Bioluminescence Imaging (BLI)

Tumor growth and luciferase knockdown of animals were monitored on a Bruker Carestream In-Vivo Xtreme (Billerica, MA, USA). Tumor growth was monitored every 2 weeks until the 6-week time point. Animals designated to BLI studies were injected I.P. with 100 µL of a freshly prepared 15 mg/mL D-Luciferin (System Bioscience, San Francisco, USA) stock solution in PBS per 10 g of their body weight. After ten minutes, mice were treated with 3% isoflurane until sufficiently sedated. Maintenance isoflurane was used during imaging to keep the mice sedated. BLI images were taken with a three-minute exposure under high sensitivity and aperture of the lens set at an f-stop of 1.1. Simultaneously, X-ray images were taken under standard 1.2 second exposure. Images were transferred to ImageJ and regions of interest (ROIs) were drawn around the tumor and metastases to determine luciferase expression. Values were normalized to day 0 luciferase expression and analysis was performed by GraphPad Prism 5.0 software.