In vivo imaging.

NSG female mice were injected intravenous (IV) with 5×10⁴ human MOLM-13-Luc luciferase cells. Xenogen IVIS bioluminescence (BLI) imaging platform (PerkinElmer) was used to acquire whole body BLI for each animal to confirm tumor establishment. Mice were injected intraperitoneal (IP) with 150 mg/kg of luciferin based on body weight and imaged 10 to15 minutes later as per manufactures instructions. Whole body [¹⁸F]FLT-PET/CT imaging was conducted using sequential Siemens Focus micro-PET (200uCi [¹⁸F]FLT IV, 60 minute incubation time, followed by 10 minute static scan) and Gamma Medica FLEX micro-CT (80 kVp, 440 uA). Nine days after tumor injection, mice with tumor burden were randomized into three treatment groups (n = 10 per group): vehicle alone (7 days daily, oral (PO)), AMG 900 at 22 mg/kg (4 days daily, PO), and AMG 900 at 12.6 mg/kg (7 days daily, PO). [¹⁸F]FLT measurements were recorded at baseline (one day before treatment) and on days 5, 8, 11, and 14 post-treatment start. BLI measurements were collected on day 1 (before dosing on the day of treatment initiation) and on days 6, 9, 12, 15 post-treatment start. Data is represented as mean whole body BLI signal (photons/second, as per Xenogen instructions) or mean skeleton [¹⁸F]FLT signal ± SEM. To quantify skeleton [¹⁸F]FLT signal, a custom analysis program created by inviCRO Inc. (Boston, MA) was used to capture the whole body skeletal bone volume based on CT segmentation algorithm. CT derived skeleton regions-of-interest were used to quantitate the percentage of injected [¹⁸F]FLT dose per gram of tissue (%ID/g). Statistical significance for [¹⁸F]FLT and BLI was determined by comparing treated groups versus vehicle-control group using paired t-test. Study and image analysis was performed by inviCRO Inc..

Details about high-content cell imaging and flow cytometry (FC) assays are described in the Supplementary Materials and Methods.