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: 10327852
• 负责人: Betty Kim
• 金额: $ 88.33万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327852
• 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; Publications; 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; detection sensitivity; 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.

摘要 细胞外小泡(EV)，如微泡和外周小体，是由 体内的细胞。EV存在于所有被测试的生物液体中(如血液、尿液、脑脊液)和 含有多种生物分子，包括DNA、RNA、蛋白质和代谢物，并被认为是 小区间通信系统的一部分。尽管它们很重要，但目前分离和分离的方法 描述电动汽车的特性在技术上是具有挑战性的。隔离方法通常繁琐且不可重现， 而这种表征依赖于诸如聚合酶链式反应(PCR)、下一代 测序(NGS)和质谱学(MS)，它们只提供整个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的分子信息就会丢失。鉴于电动汽车的异质性，它是 迫切需要在单个EV水平上研究EV介导的细胞间信号传递过程，以获得 关于它们在促进耐药、免疫抑制、上皮到血管内皮细胞转化方面作用的重要见解 间充质转化(EMT)、癌症转移和恶病质。因此，迫切需要发展 对单个电动汽车中的分子含量提供准确而高效的分析的技术。我们 提出了一种使用尺寸排除层析的集成系统，将生物体液中的电动汽车首次分选到井- 定义了基于大小的亚种群，然后将每个亚种群分配到一组平行的微流体中 通道，其中每个通道都图案化了许多用抗体连接的微域 含有分子信标(MBS)的脂质体纳米粒用于富集特异性分离/捕获 富含膜蛋白/多肽的单个EVS亚群，并同时鉴定特异性 当捕获的EV与脂质体融合时，通过MB-RNA杂交靶向RNA。荧光- 还可以将标记的抗体添加到每个微通道以量化目标膜蛋白含量 被抓获的单辆电动汽车。 这项新技术的开发和可行性论证将在UG3阶段进行 通过使用表征良好的合成囊泡(SVS)对选定的RNA和蛋白质靶标进行小规模阵列， 从胶质母细胞瘤(GBM)细胞系释放EVS，并在正常供体血清中加入EVS。在UH3阶段， 我们计划使用GMP类型的生物芯片制造来扩大生物芯片系统的设计，以实现高通量。这个 这项新技术的适用性将在来自GBM细胞系以及血清和脑内的EVS中得到验证 GBM患者脑脊液(CSF)。在这两个阶段，将研究基于EV的细胞间通信 确定特定的GBM EV亚群是否以及如何参与GBM内的免疫调节。 我们已经组建了一支在纳米生物技术方面拥有丰富知识和经验的多学科团队， 微流体、EV表征、微/纳米制造、EV RNA图谱和生物标记物发现 诊断和治疗，生物统计分析。建议的目标和里程碑如下： UG3阶段特定目标1：开发一种捕获和表征特定EV的生物芯片 单个EV水平的亚群。具体目标2：比较以电动汽车为基础的单一模式的结果 测量和传统的基于总电动汽车的平均测量。量化里程碑：(一) 分离、分离和定量分析单个SVS和EVS中选定的mRNA和miRNA靶标 与传统的超速离心法和抗体法相比，&gt；90%的重复性和更好的EV浓缩 微流体学方法；(Ii)鉴定一个或多个EV亚群，用于高灵敏度检测GBM细胞- (Iii)鉴定可能参与免疫调节的一个或多个GBM EV亚群。 UH3阶段特定目标1：生物芯片制造的规模。具体目标2：执行电动汽车 银屑病患者临床标本分析。量化里程碑：(一)分类、分离和 血液和脑脊液中单个EV的mRNA/miRNA和膜蛋白靶标的定量分析 样品的重复性为90%，EV浓缩效果是常规超速离心法的5倍 基于抗体的微流体学方法；(Ii)&lt；总共120个GBM中10%的假阳性/阴性预测 患者和非患者样本；(Iii)确定一个或多个GBM EV亚群可能涉及 免疫抑制和/或与较差的临床结果相关。