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

描述(由申请人提供)：对人类健康和疾病的详细了解需要探索细胞行为的方法，因为它们发生在完整的器官结构和活着的受试者中。近年来，成像领域出现了各种技术，使各种生物特征能够被可视化和实时跟踪。虽然功能强大，但这些方法在很大程度上仅限于在微观层面上监测细胞行为。在更大的空间尺度上可视化细胞功能--包括 参与癌症的进展和转移--需要新的成像工具。我们工作的长期目标是开发宏观的、活的生物体中的多细胞跟踪的一般策略。 这一应用的目标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显示啮齿动物模型中少量细胞的强大技术。这项技术使用的酶(荧光素酶)在与小分子底物(荧光素)孵育时产生光。自然界中存在几对荧光素酶-荧光素蛋白，其中许多已经被用来跟踪整个动物的细胞。不幸的是，用于体内成像的最佳荧光素酶使用相同的底物，因此不能用于区分单个受试者的多种细胞类型。我们的中心假设是，萤火虫荧光素酶的底物结合界面可以被重新设计，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，当互补酶-底物伙伴相互作用时，将产生强劲的发光。在强大的初步数据的指导下，我们的工作将包括以下具体目标：1)合成和鉴定发光荧光素；2)产生互补的荧光素酶并筛选正交对；3)用正交探针成像肿瘤的异质性。在第一个目标下，我们将利用我们实验室开发的发散化学来获得发光小分子。在第二个目标中，我们将采用诱变和筛选相结合的方法来鉴定与合成分子催化发光的荧光素酶。在第三个目标中，酶-底物对将被用来解决不同细胞亚群在异质肿瘤模型中的作用。我们的方法是高度创新的，因为它结合了化学和生物技术的独特混合，填补了成像能力方面的长期空白。这项拟议的研究意义重大，因为生物发光工具将实现对细胞网络的直接询问，这是现有工具集目前无法实现的。这样的研究将提供一些肿瘤异质性的第一批宏观图像，并可能从根本上改变现有的癌症进展和治疗方法的观点。此外，与其他成像技术类似，生物发光探测器可能会在广泛的领域引发新的发现。

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.