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

自生物发光在疾病治疗中连续实时体外/体内临床前脑成像的应用

• 批准号: 10292106
• 负责人: Larry J. Millet
• 金额: $ 45.86万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292106
• 关键词: 3-Dimensional; Address; Affect; Animal Model; Animals; Antineoplastic Agents; Astrocytes; Bacterial Luciferases; Biodistribution; Biological Assay; Biological Availability; Bioluminescence; Blood - brain barrier anatomy; Brain; Brain Neoplasms; Brain imaging; Cell Line; Cell model; Cells; Central Nervous System Neoplasms; Coculture Techniques; Complex; Conscious; Coupling; Dependence; Development; Disabled Persons; Disease; Drug Kinetics; Drug Screening; Environment; Environment Design; Evaluation; Excision; Fireflies; Generations; Genetic; Glioblastoma; Health; Human; Human Cell Line; Image; Imaging technology; Improve Access; In Situ; In Vitro; Injections; Kinetics; Knowledge; Light; Luciferases; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of brain; Mammalian Cell; Measurement; Metabolic; Modality; Modeling; Molecular; Monitor; Neurobiology; Operative Surgical Procedures; Output; Pathway interactions; Patients; Phenotype; Prediction of Response to Therapy; Primary Neoplasm; Process; Radiation; Reaction; Renilla; Reporter; Research; Research Personnel; Response Elements; Rodent Model; Role; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Speed; Survival Rate; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Studies; Time; Treatment Efficacy; Treatment Protocols; Treatment outcome; Tumor Burden; Tumor Pathology; Variant; Visualization; WNT Signaling Pathway; Xenograft procedure; base; chemical addition; chemotherapy; combat; cytotoxic; design; drug candidate; drug discovery; drug testing; efficacy evaluation; efflux pump; high throughput screening; imaging approach; imaging capabilities; imaging study; imaging system; improved; in vivo; in vivo evaluation; innovation; mouse model; neoplastic cell; novel; novel therapeutics; optical imaging; pre-clinical; precision medicine; response; screening; small molecule; small molecule libraries; stem cells; therapeutically effective; therapy outcome; therapy resistant; tool; treatment strategy; tumor; tumor microenvironment; undergraduate student; uptake

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.

项目摘要：此项目提案旨在开发自主生物发光（Autobiobiolumincenate） 患者衍生的胶质母细胞瘤（GBM）细胞和啮齿动物模型，无底物，连续和 对治疗效率的无创评估，以实现有效，有效的转化GBM研究。 GBM 是人类癌症中最致命的癌症之一，只有不到3％的患者在五年内生存。到 协助与GBM的斗争，有助于无创和的生物发光成像技术 肿瘤动态的纵向可视化已成为转化努力的宝贵工具 了解GBM进展的分子机制以及对预疗法的新疗法的评估 临床小动物模型。但是，依赖常规的现有生物发光成像方法 荧光素酶报告基因（Firefly，高斯，Renilla等）被残障用于大脑成像研究，因为 他们要求将动物受试者注入一个光激活的底物 测量。特别是对于大脑成像，该化学基材的无关添加 成像端点，因为其生物分布和生物利用度受到血脑屏障的干扰 以及大脑的流出泵送机制。尽管研究是致力于新颖的合成 生物发光反应底物可以改善对大脑环境的访问，我们从事 通过消除完全添加底物的需求，消除了不同的方法。我们的技术利用了 开发合成荧光素酶（LUX）盒，可有效地表达哺乳动物的生物发光 细胞独立于任何无关添加的光激活底物。这些细胞能够自同时化 来自细胞内内源代谢产物的所有必要底物，因此能够自我 在本构和可诱导的遗传控制下产生“自动发光”信号。在大脑内 环境，这些细胞超越了传统的生物发光成像，最终使连续， 神经生物学过程的无创和真实的实时可视化。在这项研究工作中，我们 提出表达患者衍生的GBM细胞系中自动脑性的提议并验证其应用 高通量的体外药物发现测定法和体内啮齿动物模型的潜力。我们将具体 开发和表征2D，3D和3D星形胶质细胞共培养分析，创建特定于特定的信号通路 定向小分子筛选的自发光细胞模型，并建立原位 用于体内化学治疗剂体内评估的自发异种移植小鼠模型，我们将在两者中测试 有意识和麻醉的主题。创新的自动发光细胞和动物模型开发了 在这个项目中，将改善胶质母细胞瘤药物筛查和测试的现状 在旨在智能的研究环境中开发新型胶质母细胞瘤疗法 刺激和挑战本科生研究人员。