Characterization of Biological Nanoparticle Subsets

在过去的一年里，我们开发了一条全面的电动汽车分析管道，我们称之为多路至单一电动汽车分析(Mt-SEA)管道。这条Mt-SEA管道包括多路高维EV调查以及高分辨率单EV研究，包括用于荧光检测和校准、计数和高通量分选的NanFACS协议，以通过NanFACS从血浆和其他生物体液中分选EV亚组。使用多个不同的囊泡种群，我们发现NanFACS分选唯一能够产生高保真的EV亚群(纯度为95%-97%)，具有功能的生物货物(RNA和蛋白质)。我们目前的重点是将这种方法专门应用于临床重要研究，用于癌症疾病负担、转移潜能和治疗反应的研究。此外，我们还设计了一类新的标记(分子纳米标记)，可以与纳米FAC一起使用，不仅可以改进EV表位的检测，还可以通过流式细胞仪进行单分子计数。我们为这项纳米FACS项目组装并继续改进的专门仪器、专业知识和生物样品管道是NCI的独特和独特的资产。

During the past year, we have developed a comprehensive pipeline for EV analysis, which we refer to as the Multiplex-to-Single-EV-Analysis (Mt-SEA) pipeline. This Mt-SEA pipeline includes multiplex high dimensional EV surveys as well as high resolution single EV studies, including nanoFACS protocols for fluorescence detection and calibration, for counting, and for high-throughput sorting, to sort EV subsets from plasma and other biofluids by nanoFACS. Using multiple distinct vesicle populations, we find that nanoFACS sorting is uniquely able to produce high fidelity EV subsets (95-97% pure), with functional biological cargo (RNA and protein). Our current focus is exclusively on application of this method for clinically important studies, for the study of cancer disease burden, metastatic potential, and responses to treatment. Furthermore, we have devised a new class of labels (Molecular NanoTags) that can be used with nanoFACS, not only to improve the detection of EV epitopes, but also to enable single molecule counting by flow cytometry. The specialized instrumentation, expertise, and biospecimen pipeline that we have assembled and are continuing to improve for this nanoFACS program is a unique and distinctive asset for NCI.