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

项目概要：本项目拟开发自主生物发光（autobioluminescent） 患者来源的胶质母细胞瘤（GBM）细胞和啮齿动物模型能够无基质、连续和 非侵入性评估治疗效果，以实现有效和高效的转化GBM研究。GBM 是人类最致命的癌症之一，只有不到3%的患者存活超过五年。到 协助对抗GBM，生物发光成像技术，促进非侵入性， 肿瘤动力学的纵向可视化已经作为转化努力的有价值的工具， 了解GBM进展的分子机制和评价新的治疗方法， 临床小动物模型。然而，现有的依赖于常规生物发光成像的生物发光成像方法， 荧光素酶报告系统（萤火虫、Gaussia、海肾等）在脑成像研究方面有障碍， 他们要求动物受试者在每次注射之前注射光激活基质 测量.特别是对于脑成像，这种化学底物的额外添加会使 因为其生物分布和生物利用度受到血脑屏障的干扰， 和大脑的外排泵机制虽然研究正在致力于合成小说 生物发光反应基板与改善进入大脑环境，我们已经从事了一项 完全不同的方法，完全消除了添加底物的需要。我们的技术利用了 在哺乳动物中有效表达生物发光的合成荧光素酶（lux）盒的开发 细胞独立于任何外源性添加的光激活底物。这些细胞能够自我合成 所有必需的底物来自细胞内内源性代谢物，因此能够自我- 在组成型和诱导型遗传控制下产生“自体发光”信号。大脑内 环境中，这种细胞超越了传统的生物发光成像，最终能够连续， 非侵入性的，真实的神经生物学过程的实时可视化。在这项研究中，我们 建议在患者来源的GBM细胞系中表达自身生物发光，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。我们将特别 开发和表征2D、3D和3D星形胶质细胞共培养试验， 用于靶向小分子筛选的自生物发光细胞模型，并建立原位 自体生物发光异种移植小鼠模型，用于体内评价化疗药物，我们将在两种模型中进行测试。 清醒和麻醉的实验对象创新的自体发光细胞和动物模型开发 本项目将改善胶质母细胞瘤药物筛选和试验的现状， 在一个研究环境中开发新型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.