Microfluidics Array Based Sorting, Isolation, and RNA Analysis in Single Extracellular V csicles

基于微流体阵列的单个细胞外 V 颗粒的分选、分离和 RNA 分析

• 批准号: 9811934
• 负责人: Betty Kim
• 金额: $ 40.23万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-10 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811934
• 关键词: Antibodies; Biological; Biometry; Blood; Brain Neoplasms; Cachexia; Cell Communication; Cell Line; Cells; Cerebrospinal Fluid; Clinic; Clinical; Communication Research; DNA; Detection; Development; Diagnosis; Doctor of Philosophy; Drug resistance; Evaluation; Florida; Fluorescence; Fluorescence Microscopy; Glioblastoma; Heterogeneity; Human; Human Cell Line; Immune; Immunosuppression; Individual; Institutes; Joints; Knowledge; Label; Liposomes; Liquid substance; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Messenger RNA; Methods; MicroRNAs; Microfluidics; Molecular; Molecular Analysis; Molecular Sieve Chromatography; Neoplasm Metastasis; Ohio; Outcome; Patients; Pattern; Peptides; Phase; Polymerase Chain Reaction; Process; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA analysis; Research; Research Personnel; Resolution; Sampling; Serum; Signal Transduction; Site; Sorting - Cell Movement; System; Systems Biology; Techniques; Technology; Testing; Tissue Sample; Tissues; Tumor Biology; Ultracentrifugation; Universities; Urine; Validation; Vesicle; Yang; antibody conjugate; base; biochip; biomarker discovery; design; epithelial to mesenchymal transition; exosome; experience; extracellular; extracellular vesicles; high throughput analysis; immunoregulation; insight; macrophage; microvesicles; multidisciplinary; nanobiotechnology; nanofabrication; nanoparticle; new technology; next generation sequencing; protein metabolite; response; scale up; tumor immunology; vesicular release

Abstract Extracellular vesicles (EVs) such as microvesicles and exosomes are small membrane vesicles released by cells in the body. EVs are present in all biological fluids tested (e.g., blood, urine, cerebral spinal fluid) and contain various biomolecules including DNAs, RNAs, proteins and metabolites, and have been implicated as part of the cell-cell communication systems. Despite their importance, the current methods of isolating and characterizing EVs are technically challenging. The isolation methods usually cumbersome and irreproducible, and the characterization relies on techniques like Polymerase Chain Reaction (PCR), Next Generation Sequencing (NGS), and Mass Spectroscopy (MS) which just provide an aggregate of the overall RNA/DNA and protein content. During the characterization process, EVs are broken down to obtain their internal contents. Consequently, the molecular information at individual EV is lost. Given the heterogeneity of EVs, it is imperative to study EV-mediated intercellular signaling processes at the single EV level in order to gain important insights of their effects on promoting drug resistance, immunosuppression, epithelial-to- mesenchymal transition (EMT), cancer metastasis, and cachexia. Therefore, there is a critical need to develop technologies that provide accurate and efficient analysis of the molecular content within individual EVs. We propose an integrated system using a size exclusion chromatography to first sort the EVs in biofluids into well- defined size-based subpopulations, and then distribute each subpopulation into a set of parallel microfluidic channels where each channel is patterned with a number of microdomains tethered with antibody-conjugated liposomal nanoparticles containing molecular beacons (MBs) for enriched isolating/capturing of specific membrane protein/peptide-rich single EVs in the subpopulation, and simultaneously identification of specific RNA targets via MB-RNA hybridization when the captured EVs are fused with liposomes. Fluorescence- labelled antibodies may also be added to each microchannel to quantify the target membrane protein content of the captured single EVs. The development and feasibility demonstration of this novel technology will be conducted in the UG3 phase with a small-scale array for selected RNA and protein targets using well-characterized synthetic vesicles (SVs), EVs released from glioblastoma (GBM) cell lines, and spiked EVs in normal donor serum. In the UH3 phase, we plan to scale-up the biochip system design for high-throughput using the GMP type biochip fabrication. The applicability of this new technology will be validated for EVs from GBM cell lines as well as serum and cerebral spinal fluid (CSF) from GBM patients. In both phases, EV-based cell-cell communication will be investigated to determine if and how specific GBM EV subpopulations are involved in immune-regulation within GBMs. We have assembled a multi-disciplinary team with extensive knowledge and experience in nanobiotechnology, microfluidics, EV characterization, micro/nano-fabrication, EV RNA profiling and biomarker discovery, GBM diagnosis and treatment, and biostatistical analysis. The proposed aims and milestones are given as follows: UG3 Phase- Specific Aim 1: Development of a biochip to capture and characterize specific EV subpopulations at single EV level. Specific Aim 2: Comparison of results between the single EV-based measurements and conventional total EV-based averaged measurements. Quantitative Milestones: (i) Sorting, isolation and quantitative analysis of selected mRNA and miRNA targets in single SVs and EVs with >90% repeatability and better EV enrichment than conventional ultracentrifugation and antibody-based microfluidics methods; (ii) Identifying one or more EV subpopulations for high sensitivity detection of GBM cell- derived EVs; (iii) Identifying one or more GBM EV subpopulations which may involve in immuno-regulation. UH3 Phase- Specific Aim 1: Scaleup of the biochip manufacturing. Specific Aim 2: To perform EV analysis on clinical samples from GBM patients. Quantitative Milestones: (i) Sorting, isolation and quantitative analysis of mRNA/miRNA and membrane protein targets in single EVs from both blood and CSF samples with >90% repeatability and 5-fold better EV enrichment than conventional ultracentrifugation and antibody-based microfluidics methods; (ii) <10% false positive/negative prediction from a total of 120 GBM patients and non-patient samples; (iii) Identifying one or more GBM EV subpopulations which may involve in immunosuppression and/or associated with worse clinical outcomes.

