Multimodality Imaging Methods for Assessing Retinoblastoma Orthotopic Xenograft Growth and Development

et al. (2014) Multimodality Imaging Methods for Assessing Retinoblastoma Orthotopic Xenograft Growth and Development. PLoS ONE 9(6): e99036. doi:10.1371/journal.pone.0099036 Multimodality Imaging Methods for Assessing Retinoblastoma Orthotopic Xenograft Growth and Development Timothy W. Corson 0 1 Brian C. Samuels 0 1 Andrea A. Wenzel 0 1 Anna J. Geary 0 1 Amanda A. Riley 0 1 Brian P. McCarthy 0 1 Helmut Hanenberg 0 1 Barbara J. Bailey 0 1 Pamela I. Rogers 0 1 Karen E. Pollok 0 1 Gangaraju Rajashekhar 0 1 Paul R. Territo 0 1 Sanjoy Bhattacharya, Bascom Palmer Eye Institute, University of Miami School of Medicine, United States of America 0 Current address: Department of Ophthalmology, University of Alabama at Birmingham , Birmingham, Alabama , United States of America 1 1 Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 3 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 4 Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, Indiana, United States of America, 5 Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 6 Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 7 Eastern University , St. Davids , Pennsylvania, United States of America, 8 Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 9 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 10 Herman B Wells Center for Pediatric Research, Department of Pediatrics, Section of Pediatric Hematology/Oncology, Riley Hospital for Children at Indiana University Health , Indianapolis , Indiana, United States of America, 11 Indiana Center for Vascular Biology and Medicine, Indiana University School of Medicine , Indianapolis, Indiana , United States of America Genomic studies of the pediatric ocular tumor retinoblastoma are paving the way for development of targeted therapies. Robust model systems such as orthotopic xenografts are necessary for testing such therapeutics. One system involves bioluminescence imaging of luciferase-expressing human retinoblastoma cells injected into the vitreous of newborn rat eyes. Although used for several drug studies, the spatial and temporal development of tumors in this model has not been documented. Here, we present a new model to allow analysis of average luciferin flux (F ) through the tumor, a more biologically relevant parameter than peak bioluminescence as traditionally measured. Moreover, we monitored the spatial development of xenografts in the living eye. We engineered Y79 retinoblastoma cells to express a lentivirally-delivered enhanced green fluorescent protein-luciferase fusion protein. In intravitreal xenografts, we assayed bioluminescence and computed F , as well as documented tumor growth by intraocular optical coherence tomography (OCT), brightfield, and fluorescence imaging. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated in orthotopic xenografts. By OCT, xenografts were dense and highly vascularized, with well-defined edges. Small tumors preferentially sat atop the optic nerve head; this morphology was confirmed on histological examination. In vivo, F in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of F modeling and in vivo intraocular imaging allows both quantitative and high-resolution, non-invasive spatial analysis of this retinoblastoma model. This technique will be applied to other cell lines and experimental therapeutic trials in the future. - Funding: This work was supported in part by the Indiana Clinical and Translational Sciences Institute funded, in part by the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (TR000006 and TR000163; TWC, BCS and GR), by R01 CA138798 (BJB and KEP) and by an Alcon Research Institute Young Investigator Award (TWC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journals policy on Competing Interests and have the following conflicts: TWC has received research funding (unrelated to the current study) and travel support from Phoenix Research Laboratories, Inc., manufacturer of a piece of equipment used in the study. No author has any other conflict of interest to disclose in relation to this study. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. The pediatric ocular tumor retinoblastoma is the prototypic genetic cancer [1]. It is initiated in most cases by mutation of both alleles of the RB1 gene, the first tumor suppressor gene to be cloned, although some retinoblastomas initiate without RB1 mutation [2]. In recent years, genetic characterization of retinoblastomas beyond loss of RB1 has provided multiple potential targets for therapeutic intervention (reviewed in [3]), including the oncogenes KIF14 [4], MYCN [2], E2F3 [5], DEK [5], MDM4 [6] and SYK [7], the tumor suppressor cadherin-11 [8], and the oncomiR cluster 17,92 [9]. However, targeted therapeutics for retinoblastoma have yet to transition into the clinic. Currently, the standard of care for this cancer involves laser therapy or cryotherapy for small tumors, often with systemic cytotoxic chemotherapy. Treatment of large tumors often requires enucleation of the eye or the use of external beam radiation; however, patients subjected to radiation therapy incur a lifetime risk of treatment toxicity [1]. As molecular targeted therapies become a possibility for retinoblastoma, effective animal models are needed for testing these therapies in vivo [10]. Although genetically modified mice are popular models for retinoblastoma, the complex derivation of such models and lack of some shared characteristics with the human tumor [11] have led to considerable interest in xenograft models of this cancer. In recent years, bioluminescence imaging (BLI) has been combined with orthotopic retinoblastoma xenografts to document tumor growth in vivo [12,13]. One model involves intravitreal injection of luciferase-expressing Y79 retinoblastoma cells into the eyes of newborn (postnatal day 0, P0), wild type rats [12]. This neonate model offers two key advantages: 1) a developmentally appropriate host environment for these pediatric tumor cells, and 2) a naturally immuno (...truncated)