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.

抽象的细胞外囊泡（EV），例如微泡和外泌体是释放的小膜囊泡体内细胞。 EV存在于所有测试的生物液中（例如，血液，尿液，脑脊髓液）和包含包括DNA，RNA，蛋白质和代谢物在内的各种生物分子，并被认为是细胞电池通信系统的一部分。尽管它们的重要性，但目前的隔离和电动汽车的表征在技术上具有挑战性。隔离方法通常很麻烦且不可复制，表征依赖于聚合酶链反应（PCR）等技术，下一代测序（NGS）和质谱（MS），仅提供整体RNA/DNA的骨料和蛋白质含量。在表征过程中，电动汽车被分解以获得其内部内容。因此，丢失了单个EV的分子信息。鉴于电动汽车的异质性，这是必须在单个EV水平上研究EV介导的细胞间信号传导过程以获得对促进耐药性，免疫抑制，上皮到上皮的影响的重要见解间充质转变（EMT），癌症转移和恶病质。因此，迫切需要发展提供对单个EV中分子含量的准确有效分析的技术。我们使用尺寸排除色谱法提出一个集成系统，将生物流体中的电动汽车首先排序到井中定义的基于尺寸的亚群，然后将每个亚群分配到一组平行的微流体中每个通道都用抗体偶联的许多微域图案的通道含有分子信标（MB）的脂质体纳米颗粒，用于富集/捕获特定的分离/捕获亚群中的膜蛋白/肽富含富含的单eV，并同时识别特定当捕获的EV与脂质体融合时，通过MB-RNA杂交进行RNA靶标。荧光标记的抗体也可以添加到每个微通道中，以量化靶膜蛋白含量捕获的单电动汽车。该新技术的开发和可行性证明将在UG3阶段进行使用良好的合成囊泡（SVS），具有小尺度的阵列，用于选定的RNA和蛋白质靶标从胶质母细胞瘤（GBM）细胞系释放的电动汽车和正常供体血清中的EVS。在UH3阶段，我们计划使用GMP类型的生物芯片制造来扩展用于高通量的生物芯片系统设计。这该新技术的适用性将用于GBM细胞系以及血清和脑的电动汽车的验证 GBM患者的脊柱液（CSF）。在这两个阶段中，将研究基于EV的细胞电池通信确定特定的GBM EV亚群是否以及如何参与GBM内的免疫调节。我们已经组建了一个具有广泛知识和纳米属技术的知识和经验的多学科团队，微流体，电动汽车表征，微/纳米制作，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：开发生物芯片以捕获和表征特定的EV 单eV水平的亚群。特定目的2：基于单个EV之间的结果比较测量和常规的基于EV的总平均测量值。定量里程碑：（i）对单个SV和EV中选定的mRNA和miRNA靶标的分类，分离和定量分析， > 90％的可重复性和比常规超速离心和基于抗体的EV富集更好微流体方法；（ii）确定一个或多个EV亚群来高灵敏度检测GBM细胞 - 派生的电动汽车；（iii）识别可能涉及免疫调节的一个或多个GBM EV亚群。 UH3相位特定目标1：生物芯片制造的规模。特定目标2：执行EV 分析GBM患者的临床样本。定量里程碑：（i）分类，隔离和来自血液和CSF的单个EV中的mRNA/miRNA和膜蛋白靶标的定量分析与传统的超速离心和相比基于抗体的微流体方法；（ii）<10％的假阳性/阴性预测总计120 GBM 患者和非患者样本；（iii）确定可能涉及的一个或多个GBM EV亚群免疫抑制和/或与临床结果较差有关。