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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

基本信息

批准号：
10487539
负责人：
Betty Kim
金额：
$ 86.56万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-10 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10487539
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

项目摘要

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.

摘要细胞外囊泡（EV）如微囊泡和外来体是由微囊泡释放的小膜囊泡。体内的细胞。EV存在于所有测试的生物流体中（例如，血液、尿液、脑脊液）和含有各种生物分子，包括DNA、RNA、蛋白质和代谢物，并被认为是细胞间通讯系统的一部分。尽管它们很重要，但目前的分离和表征EV在技术上具有挑战性。分离方法通常繁琐且不可重复，并且表征依赖于诸如聚合酶链式反应（PCR）、下一代聚合酶链式反应（PCR）、聚合酶链式反应（PCR）、聚合酶链式反应（PCR）和聚合酶链式反应（PCR）等技术。测序（NGS）和质谱（MS），它们仅提供总体RNA/DNA的聚集体和蛋白质含量。在表征过程中，EV被分解以获得其内部内容。因此，单个EV的分子信息丢失。考虑到电动汽车的异质性，必须在单个EV水平上研究EV介导的细胞间信号传导过程，以获得重要的见解，他们的影响，促进耐药性，免疫抑制，上皮细胞，间质转化（EMT）、癌症转移和恶病质。因此，迫切需要发展这些技术可以提供对单个EV内分子含量的准确和有效分析。我们提出了一种使用尺寸排阻色谱法的集成系统，以首先将生物流体中的EV分选到井中，定义的基于大小的亚群，然后将每个亚群分配到一组平行的微流体其中每个通道被图案化有许多微区，所述微区与抗体缀合的含有分子信标（MB）的脂质体纳米颗粒，用于富集分离/捕获特异性膜蛋白/肽丰富的单EV的亚群，并同时鉴定特异性当捕获的EV与脂质体融合时，RNA通过MB-RNA杂交靶向。荧光- 标记的抗体也可以加入到每个微通道中捕获的单个电动汽车。这项新技术的开发和可行性论证将在UG 3阶段进行通过使用良好表征的合成囊泡（SV）的用于选定RNA和蛋白质靶的小规模阵列，从胶质母细胞瘤（GBM）细胞系释放的EV，以及在正常供体血清中掺入的EV。在UH 3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以实现高通量。的这项新技术的适用性将被验证用于来自GBM细胞系以及血清和大脑的EV。来自GBM患者的脊髓液（CSF）。在这两个阶段中，将研究基于EV的蜂窝通信，确定特定GBM EV亚群是否以及如何参与GBM内的免疫调节。我们已经组建了一个多学科的团队，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学分析。拟议的目标和里程碑如下： UG 3阶段-特定目标1：开发生物芯片以捕获和表征特定EV 单EV水平的亚群。具体目标2：基于EV的单一测量值和常规的基于EV的总平均测量值。定量里程碑：（i）在单个SV和EV中选择的mRNA和miRNA靶标的分选、分离和定量分析， >90%的重复性和比传统超离心和基于抗体的更好的EV富集（ii）鉴定用于高灵敏度检测GBM细胞的一种或多种EV亚群; （iii）鉴定可能参与免疫调节的一个或多个GBM EV亚群。 UH 3阶段-特定目标1：生物芯片制造的规模扩大。具体目标2：执行EV 分析来自GBM患者的临床样品。定量分析：（一）分类、分离和来自血液和CSF的单个EV中的mRNA/miRNA和膜蛋白靶标的定量分析样品具有>90%的重复性和比常规超离心高5倍的EV富集，基于抗体的微流体方法;（ii）总计120个GBM中假阳性/阴性预测<10% （iii）鉴定一种或多种GBM EV亚群，所述亚群可能涉及与GBM EV相关的免疫应答; 免疫抑制和/或与更差的临床结果相关。