Athymic nude foxn1nu nu mice

Subcutaneous tumours were generated in 6- to 7-week-old male Athymic Nude Foxn1nu/nu mice (ENVIGO) by injecting 2 × 10⁶ MIA PaCa-2 or PANC-1 cells embedded in Matrigel 1:1 (BD Biosciences) into each flank. Tumours were measured at least twice weekly, and the tumoural volumes calculated using the formula, V = D × d² × π ÷ 6 V. Mice were randomly assigned to either group for treatment. Viruses (4 × 10¹⁰ vp/animal) were administered intravenously in physiological saline solution once tumours achieved a median volume of 100 mm³. The experiment remained blinded until the conclusion of the study. Animals were euthanized 45–48 days after virus administration, and tumours were collected and flash-freezed in liquid nitrogen. All animal procedures met the guidelines of European Community Directive 86/609/EEC and were approved by the ethical committee (CEEA-University of Barcelona) and by the local authorities of the Generalitat de Catalunya.

All animal experiments were carried out with the approval of the Ethical Committees of the University of Alcalá and the Comunidad de Madrid (UAH-CAM, procedure PROEX 178/17), in accordance with the Spanish institutional regulation (RD 53/2013) for the housing, care and use of experimental animals and met the European Community directives regulating animal research. Athymic nude-Foxn1 (nu/nu) mice (aged 4 weeks) were purchased from Envigo RMS (Barcelona, Spain) and housed under pathogen-free conditions with access to food and water ad libitum. BHT101 cells (5 × 10⁶ in 200 mL of in serum-free DMEM medium) were injected subcutaneously into both flanks of the mice. When tumours reached a volume of 70 mm³, the mice were randomly divided into four experimental groups (n = 6 in each group). The six mice in each group were administered interperitoneally for 12 days with DMSO, PLX4720 (25 mg/kg/day), Chloroquine (CQ) (60 mg/kg/day), or a combination of PLX4720 and CQ. The tumour size was measured every day with a digital callipers and volume calculated as (length × width²)/2. At the end of the study, the mice were euthanized by placing them in a CO₂ gas-filled chamber, and the tumours were dissected, weighed and photographed.