Quantitative Imaging of Cancer Drug Resistance via Radioluminescence Microarrays

描述(由申请人提供)：越来越明显的是，细胞群体在其功能、疾病状态和对治疗的反应方面是不同的。肿瘤的异质性是导致耐药的主要因素之一。现在，人们对在单细胞水平上操作的表征方法投入了极大的兴趣，而不是只能测量给定人群的平均性质的整体分析。长期以来，荧光方法一直被用来测量单个活细胞的分子过程。然而，对于荧光探测来说，大量的小分子仍然是不可见的。这些分子缺乏固有的荧光，在不改变其生化活性的情况下无法进行荧光标记。对小分子的精确和灵敏的定量(例如，在药物药代动力学研究中)仍然是放射性核素检测方法(闪烁计数、放射自显影和正电子发射断层扫描)的领域，因为在大多数情况下，放射性标记可以保持生化活性。最近的发现，放射性核素分子也可以在显微镜下的细胞水平上成像，这代表着与以前所知的根本背道而驰。这种名为放射发光显微镜的新方法可以测量放射性核素分子在单个活细胞中的积累。虽然这项技术已经在各种应用中得到了演示，但这项技术仍处于初级阶段。在这项提案中，我们提出了几项根本性的改进，使我们能够在一次采集中测量多达1000个单个细胞的放射性核素探针摄取。研究大型细胞的研究 出于统计原因和稀有细胞亚群的存在，数字是必要的。基于令人鼓舞的初步结果，我们提出了一种新的仪器设计，称为放射发光微阵列，可以实现这一目标。这一新的设计结合了几项根本性的改进，将把放射发光显微镜转变为具有许多潜在应用的高通量研究的多功能工具。在目标1中，我们将开发一种名为放射发光微阵列的新设备。该设备包括用于高分辨率成像的微米薄闪烁体、用于优化细胞放置的微孔阵列、用于可重复样品制备的流体平台以及用于多模式成像的改进的荧光附加组件。在目标2中，我们将实现放射发光显微镜图像的实时显示和自动分析软件。最后，在目标3中，我们将通过研究小分子非荧光化疗药物5-氟尿嘧啶(5-FU)与单个活细胞的相互作用来验证整个方法。利用放射性形式的药物([18F]5-FU)，我们将确定5FU如何在不同种类的癌细胞中分布。荧光显微镜将被用来根据细胞周期中的位置或干细胞状态等因素将细胞分配到不同的亚群。综上所述，该项目将开发一种新的、具有无与伦比的能力的新仪器，它将应用于加深我们对异质癌细胞群体中耐药的理解。

DESCRIPTION (provided by applicant): It is becoming increasingly apparent that cell populations are heterogeneous in their functions, disease states and response to therapy. Tumor heterogeneity is one of the main factors contributing to acquire drug resistance. Substantial interest is now devoted to characterization methods that operate at the single-cell level, as opposed to bulk analyses that can only measure average properties over a given population. Fluorescence methods have long been used to measure molecular processes in single living cells. However, a vast number of small molecules remain invisible to fluorescence probing. These molecules lack inherent fluorescence and cannot be fluorescently labeled without altering their biochemical activity. Precise and sensitive quantitation of small molecules (e.g. in drug pharmacokinetics studies) remains the domain of radionuclide detection methods (scintillation counting, autoradiography, and positron emission tomography) since radiolabeling in most cases can preserve biochemical activity. The recent finding that radionuclide molecules too can be imaged at the cellular level in a microscope represents a radical departure from what was previously known. This new method, called radioluminescence microscopy, can measure the accumulation of a radionuclide molecule in single living cells. While the technique has been demonstrated for a variety of applications, the technology is still in its infancy. In this proposa, we are proposing several radical improvements that will allow us to measure radionuclide probe uptake in up to 1000 individual cells, in a single acquisition. Studies that investigate large cell numbers are necessary for statistical reasons and due to the existence of rare cell subpopulations. Based on encouraging preliminary results, we are proposing a new instrument design called the radioluminescence microarray that can achieve this goal. This new design incorporates several radical improvements that will transform radioluminescence microscopy into a versatile tool for high-throughput studies with many potential applications. In Aim 1, we will develop a new device called the radioluminescence microarray. This device includes a micrometer-thin scintillator for high-resolution imaging, a microwell array for optimized cell placement, a fluidics platform for repeatable sample preparation, and an improved epifluorescence add-on for multi-modality imaging. In Aim 2, we will implement software for real-time display and automated analysis of radioluminescence microscopy images. Last, in Aim 3, we will validate the overall approach by investigating the interaction with single living cells of -fluorouracil (5-FU), a small-molecule non-fluorescent chemotherapeutic agent. Using the radioactive form of the drug ([18F] 5-FU), we will determine how 5FU distributes in heterogeneous cancer cell populations. Fluorescence microscopy will be used to assign cells to different subpopulations according to factors such as position in the cell cycle or stem-cell status. In summary, this project will develop new a new instrument with unmatched capabilities, which will be applied to deepening our understanding of drug resistance in heterogeneous cancer cell populations.