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：该项目建议开发自主生物发光（自动发光） 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D 对治疗性的无创评估，以实现有效和有效的转化GBM研究。 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。 协助与GBM的斗争，有助于无创和的生物发光成像技术 肿瘤动力学的纵向可视化已成为转化源的宝贵工具，以更好地 了解GBM进展的分子机制以及对预生产物的新疗法的评估 但是，临床小动物模型。 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 他们要求动物受试者在每一个之前注入光激活的底物 尤其是大脑成像的测量 成像端点，因为其生物分布和生物幻灯片可干扰血液障碍障碍 大脑的流出泵送机制。 具有改善访问大脑环境的生物发光反应底物，我们有英语 完全不同的方法是完全添加底物的方法。 开发哺乳动物中表达生物发光的合成卢比夫骚扰（LUX）盒式磁带 细胞独立于任何无关的添加 来自细胞内内源代谢产物的所有必要底物，因此能够自我 在脑部内部遗传控制下产生“自动发光”信号 环境，这些细胞超越了公亮召集的涂层成像，最终是连续的， 神经生物学过程的无创和真实的实时可视化。 提议在患者衍生的GBM细胞lin中表达自传发光并验证其应用 高通量的体外药物中的潜力发现了我们将专门进行的Assassays和体内啮齿动物模型 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的体内评估的自动发光的异种小鼠模型，我们将在两者中测试 有意识和麻醉的受试者。 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，并促进您 在研究环境中开发新颖的胶质母细胞瘤疗法 刺激和挑战地下学生研究人员。