Application of autobioluminescence toward continuous and real-time in vitro/in vivo pre-clinical brain imaging for disease therapeutics

项目简介：本项目拟自主开发生物发光材料(自体发光材料) 患者来源的胶质母细胞瘤(GBM)细胞和啮齿动物模型能够无底物，连续，和 对治疗效果的非侵入性评估，以实现有效和高效的转化性GBM研究。GBM 是人类最致命的癌症之一，只有不到3%的患者能存活五年以上。至 协助抗击GBM，生物发光成像技术可促进非侵入性和 肿瘤动力学的纵向可视化已成为翻译工作中的宝贵工具，以更好地 了解肾小球基底膜进展的分子机制及新疗法的评价 临床小动物模型。然而，现有的生物发光成像方法依赖于传统的 荧光素酶报告系统(Frefly、Gaussia、Renilla等)在脑成像研究方面有障碍，因为 他们要求受试者在每次实验前注射一种光激活底物。 测量。特别是对于大脑成像来说，这种化学底物的外来添加会使人感到困惑。 成像终点，因为其生物分布和生物利用度受到血脑屏障的干扰 以及大脑的外流泵送机制。尽管研究致力于小说的合成 生物发光反应底物通过改善对大脑环境的访问，我们从事了一项 完全不同的方法，完全不需要添加底物。我们的技术利用 在哺乳动物中高效表达生物发光的合成荧光素酶(Lux)盒的研制 细胞独立于任何外来的光激活底物的添加。这些细胞能够自我合成 所有必需的底物都来自细胞内的内源性代谢产物，因此能够自我 在结构性和诱导性基因控制下产生“自体发光”信号。在大脑中 在环境中，这种细胞超越了传统的生物发光成像，最终实现了连续、 神经生物学过程的非侵入性和真实的实时可视化。在这项研究工作中，我们 建议在患者来源的GBM细胞系中表达自体生物发光并验证其应用 在高通量体外药物发现试验和体内啮齿动物模型中的潜力。我们将具体地 开发和表征2D、3D和3D星形胶质细胞共培养试验，创建信号通路特异性 用于靶向小分子筛选的自体生物发光细胞模型，并建立原位 用于体内评价化疗药物的自体生物发光异种移植小鼠模型，我们将在两者中测试 有意识的和麻醉的受试者。开发创新的自体生物发光细胞和动物模型 该项目将改善胶质母细胞瘤药物筛选和检测的现状，促进 在智力设计的研究环境中开发新的胶质母细胞瘤治疗药物 激励和挑战本科生研究人员。

Project Summary: This project proposes to develop autonomously bioluminescent (autobioluminescent) patient-derived glioblastoma (GBM) cellular and rodent models capable of substrate-free, continuous, and noninvasive assessment of therapeutic efficacy to enable effective and efficient translational GBM research. GBM is one of the most lethal of human cancers, with less than 3% of patients surviving beyond a five-year period. To assist in the battle against GBM, bioluminescent imaging technologies that facilitate the noninvasive and longitudinal visualization of tumor dynamics have served as valuable tools in translational efforts to better understand the molecular mechanisms of GBM progression and the evaluation of novel therapeutics in pre- clinical small animal models. However, existing bioluminescent imaging approaches that rely upon conventional luciferase reporter systems (firefly, Gaussia, Renilla, etc.) are handicapped for brain imaging studies because they require that the animal subject be injected with a light-activating substrate prior to each and every measurement. For brain imaging in particular, the extraneous addition of this chemical substrate confounds imaging endpoints because its biodistribution and bioavailability is interfered with by the blood-brain barrier and the brain’s efflux pumping mechanisms. Although research is being dedicated toward the synthesis of novel bioluminescent reaction substrates with improved access to the brain environment, we have engaged in an entirely different approach by eliminating the need to add substrate altogether. Our technology leverages the development of a synthetic luciferase (lux) cassette that efficiently expresses bioluminescence in mammalian cells independent of any extraneous addition of a light-activating substrate. These cells are able to self-synthesize all of the requisite substrates from intracellular endogenous metabolites and are therefore capable of self- generating ‘autobioluminescent’ signals under both constitutive and inducible genetic controls. Within the brain environment, such cells go beyond conventional bioluminescent imaging to ultimately enable continuous, noninvasive, and authentic real-time visualization of neurobiological processes. In this research effort, we propose to express autobioluminescence in patient-derived GBM cell lines and validate their application potential in high-throughput in vitro drug discovery assays and in in vivo rodent models. We will specifically develop and characterize 2D, 3D, and 3D astrocyte co-culture assays, create signaling pathway-specific autobioluminescent cellular models for targeted small molecule screening, and establish an orthotopic autobioluminescent xenograft mouse model for in vivo evaluation of chemotherapeutics that we will test in both conscious and anesthetized subjects. The innovative autobioluminescent cellular and animal models developed in this project will improve the status quo of glioblastoma drug screening and testing and facilitate the development of novel glioblastoma therapeutics within a research environment designed to intellectually stimulate and challenge undergraduate student researchers.