Acoustofluidic Separation (AFS), Purification and Raman Spectral Fingerprinting of Single EVs: From Cell of Origin to Target Cell and Biofluids

单个 EV 的声流分离 (AFS)、纯化和拉曼光谱指纹识别：从起源细胞到目标细胞和生物流体

• 批准号: 10009490
• 负责人: Ji Yeong An
• 金额: $ 41.62万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2021-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009490
• 关键词: Abbreviations; Apoptotic; Area Under Curve; Biological Markers; Blood; Cancer Detection; Cells; Clinical; Communication; Communication Programs; Communication Research; Communities; Coupling; Credentialing; Detection; Development; Diagnostic; Disease; ERCC2 gene; Early Diagnosis; Fingerprint; Foundations; Funding; Goals; Heterogeneity; Homing; Human; Immunoblotting; Label; Malignant Neoplasms; Manuscripts; Metabolic Diseases; Molecular Analysis; Non-Invasive Cancer Detection; Organ; Outcome; Performance; Phase; Preparation; Procedures; Protocols documentation; Public Health; Publishing; RNA; Raman Spectrum Analysis; Reproducibility; Saliva; Salivary; Salivary Glands; Seminal; Sorting - Cell Movement; Source; Specificity; Surface; System; Systemic disease; TSG101 gene; Technology; Testing; Therapeutic; Tissues; Transducers; United States National Institutes of Health; base; biomarker development; exosome; extracellular; extracellular vesicles; human tissue; innovative technologies; liquid biopsy; malignant stomach neoplasm; microvesicles; nervous system disorder; new technology; novel; scale up; tool

Project Summary/Abstract: This UG3/UH3 application is responsive to the NIH Common Fund initiative RFA-RM-18-028 “Advancing Extracellular RNA (exRNA) Communication Research: Towards Single Extracellular Vesicle (EV) Sorting, Isolation, and Analysis of Cargo”. This proposal is built on a foundation of a 5-year NIH Common Fund “Extracellular RNA Communication Consortium Stage 1 (ERCC1)” project to develop salivary exRNA biomarkers for gastric cancer detection where a panel of highly discriminatory salivary extracellular RNA (exRNAs) have been developed (discovered and definitively validated) for gastric cancer, scientifically and translationally credentialed salivary exRNA for systemic disease detection. This ERCC Stage 2 project is to develop an innovative technology, AcoustoFluidic Separation (AFS) coupling with Surface Enhanced Raman Spectroscopy (SERS), towards single EV isolation and to characterize exRNA cargos associated with specific EV subpopulations based on the cells of origin, intended target cells and biolfuids. Eight Specific Aims with twelve quantitative milestones in two phases (UG3, UH3) are in place to test the central hypothesis that exosomes are the EV from gastric cancer tissue/cells of origin harboring the nine validated discriminatory salivary exRNA biomarkers, transported through vasculature, homing into salivary glands (target organ) and into saliva. Four Specific Aims in the UG3 Phase are to develop the AFS technology as a standard operating procedure (SOP) for rigor and reproducibility (Aim 1); SERS development for EV fingerprinting and co-localization of exRNA targets to singe EV (Aim 2); two independent “Rigor and Reproducibility Labs (R&R Labs)” to evaluate the AFS SOP (Aim 3) and approaches to share strategies, protocols, tools with broader scientific community with DMRR (Aim 4). In the UH3 Phase four Specific Aims are in place to optimize, refine and scale up the AFS SOP (Aim 5); perform sorting, tracking of validated salivary exRNA biomarkers for gastric cancer detection from cells of origin, to blood, to salivary glands and to saliva (Aim 6); “R&R Labs” to optimize AFS to other human biofluids (Aim 7) and approaches to share strategies, protocols, tools with broader scientific community with DMRR (Aim 8). Completing these Aims and goals based on the outcome of the ERCC1 project is logical and highly impactful as the outcomes of the ERCC2 project will deliver a set of novel technologies, AFS in tandem with SERC, for rapid, high yield (6X over current technologies) and single EV level isolation for salivary biomarker development for systemic disease detection. These will constitute the foundation of “exRNA Saliva Liquid Biopsy (exRNA- SLB)” where the diagnostic and therapeutic functionality of exRNAs can be fully realized when the range of EV subpopulations from a given cell source can be characterized and analyzed for molecular cargos. 1

项目概要/摘要： 此UG 3/UH 3申请是对NIH共同基金倡议RFA-RM-18-028“推进 细胞外RNA（exRNA）通讯研究：朝向单个细胞外囊泡（EV）分选， 货物的分离和分析”。该提案是建立在一个为期5年的NIH共同基金的基础上的。 “细胞外RNA通讯联盟第1阶段（ERCC 1）”项目开发唾液exRNA生物标志物 对于胃癌检测，其中一组高度区分的唾液细胞外RNA（exRNA）具有 在科学和预防方面，已开发（发现并最终验证）用于胃癌 用于系统性疾病检测的合格唾液exRNA。 该ERCC第二阶段项目是开发一种创新技术，声流分离（AFS）耦合 与表面增强拉曼光谱（Sers），对单一的EV分离和表征exRNA 基于来源细胞、预期靶细胞和生物流体，与特定EV亚群相关的货物。 在两个阶段（UG 3，UH 3）中有八个具体目标和十二个定量里程碑，以测试 中心假设，即外泌体是来自携带9种肿瘤细胞的胃癌组织/细胞的EV。 经验证的区别性唾液exRNA生物标志物，通过脉管系统转运，归巢到唾液中， 腺体（靶器官）和唾液。UG 3阶段的四个具体目标是开发AFS技术 作为标准操作程序（SOP）的严格性和再现性（目标1）; EV的Sers开发 exRNA靶标的指纹和共定位以单一EV（Aim 2）;两个独立的“Rigor和 生殖实验室（R&R实验室）”，以评估AFS SOP（目标3）和分享战略的方法， 协议，工具与更广泛的科学界与DMRR（目标4）。在UH 3阶段，四个具体目标是 优化、完善和扩大AFS SOP（目标5）;对经验证的唾液进行分类、跟踪 exRNA生物标志物用于胃癌检测，从细胞起源，血液，唾液腺和唾液（目的 6）;“R&R实验室”，以优化AFS到其他人类生物流体（目标7）和方法，以分享战略，协议， 与更广泛的科学界一起使用DMRR工具（目标8）。 根据ERCC 1项目的结果完成这些宗旨和目标是合乎逻辑的，并且具有高度影响力 由于ERCC 2项目的成果将提供一套新颖的技术，AFS与SERC协同工作， 用于唾液生物标志物开发的快速、高产率（比现有技术高6倍）和单EV水平分离 用于系统性疾病检测。这些将构成“exRNA唾液液体活检（exRNA- SLB）”，其中当EV的范围被改变时，exRNA的诊断和治疗功能可以被完全实现。 可以表征来自给定细胞来源的亚群并分析分子货物。 1