Xenograft Model for Retinoblastoma

Animal protocols were in accordance with local and national guidelines. All animals were handled in agreement with the standard operating procedures of the University Health Network and all procedures were approved by the Animal Care Committee of the University Health Network, Toronto. IVT injections were performed under general anesthesia using isoflurane. Human RB cell lines were prepared at 25,000 cells/μL in sterile PBS with 10% matrigel (BD Bioscience, Mississauga, ON, Canada) and 5% trypan blue, then 2 μL of the mixture were injected into the right vitreous of 3–4 weeks-old NOD-Scid mice. Uninjected left eye served as a negative control. We used three animals per dose tested (unless specified otherwise). After seven days, MLN4924 or PBS was injected in tumor-bearing eyes at the indicated doses. Luciferase⁺ tumor cells were tracked live by i.p injection of d-luciferin at 150 mg/kg for 10 min and radiance total flux (photons/second) tracked with the Xenogen IVIS Imaging System 100 (PerkinElmer, Woodbridge, ON, Canada). For other RB lines, tumor volume was evaluated on sections as described^{44 (link)}. A human marker (intact mitochondria antibody, MAB1273, Millipore, Etobicoke, ON, Canada) was used to delineate tumor cells.