High-throughput radionuclide counting and sorting of single cells

描述(由申请人提供)：该项目的第一个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。这一目的的基本原理是，目前形式的流式细胞仪只能通过检测单细胞的荧光发射来询问细胞状态，这一过程排除了既不具有固有荧光也不能被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个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。许多小分子可以用发射β的放射性核素标记，如11C、18F、32P、35S、64Cu和124I，这使得所提出的方法在可利用的分子范围内几乎是通用的。然而，检测流式细胞仪中的放射性核素是一个重大挑战。由于高吞吐量，每个电池只能测量几毫秒，这对于发生大量的放射性衰变来说太短了。因此，我们计划使用光刺激荧光粉(PSP)来物理记录和存储每个电池在长时间曝光后发生的放射性衰变的数量。利用微流控技术，我们将放射性单细胞和PSP微晶包裹在海藻酸钙液滴中。这将确保PSP晶体与单个细胞唯一地关联。在放射性核素标记完全衰变后，这些液滴将流经流式细胞仪，以回收储存在PSP微晶体内的能量，这与每个细胞内发生的放射性衰变次数成正比。因此，这种方法将使我们能够测量多达100,000个单细胞的放射性核素摄取。该项目的第二个目标是开发一种补充方法，用于测量单细胞膜上放射性标记分子的动态交换。测量单个细胞内小分子随时间变化的摄取将使我们能够定量地估计细胞内各种膜转运体和酶的流入和流出速率，从而估计其数量和活性。然而，这需要以统计稳健的方式随着时间的推移重复测量相同的单个细胞。因此，利用微流控技术，我们计划开发一种流体动力学细胞捕获阵列，该阵列结合到透明闪烁板上，能够方便和灵敏地定量大约500个单细胞中随时间变化的放射性核素浓度。总之，这两个互补的研究目标将为研究单个癌细胞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)。

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).