Male balb c nu nu mice

Male BALB/c nu/nu mice (20 g, 6 weeks old) were purchased from Orient Bio (Gyeonggi‐do). 5 × 10⁶ SNU‐1327 cells with Matrigel (BD Biosciences) were subcutaneously injected into the mice. The mice were randomly divided into four groups. After 100 mm³ tumor sizes, the mice were treated with the vehicle control (0.5% Tween 80 in PBS for oral treatment, 0.1% DMSO in PBS for intraperitoneal treatment), oral gefitinib (5 mg/kg, daily), intraperitoneal PAO (0.2 mg/kg, every 2 days), or the combined drugs for 3 weeks and checked the tumor volume twice per week. Tumor volumes were calculated by the following formula: volume (mm³) = width² × length/2. All procedures were performed according to the guidelines of the Institutional Animal Care and Use and IRB committees (KUIACUC‐2015‐13) at Korea University (Seoul, Korea).

U87-MG cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 4 mM L-glutamine, 4,500 mg/L glucose, 1 mM sodium pyruvate, 1.5 g/mL sodium bicarbonate, 10% FBS and antibiotic-antimycotic (Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere of 5% CO₂. U87-MG cells were cultured for 2–3 days without changing the medium and were harvested with trypsin-EDTA in PBS. Male Balb/c nu/nu mice, obtained at 6–7 weeks of age (Orient Bio Inc., Seongnam, Korea), were subcutaneously injected in the right shoulder with 5 × 10⁶ U87-MG cells suspended in 100 μL of 100 mM PBS (pH 7.4). Animal protocols were approved by the Seoul National University Bundang Hospital Animal Care and Use Committee and the methods were carried out in accordance with the approved guidelines. We also performed calliper measurements of the longest perpendicular tumour diameters.