13 and TC-13) and pLenti-H1-(shRNA-Neg-control)-Rsv(RFP-Bsd) plasmids (BTIC-18 and TC-18). Lentivirally transduced BTICs were grown as spheroids in agarose-coated 96-well plates (10,000 cells/well) 48 h prior to implantation. OBSC were prepared according to Gogolla et al. [44] with individual modifications as described [12]. We implanted one spheroid per slice and used three technical replicates. Spheroid diameter ranged between 3 and 8 × 10⁻⁵ µm² depending on initial cell size. Two days were sufficient to form cell spheroids that were transferrable by pipette. The lentiviral transduction did not affect proliferation or migration compared to non-transduced counterparts.

Metformin hydrochloride (Sigma-Aldrich #PHR1084, St. Louis, MI, USA) was dissolved in DMEM low glucose (5.5 mM) medium and used at indicated doses.