In vivo imaging and characterization of an orthotopic retinoblastoma xenograft model

Abstract

Purpose: Xenografts of human retinoblastoma cells are an important system in which to test novel therapeutics for this pediatric ocular cancer. One recently developed orthotopic xenograft model involves injection of a luciferase-expressing Y79 human retinoblastoma cell line into the vitreous of newborn wild-type rats. Use of neonates takes advantage of the fact that these animals are naturally immunonaïve, and also allows for intraocular tumor growth at an age that is developmentally appropriate for retinoblastoma. We aimed to better characterize tumor growth in this model by combining both bioluminescence and intraocular fluorescence imaging of developing xenografts.

Methods: We engineered Y79 retinoblastoma cells to overexpress a luciferase-EGFP fusion. We assayed cell line bioluminescence in vitro using a plate reader and fluorescence with both a plate reader and a Typhoon laser scanner. PBS vehicle, 1,000, or 10,000 cells were injected into the vitreous of newborn Sprague-Dawley rats (N=30). Over a 28 day period, in vivo bioluminescence and fluorescence imaging was performed using a NightOwl bioluminescence imager and Phoenix Micron III intraocular imager, respectively.

Results: We confirmed linearity of detection of both bioluminescence (luciferase activity) and fluorescence (EGFP) with increasing cell number in vitro by both plate reader and laser scanner analysis. Xenografted cells formed rapidly growing tumors in 90% of animals. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated. Despite significant corneal neovascularization, even in vehicle-injected animals, intraocular brightfield and fluorescence imaging allowed delineation of tumor growth over time, including small tumors preferentially sitting atop the optic nerve head, multifocal seeding, and large vitreous-filling tumors that were highly vascularized.

Conclusions: The combination of non-invasive bioluminescence and in vivo intraocular brightfield/fluorescence imaging allows both quantitative and high-resolution spatial analysis of this retinoblastoma model, and will be applied to other cell lines and experimental therapeutic trials in future.

Keywords: 703 retinoblastoma • 551 imaging/image analysis: non-clinical • 744 tumors