Buparlisib

Cells were maintained in humid incubators at 37 °C and 5 % CO₂. The U87 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 % fetal bovine serum, 3.2 % non-essential amino acids, 100 units/mL Penicillin/Streptomycin, 400 mol/L l-glutamine (all Lonza, Cologne, Germany) and 0.005 mg/mL Plasmocin (InvivoGen, San Diego, CA, USA). Prior to animal implantation, spheroids of U87 cells were prepared; 1000 cells were centrifuged at 2250 rpm for 30 min in 96-well plates with conical bottom, containing 0.05 % methylcellulose. After 7 days incubation, each spheroid contained 17,000 cells. Three spheroids (51,000 cells) were implanted in each animal. For in vitro assessment of buparlisib efficacy, a 10 mM stock solution was prepared by dissolving buparlisib [kindly provided by Novartis (Basel, Switzerland)] in 100 % DMSO (Sigma-Aldrich).

The kinase inhibitors Buparlisib (Pan-PI3K inhibitor) and MK-2206 (AKT1/2/3 inhibitor) were purchased from Selleck Chemicals (Absource Diagnostics GmbH, Munich, Germany). According to the manufacturer’s instructions, Buparlisib and MK-2206 were separately dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) as a stock solution at a final concentration of 10 mM. The stock solutions were stored at −80 °C and diluted into corresponding working concentrations before each experiment.

Homozygous nude rats (rnu/rnu, Rowett) were used for the experiments. The animals were fed a standard pellet diet and provided water ad libitum, and kept in a pathogen free environment at a constant temperature and humidity with standard 12/12 h light and dark cycle. The experiment was approved by the Norwegian Animal Research Authority (Bergen, Norway). All animals were anaesthetized with 3 % isoflurane gas (Abbott Laboratories, Abbot Park, IL, USA) mixed with 50 % air and 50 % O₂, and Marcaine (AstraZeneca, London, England) subcutaneously. Tumour implantation was performed as previously described [10 (link)]. Three animal studies were performed, and treatment started immediately after confirmed tumour take by MRI (U87 speroids; 10 days, P3 cell suspension; 21 days). Animals were randomly assigned to two different groups: (1) untreated controls and (2) 5 mg/kg buparlisib treatment (recommended by Novartis). buparlisib suspension was prepared in 0.5 % methyl cellulose and 0.5 % Tween20 (both Sigma-Aldrich), while control group received vehicle only (0.5 % methyl cellulose and 0.5 % Tween20). Both groups were treated 5 days a week and received 10 mL/kg solution by oral gavage, using malleable oral dosing needles (Scanbur, Karlslunde, Denmark). Animals were weighed five times a week, inspected daily and euthanized by CO₂ inhalation at the onset of symptoms.