Expanding the bioluminescent toolbox for multi-cellular imaging of tumor heteroge

扩展肿瘤异质性多细胞成像的生物发光工具箱

• 批准号: 8839799
• 负责人: Jennifer Prescher
• 金额: $ 27.13万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839799
• 关键词: Address; Animals; Behavior; Binding; Biological; Biological Assay; Biological Process; Bioluminescence; Cell Count; Cell physiology; Cells; Chemicals; Chemistry; Collection; Communities; Complex; Data; Development; Diagnostic; Disease; Electronics; Engineering; Enzymes; Firefly Luciferases; Goals; Health; Heterogeneity; Hour; Human; Image; Imaging Device; Imaging Techniques; Imaging technology; In Vitro; Knowledge; Length; Life; Light; Luciferases; Malignant Neoplasms; Methods; Microscopic; Mission; Modeling; Modification; Molecular; Monitor; Mutagenesis; Nature; Optics; Organ; Organism; Population; Process; Proteins; Public Health; Publishing; Research; Rodent Model; Role; Science; Solid; Structure; Techniques; Technology; Therapeutic; Time; Visible Radiation; Work; analog; base; bioluminescence imaging; burden of illness; cancer imaging; cell motility; cell type; cellular imaging; design; enzyme substrate; human disease; imaging agent; imaging probe; immune function; in vivo; in vivo imaging; innovation; insight; light emission; luciferin; migration; mutant; novel; optical imaging; scaffold; screening; small molecule; tool; tumor; tumor growth; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales-including those involved in cancer progression and migration-requires new imaging tools. The long-term goal of our work is to develop general strategies for macroscopic, multi-cell tracking in living organisms. The objective of this application is to engineer novel bioluminescent tools for multi-cellular imaging in vivo. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging utilize the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. Our central hypothesis is that the substrate-binding interface of firefly luciferase can be re-engineered to generate a panel of mutant enzymes that accept chemically distinct luciferins. When the mutants and analogs are mixed together, robust light emission will be produced when complementary enzyme-substrate partners interact. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Synthesize and identify light-emitting luciferins; 2) Generate complementary luciferases and screen for orthogonal pairs; and 3) Image tumor heterogeneity with orthogonal probes. Under the first aim, we will utilize divergent chemistries developed in our lab to access light-emitting small molecules. In the second aim, we will employ a combination of mutagenesis and screening assays to identify luciferase enzymes that catalyze light emission with the synthesized molecules. In the third aim, the enzyme-substrate pairs will be utilized to address the roles of distinct cellular subsets in heterogeneous tumor models. Our approach is highly innovative, as it combines a unique blend of chemical and biological techniques to fill a long-standing void in imaging capabilities. The proposed research is significant, as the bioluminescent tools will enable the direct interrogation of cell networks not currently possible with existing toolsets. Such studies will provide some of the first macroscopic images of tumor heterogeneity and may fundamentally change existing views on cancer progression and therapeutic approaches. Additionally, similar to other imaging technologies, the bioluminescent probes will likely inspire new discoveries in a broad spectrum of fields.

描述（由申请人提供）：对人类健康和疾病的详细了解需要方法来探测在完整器官结构和活体受试者中发生的细胞行为。近年来，成像界出现了一些技术，可以实时可视化和跟踪不同的生物特征。虽然这些方法很强大，但很大程度上仅限于在微观水平上监测细胞行为。在更大的空间尺度上可视化细胞功能——包括那些 参与癌症进展和迁移 - 需要新的成像工具。我们工作的长期目标是开发生物体宏观、多细胞追踪的通用策略。 该应用的目的是设计用于体内多细胞成像的新型生物发光工具。生物发光成像是一种用于可视化啮齿动物模型中少量细胞的强大技术。该技术采用酶（荧光素酶），与小分子底物（荧光素）一起孵育后产生光。自然界中存在几种荧光素酶-荧光素对，其中许多已适用于追踪整个动物的细胞。不幸的是，用于体内成像的最佳荧光素酶利用相同的底物，因此不能用于区分单个受试者中的多种细胞类型。我们的中心假设是，萤火虫荧光素酶的底物结合界面可以重新设计，以产生一组接受化学上不同的荧光素的突变酶。当突变体和类似物混合在一起时，互补的酶-底物伴侣相互作用时会产生强烈的光发射。在强有力的初步数据的指导下，我们的工作将涵盖以下具体目标：1）合成和识别发光荧光素； 2) 生成互补荧光素酶并筛选正交对； 3) 使用正交探针对肿瘤异质性进行成像。在第一个目标下，我们将利用我们实验室开发的不同化学物质来获取发光小分子。在第二个目标中，我们将采用诱变和筛选分析相结合的方法来鉴定利用合成分子催化光发射的荧光素酶。在第三个目标中，酶-底物对将用于解决异质肿瘤模型中不同细胞亚群的作用。我们的方法具有高度创新性，因为它结合了化学和生物技术的独特结合，填补了成像能力方面长期存在的空白。拟议的研究意义重大，因为生物发光工具将能够直接询问细胞网络，而这是现有工具集目前无法实现的。此类研究将提供肿瘤异质性的一些第一批宏观图像，并可能从根本上改变对癌症进展和治疗方法的现有看法。此外，与其他成像技术类似，生物发光探针可能会激发广泛领域的新发现。