抽象的 细胞外囊泡（EV），例如微泡和外泌体，是由细胞释放的小膜囊泡 体内的细胞。 EV 存在于所有测试的生物液体（例如血液、尿液、脑脊液）中，并且 含有各种生物分子，包括 DNA、RNA、蛋白质和代谢物，并被认为是 细胞间通信系统的一部分。尽管它们很重要，但目前的隔离和 表征电动汽车在技术上具有挑战性。分离方法通常繁琐且不可重复， 表征依赖于聚合酶链式反应 (PCR)、下一代等技术 测序 (NGS) 和质谱 (MS) 仅提供整体 RNA/DNA 的聚合 和蛋白质含量。在表征过程中，电动汽车被分解以获得其内部内容。 因此，单个 EV 的分子信息丢失。鉴于电动汽车的异质性， 必须在单一 EV 水平上研究 EV 介导的细胞间信号传导过程，以获得 关于它们对促进耐药性、免疫抑制、上皮细胞间相互作用的影响的重要见解 间质转化（EMT）、癌症转移和恶病质。因此，迫切需要开发 能够对单个电动汽车内的分子内容进行准确有效的分析的技术。我们 提出了一种使用尺寸排阻色谱法的集成系统，首先将生物流体中的 EV 分类到井中 定义基于尺寸的子群，然后将每个子群分配到一组并行的微流体中 通道，其中每个通道都由许多与抗体缀合的微域图案化 含有分子信标 (MB) 的脂质体纳米粒子，用于富集分离/捕获特定的 亚群中富含膜蛋白/肽的单一 EV，并同时鉴定特定的 当捕获的 EV 与脂质体融合时，通过 MB-RNA 杂交实现 RNA 靶向。荧光- 标记抗体也可以添加到每个微通道中以量化目标膜蛋白含量 捕获的单个电动汽车的数量。 该新技术的开发和可行性论证将在UG3阶段进行 使用经过充分表征的合成囊泡 (SV) 对选定的 RNA 和蛋白质靶标进行小规模阵列， 胶质母细胞瘤 (GBM) 细胞系释放 EV，并在正常供体血清中掺入 EV。在UH3阶段， 我们计划使用 GMP 型生物芯片制造来扩大生物芯片系统设计的高通量。这 这项新技术的适用性将针对 GBM 细胞系以及血清和脑细胞的 EV 进行验证 GBM 患者的脊髓液 (CSF)。在这两个阶段中，将研究基于电动汽车的细胞间通信，以 确定特定 GBM EV 亚群是否以及如何参与 GBM 内的免疫调节。 我们组建了一支多学科团队，在纳米生物技术方面拥有丰富的知识和经验， 微流体、EV 表征、微/纳米制造、EV RNA 分析和生物标志物发现、GBM 诊断和治疗以及生物统计分析。拟议的目标和里程碑如下： UG3 阶段特定目标 1：开发生物芯片来捕获和表征特定 EV 单一 EV 水平的亚群。具体目标2：基于单一EV的结果比较 测量和传统的基于 EV 的平均测量。定量里程碑：(i) 对单个 SV 和 EV 中选定的 mRNA 和 miRNA 靶标进行分选、分离和定量分析 比传统超速离心和基于抗体的方法具有 >90% 的重复性和更好的 EV 富集 微流体方法； (ii) 鉴定一种或多种 EV 亚群以用于 GBM 细胞的高灵敏度检测 衍生电动汽车； (iii)鉴定可能参与免疫调节的一种或多种GBM EV亚群。 UH3 阶段特定目标 1：扩大生物芯片制造规模。具体目标 2：执行 EV GBM 患者的临床样本分析。定量里程碑：(i) 分类、隔离和 对血液和脑脊液中单个 EV 中的 mRNA/miRNA 和膜蛋白靶标进行定量分析 与传统超速离心相比，样品的重复性 >90%，EV 富集效果好 5 倍 基于抗体的微流体方法； (ii) 总共 120 个 GBM 的假阳性/阴性预测 <10% 患者和非患者样本； (iii) 识别一个或多个可能涉及的 GBM EV 亚群 免疫抑制和/或与较差的临床结果相关。