High-throughput radionuclide counting and sorting of single cells

单细胞的高通量放射性核素计数和分选

• 批准号: 8850698
• 负责人: Guillem Pratx
• 金额: $ 24.44万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-18 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8850698
• 关键词: Antibodies; Autoradiography; Binding; Biochemical; Biochemical Process; Biological Assay; Biomedical Research; Cancer Biology; Cell Separation; Cells; Classification; Detection; Encapsulated; Ensure; Enzymes; Flow Cytometry; Fluorescence; Goals; Health; Heterogeneity; Immobilization; Immobilized Cells; Immune; In Vitro; Individual; Investigation; Label; Link; Liquid substance; Lymphoma; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Transport Proteins; Metabolic; Methodology; Methods; Microbiology; Microfluidics; Microscopy; Molecular; Molecular Analysis; Outcome; Pharmaceutical Preparations; Phenotype; Ploidies; Population; Process; Property; Proteomics; RNA Sequences; Radioactive; Radioisotopes; Radiolabeled; Research; Research Project Grants; Resolution; Sampling; Scintillation Counting; Signal Transduction; Sorting - Cell Movement; Surface; Suspension substance; Suspensions; System; Techniques; Technology; Therapeutic Agents; Time; Tumor Biology; Variant; anticancer research; base; calginat; cancer cell; cancer diagnosis; cancer therapy; design; fluorophore; imaging agent; improved; in vivo; innovation; luminescence; millisecond; novel; novel strategies; phosphorescence; radiotracer; single cell analysis; single molecule; small molecule; stem; temporal measurement; tool; transcriptome sequencing; tumor; uptake

DESCRIPTION (provided by applicant): The first aim of this project is to develop an innovative methodology for measuring radionuclide uptake in single cells using a standard flow cytometer. The rationale for this aim is that flow cytometry in its current form can only interrogate cellular states by detecting fluorescence emissions from single cells, a process that excludes small-molecule compounds that are neither intrinsically fluorescent nor can be labeled with a fluorophore. The novel method we plan to develop is aimed at studying how single cells interact with any small molecule in the context of improving our understanding of fundamental cancer biology as well as developing new molecular agents for cancer diagnosis and treatment. Many small molecules can be labeled with beta-emitting radionuclides such as 11C, 18F, 32P, 35S, 64Cu, and 124I, which make the proposed approach almost universal with respect to the range of molecules that can be utilized. However, detecting radionuclides within a flow cytometer poses a major challenge. Due to the high throughput, each cell can only be measured for a few milliseconds, which is too short for a significant number of radioactive decays to occur. Thus, we plan to use photostimulable phosphors (PSPs) to physically record and store the number of radioactive decays that occur within each single cell over a prolonged exposure. Using microfluidics technology, we will encapsulate radioactive single cells and PSP microcrystals inside calcium-alginate droplets. This will ensure that PSP crystals are uniquely associated with a single cell. After complete decay of the radionuclide label, these droplets will be flowed through a flow cytometer to retrieve the energy stored inside the PSP microcrystals, which is directly proportional to the number of radioactive decays that occurred within each single cell. Hence, this approach will allow us to measure radionuclide uptake in up to 100,000 single cells. The second aim of this project is to develop a complementary approach for measuring the dynamic exchange of radiolabeled molecules across the membrane of single cells. Measuring the time-varying uptake of small molecules inside of single cells will allow us to quantitatively estimate influx and efflux rates and thus the amount and activity of various membrane transporters and enzymes within the cells. However, this requires that the same single cells be measured repeatedly over time in a statistically robust fashion. Thus, using microfluidic technology, we plan to develop a hydrodynamic cell-trapping array that is bonded to a transparent scintillator plate to enable facile and sensitive quantitation of the time-varying concentration of a radionuclide in approximately 500 single cells. Together, these two complementary research aims will open entirely new research avenues for studying normal and abnormal molecular processes in single cancer cells, with high throughput (aim 1) and high temporal resolution (aim 2).

描述（由申请人提供）：该项目的首要目标是开发一种使用标准流式细胞仪测量单细胞放射性核素摄取的创新方法。这一目标的基本原理是，目前形式的流式细胞术只能通过检测单个细胞的荧光发射来询问细胞状态，这一过程排除了既不本质上荧光也不能用荧光团标记的小分子化合物。我们计划开发的新方法旨在研究单细胞如何与任何小分子相互作用，以提高我们对基础癌症生物学的理解，并开发用于癌症诊断和治疗的新分子制剂。许多小分子可以用 11C、18F、32P、35S、64Cu 和 124I 等 β 发射放射性核素标记，这使得所提出的方法在可利用的分子范围方面几乎是通用的。然而，在流式细胞仪内检测放射性核素提出了重大挑战。由于高通量，每个细胞只能测量几毫秒，这对于发生大量放射性衰变来说太短了。因此，我们计划使用光刺激磷光体（PSP）来物理记录和存储长时间暴露在每个单细胞内发生的放射性衰变的数量。利用微流体技术，我们将放射性单细胞和 PSP 微晶封装在海藻酸钙液滴内。这将确保 PSP 晶体与单个细胞唯一关联。放射性核素标记完全衰变后，这些液滴将流过流式细胞仪，以检索 PSP 微晶内存储的能量，该能量与每个单细胞内发生的放射性衰变的数量成正比。因此，这种方法将使我们能够测量多达 100,000 个单细胞中放射性核素的吸收。该项目的第二个目标是开发一种补充方法来测量单细胞膜上放射性标记分子的动态交换。测量单细胞内小分子随时间变化的吸收将使我们能够定量估计流入和流出速率，从而定量估计细胞内各种膜转运蛋白和酶的数量和活性。然而，这需要以统计稳健的方式随时间重复测量相同的单细胞。因此，我们计划使用微流体技术开发一种流体动力细胞捕获阵列，该阵列粘合到透明闪烁体板上，以便能够对大约 500 个单细胞中放射性核素随时间变化的浓度进行轻松、灵敏的定量。这两个互补的研究目标将为研究单个癌细胞中的正常和异常分子过程开辟全新的研究途径，具有高通量（目标 1）和高时间分辨率（目标 2）。