Ln405

HEK293 and human glioblastoma cell lines LN405, T98G, U87 were obtained from the American Type Culture collection (LGC Standards GmbH; Wesel, Germany). Cells at a density of 2.5 × 10⁴ cells/ml, 200 μl per well, were seeded on microplates (Greiner, μClear, black clear bottom) in DMEM (4.5 g/L glucose, 10% FCS, 2 mM Glutamax, containing antibiotics). Medium was exchanged 24 h later for 100 μl per well of Hanks’ solution (HBSS) (1 g/L glucose, 0.37 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄, 4.2 mM NaHCO₃). P-analogs of pyruvate were added at different concentrations (0.2, 0.5, 1.0, 10 and 20 mM). 5 h later ATP levels were determined using the CellTiterGlo assay system (Promega, Heidelberg, Germany) according to manufacturer's recommendations as described previously [40 (link)]. Concentration dependence data were obtained by averaging luminescence from six wells, and % of the ATP levels in the treated vs control cells were used to characterize the effects of the analogs on cellular viability.

This study was approved by the Ethical Committee for human experimentation of IEO (European Institute of Oncology) and all patients signed an approved consent document prior to surgery. Surgical specimens of tumors were collected at the Neurosurgery Dpt. at IRCCS Istituto Clinico Humanitas and examined by a neuropathologist to verify that each case met criteria for GBM and to select a tissue fragment with high content of viable tumor tissue. Each tissue specimen was dissociated into single cell suspension and maintained as neurospheres in growth factors supplemented DMEM-F12 1:1 medium, as previously described [38 (link)]. Human GBM cell lines U87MG, A172, LN405, U118MG, T98G, DBTRG-05MG and U373 MG were purchased from American Type Culture Collection (ATCC) and maintained in DMEM (Lonza) supplemented with 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, and 10% FBS. All cell cultures were maintained at 37°C in a humidified 5% CO₂ incubator.

Five malignant human glioma cell lines, LN405, U118, SW1080, T98M and U87MG were purchased from the American Type Culture Collection (ATCC, USA) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cells were incubated at 37°C in a humidified chamber containing 5% CO₂. The cells were harvested in the logarithmic phase of growth for use in the experiments outlined below.