Orthotopic mouse lung cancer model

Athymic nude mice (8–12 week old females) were obtained from an in-house breeding colony, maintained by the veterinary staff at the University of Colorado Anschutz Medical Campus. Mice were anesthetized using isoflourane and positioned laterally such that their flank was accessible. A small skin incision was made over the ribcage to visualize the lung. Cultured, luciferase-tagged NSCLC cells (1×10⁶) were injected directly into the lung using a 28-gauge needle, inserted to a depth of 3 mm. Tumor growth was assessed every 14 days using a Living Image IVIS system. Once a mouse’s tumor reached a threshold of 5×10⁸ p/s, the mouse was randomized into a treatment group. For the HCC4006 and H1650 lines the treatment groups were as follows: Vehicle (Methocel E4M), gefitinib (6.25 mg/kg), AZ1366 (either 25 or 50 mg/kg) or a combination of gefitinib and either dose of AZ1366. For the PC9T790M line, mice were treated with either vehicle (Methocel E4M), osimertinib (6.25 mg/kg), AZ1366 (25 mg/kg) or a combination of osimertinib and AZ1366. All drug combinations were delivered by oral gavage daily (5/7 days). Tumor growth was assessed every 14 days by IVIS imaging and mice were weighed bi-weekly. The event resulting in euthanasia was reaching moribund criteria, as evaluated by the veterinary staff. Mice were maintained in accordance with institutional guidelines, and all experimental procedures were approved by the University of Colorado Institutional Animal Care and Use Committee.