Corson Timothy W, Samuels Brian C, Wenzel Andrea A, Geary Anna J, Riley Amanda A, McCarthy Brian P, Hanenberg Helmut, Bailey Barbara J, Rogers Pamela I, Pollok Karen E, Rajashekhar Gangaraju, Territo Paul R
Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, Indiana, United States of America.
Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
PLoS One. 2014 Jun 5;9(6):e99036. doi: 10.1371/journal.pone.0099036. eCollection 2014.
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 ([Formula: see text]) 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 [Formula: see text], 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, [Formula: see text] in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of [Formula: see text] 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.
小儿眼部肿瘤视网膜母细胞瘤的基因组研究正在为靶向治疗的发展铺平道路。对于测试此类治疗方法而言，诸如原位异种移植等强大的模型系统是必要的。一种系统涉及将表达荧光素酶的人视网膜母细胞瘤细胞注入新生大鼠眼玻璃体内后的生物发光成像。尽管该模型已用于多项药物研究，但其肿瘤的空间和时间发展情况尚未有记录。在此，我们提出一种新模型，用于分析通过肿瘤的平均荧光素通量（[公式：见正文]），这是一个比传统测量的峰值生物发光更具生物学相关性的参数。此外，我们监测了活体眼中异种移植瘤的空间发展情况。我们对Y79视网膜母细胞瘤细胞进行基因工程改造，使其表达通过慢病毒递送的增强型绿色荧光蛋白 - 荧光素酶融合蛋白。在玻璃体内异种移植中，我们测定了生物发光并计算了[公式：见正文]，还通过眼内光学相干断层扫描（OCT）、明场和荧光成像记录了肿瘤生长情况。在原位异种移植中，体内生物发光、离体肿瘤大小和离体荧光信号均高度相关。通过OCT观察，异种移植瘤致密且血管高度丰富，边缘清晰。小肿瘤优先位于视神经乳头上方；组织学检查证实了这种形态。在体内，随着肿瘤受眼的尺寸限制，异种移植瘤中的[公式：见正文]呈现出平台效应。[公式：见正文]建模与体内眼内成像相结合，能够对该视网膜母细胞瘤模型进行定量且高分辨率的非侵入性空间分析。该技术未来将应用于其他细胞系和实验性治疗试验。
