4.12. Tumor Animal Models

All animal procedures were performed according to protocols approved by the Animal Experimentation Ethics Committee from the University of Barcelona (ethic code: #9850). For the subcutaneous tumor model, male Swiss nude mice, 5–6 weeks old, were kept under pathogen-free conditions with free access to standard food and water. HepG2 sorafenib-resistant cells (5 × 10⁶) or BCLC9 cells (2.5 × 10⁶) were injected subcutaneously into the flanks of mice in 100 μL DMEM without FBS, as previously reported [19,23,53]. Treatments with A-1331852 (25 mg/Kg body weight), regorafenib (30 mg/Kg), or vehicle (12.5% Cremophor, 12.5% ethanol, 75% sterile saline) were delivered daily via oral gavage. Tumors were measured periodically with a Vernier caliper, and the volume was calculated as length × width² × 0.5.