Quantitative Imaging of Cancer Drug Resistance via Radioluminescence Microarrays

通过放射发光微阵列对癌症耐药性进行定量成像

• 批准号: 8674402
• 负责人: Guillem Pratx
• 金额: $ 36.31万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8674402
• 关键词: Antineoplastic Agents; Apoptosis; Autoradiography; Behavior; Biochemical; Biochemistry; Calibration; Cell Count; Cell Cycle; Cell Fraction; Cell Line; Cells; Cellular Stress; Computer software; Computers; Coupled; Detection; Devices; Disease; Drug Efflux; Drug Kinetics; Drug resistance; Environment; Event; Fluorescence; Fluorescence Microscopy; Fluorouracil; Goals; Heterogeneity; Image; Image Analysis; Imaging Device; Imaging Techniques; Individual; Label; Lead; Life; Location; Malignant Neoplasms; Masks; Measurement; Measures; Methods; Microscope; Microscopy; Molecular; Monitor; Organism; Pattern; Pharmaceutical Preparations; Play; Population; Population Heterogeneity; Positioning Attribute; Positron-Emission Tomography; Preparation; Procedures; Process; Property; Radiation; Radioactive; Radioisotopes; Radiolabeled; Radionuclide Imaging; Research; Resistance; Resolution; Role; Sampling; Scintillation Counting; Stem cells; Techniques; Technology; Time; base; cancer cell; cancer type; cell type; chemotherapeutic agent; chemotherapy; data acquisition; design; effective therapy; high throughput screening; imaging modality; improved; infancy; instrument; interest; irinotecan; novel; public health relevance; radiotracer; resistance mechanism; response; single cell analysis; small molecule; software development; stem; tool; trafficking; tumor; tumorigenic; uptake; user-friendly

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.

描述（由申请人提供）：越来越明显的是，细胞群体的功能，疾病状态和对治疗的反应是异质的。肿瘤异质性是导致获得耐药性的主要因素之一。现在，实质性的兴趣致力于在单细胞水平上运行的表征方法，而不是只能在给定人群上衡量平均属性的批量分析。荧光方法长期以来一直用于测量单个活细胞中的分子过程。但是，大量的小分子仍然是荧光探测的看不见的。这些分子缺乏固有的荧光，并且不能在不改变其生化活性的情况下被荧光标记。小分子的精确定量（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的领域（闪烁计数，自显影和正电子发射断层扫描），因为在大多数情况下可以保留放射性标记的生物化学活性。最近的发现，即放射性核素分子也可以在显微镜中成像在细胞水平上成像，这是与以前已知的那样的根本性。这种称为辐射光学显微镜的新方法可以测量单个活细胞中放射性核素分子的积累。尽管该技术已用于各种应用，但该技术仍处于起步阶段。在此提案中，我们提出了几种根本改进，这将使我们能够在一次采集中测量多达1000个单个细胞中的放射性核素探针摄取。研究大细胞的研究 由于统计原因和由于存在罕见的细胞亚群而需要数字。基于鼓励初步结果，我们提出了一种称为“放射性发光微阵列”的新仪器设计，可以实现这一目标。这一新设计结合了几种根治性的改进，可以将放射性发光显微镜转化为具有许多潜在应用的高通量研究的多功能工具。在AIM 1中，我们将开发一种称为辐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的闪烁体，用于优化的细胞放置的微孔阵列，用于重复样品制备的流体平台以及用于多模式成像的改进的表荧光添加剂。在AIM 2中，我们将实施用于实时显示和自动分析辐射显微镜图像的软件。最后，在AIM 3中，我们将通过研究与小分子非荧光化疗剂-Fluorouracil（5-FU）的单个活细胞的相互作用来验证总体方法。使用药物的放射性形式（[18F] 5-FU），我们将确定5FU如何在异质性癌细胞种群中分布。荧光显微镜将根据细胞周期或干细胞状态等因素（例如位置）将细胞分配给不同的亚群。总而言之，该项目将开发具有无与伦比能力的新工具，该工具将应用于加深我们对异质癌细胞种群中耐药性的理解。