Foxn1nu nu nu mice

LNEN xenografts were established 5 days following the subcutaneous injection of 4 × 10⁶ NCI-H727 cells into the backs of athymic nude mice (FOXN1NU NU/NU mice, ENVIGO, Israel). Once the neoplasm size reached 130 mm³, the mice were randomized into 4 groups and treated for the next 13 days with (1) vehicle (PBS, 100 μL), (2) CQ (60 mg/kg), (3) RAD001 (3 mg/kg), or (4) CQ and RAD001 (60 mg/kg and 3 mg/kg, respectively). All treatments were administered via IP (intraperitoneal) injections. Tumor size was measured daily using a caliper, and its volume was calculated using the following equation: length × (width²)/2. We chose the drug concentrations after conducting calibration experiments with the recommended concentrations in this model, based on reviewing the literature and choosing those concentrations that suggested efficacy [15 (link)]. The animals were treated daily (except for weekends). At the completion of the experiment, the mice were euthanatized, and the tumors were excised and weighed, followed by histopathological analysis including H&E staining and validation of the neuroendocrine tumor morphology. The subcutaneous xenograft model is presented in Figure 7 (adapted from our previous publication [15 (link)]).