Vesicle Epitope Transcript sequencing (VET-seq): Droplet-based Multiomic Profiling Platform for Single Vesicle Analysis

摘要 细胞外小泡是释放到血液中的直径为30-1000 nm的双层膜结构。 全身细胞的浓度约为每毫升1010。它们的蛋白质分子含量， DsDNA寡聚体、microRNAs(MiRNAs)、mRNAs和其他分析物可能通过EV发挥多种功能作用 还可以提供关于疾病部位或起源组织的诊断报告。因此，电动汽车 可以作为癌症生物标记物的潜在来源，甚至可以提供对遗传和 肿瘤微环境的功能特征。然而，这一潜力在很大程度上仍未得到开发。 面对技术挑战。 我们提出了一种基于液滴的超高通量多组谱分析平台--囊泡表位 转录测序(VET-SEQ)，可以同时解析表面蛋白质组，内部蛋白质， 以及具有单一细胞外小泡(SEV)分辨率的广谱RNA。在目标1中，我们操纵电动汽车来 允许接触密封的电动汽车货物，并允许电动汽车分子的原位修饰。这将会产生 用于检测广谱EV分子的协议。在这里，我们还开始生成和验证定制 用于兽医序列的探头。在目标2中，我们提出了一种EV索引方法，将特定于水泡的条形码添加到EV中 能够识别其囊泡来源的分子。这一战略将显著增加 通过克服限制稀释的需要，实现了基于液滴的电动汽车分析的吞吐量。向未满足的人发表讲话 需要一种经济高效的多组SEV图谱方法，在AIM 3中，我们集成了原位修饰协议 和EV索引协议，以形成VET-SEQ工作流。我们将在电动汽车上对VET-SEQ协议进行基准测试 从人类癌细胞系和耐药模型中分离出来。VET-SEQ的EV检测下限将为 在从人血浆和血清分离的EVS的背景中确定癌细胞来源的EVS。高- VET-SEQ实现的空间SEV图谱将加深我们对分子内容物和 EVS在癌症背景下的生物学意义。这将潜在地揭示电动汽车作为 癌症生物标志物，用于癌症或复发癌症的早期检测以及监测治疗反应 癌症治疗学。 我们的多学科团队由拥有微纳米技术专业知识的科学家和临床医生组成， 多组学分析、肠道病毒生物学、计算生物学、临床肿瘤学和化验发展。

ABSTRACT Extracellular vesicles (EVs) are bilayer membrane structures of diameters 30 – 1000 nm released into the blood by cells throughout the body, at concentrations on the order of 1010 per ml. Their molecular content of proteins, dsDNA oligomers, microRNAs (miRNAs), mRNAs, and other analytes, may play multiple functional roles via EV trafficking, and may also provide a diagnostic report back on the disease site or tissue of origin. As such, EVs can serve as potential sources of cancer biomarkers, perhaps even providing insights into the genetic and functional characteristics of the tumor microenvironment. However, this potential remains largely untapped due to technical challenges. We propose to develop an ultra-high-throughput droplet-based multiomic profiling platform, Vesicle Epitope Transcript sequencing (VET-seq), that can simultaneously resolve the surface proteome, internal proteins, and broad RNA spectrum with single extracellular vesicle (sEV) resolution. In AIM 1, we manipulate EVs to permit access to encapsulated EV cargoes and allow in situ modifications of EV molecules. This will yield protocols for detecting a broad spectrum of EV molecules. Here, we also begin to generate and validate custom probes for VET-seq. In AIM 2, we propose an EV indexing approach to add vesicle-specific barcodes to EV molecules that enable the identification of their vesicle-source. This strategy will significantly increase the throughput for droplet-based EV profiling by overcoming the need for limiting dilution. To address the unmet need for a cost-effective multiomic sEV profiling method, in AIM 3, we integrate the in situ modification protocols and the EV indexing protocol to form the VET-seq workflow. We will benchmark the VET-seq protocol on EVs isolated from human cancer cell lines and drug resistance models. The EV detection limit of VET-seq will be determined for cancer-cell derived EVs in a background of EVs isolated from human plasma and serum. High- dimensional sEV profiling enabled by VET-seq will deepen our understanding of the molecular contents and biological implications of EVs in the context of cancer. This will potentially shed light into the utility of EVs as cancer biomarkers for early detection of cancer or recurrent cancer as well as for monitoring treatment response to cancer therapeutics. Our multidisciplinary team is comprised of scientists and clinicians with expertise in micro-nanotechnologies, multiomics analysis, EV biology, computational biology, clinical oncology, and assay development.