Mass spectrometry for highly sensitive and sample-sparing analysis of extracellular vesicles in liver diseases

用于肝脏疾病细胞外囊泡高灵敏度和样品节省分析的质谱法

• 批准号: 10736006
• 负责人: Alexander Revzin
• 金额: $ 70.65万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736006
• 关键词: Antibodies; Biological Markers; Blood; Cells; Circulation; Clinical; Development; Diagnosis; Disease; Disease Progression; Early Diagnosis; Fatty Liver; Fibrosis; Functional disorder; Future; Hand; Harvest; Hepatocyte; Inflammation; Label; Lipids; Liver; Liver Cirrhosis; Liver diseases; Maintenance; Malignant neoplasm of liver; Mass Spectrum Analysis; Methodology; Microfluidic Microchips; Microfluidics; Monitor; Organ; Patient Care; Patients; Physiological; Plasma; Populations at Risk; Proteins; Proteomics; Protocols documentation; Risk; Sampling; Signal Transduction; Spectrometry, Mass, Secondary Ion; Surface; Technology; Testing; Time; Tissues; Training; United States; biomarker identification; detection sensitivity; extracellular vesicles; instrumentation; lipidomics; liver biopsy; liver injury; neural network; new technology; non-alcoholic fatty liver; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; novel strategies; patient stratification; peripheral blood; risk stratification; screening; timeline; transmission process; treatment response; vesicular release

ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the United States. NAFLD is a conglomerate of the relatively nonprogressive non-alcoholic fatty liver (NAFL) and progressive non-alcoholic steatohepatitis (NASH). While hepatic steatosis is a conserved feature of both NAFL and NASH, the latter is characterized by liver injury, inflammation, fibrosis, and the risk of liver cancer and cirrhosis. At the present time, there are no effective noninvasive and scalable screening strategies to distinguish between NAFL and NASH nor monitor NASH progression. Thus, there is a significant unmet need for biomarkers which are relatively easy to obtain, can be tested repeatedly over time, can distinguish NAFL from NASH, are pathophysiologically in- formed, and help risk stratify patients with NASH. Extracellular vesicles (EVs) carry signals from diseased cells and organs and therefore represent ideal biomarkers for NAFL and NASH. However, the development of EV based biomarkers has been confounded by several challenges. First challenge relates to the large sample volume requirements of mass spectrometry approaches traditionally used for EV analysis (e.g. LC-MS/MS). This limits their utility for the analysis of scarce patient samples. Second challenge lies in the fact that EVs released from the diseased liver become diluted by circulation and represent only a small subset (~1%) of total EVs present in peripheral blood. This makes it difficult to identify biomarkers of disease progression or therapy re- sponse. In this project, we will address the aforementioned challenges by developing novel technologies: 1) nanoprojectile (NP) secondary ion mass spectrometry (SIMS) that will enable multiplexed analysis of EVs using microliters of sample and 2) microfluidic organotypic liver cultures that will allow us to harvest undiluted liver EVs from healthy or diseased tissue. Upon completion of this project, we will have in hand a powerful mass spectrometry technology for proteomic and lipidomic analysis of EVs. The identification of EV biomarkers in this project will, in the future, be parlayed into a blood-based test that will allow to diagnose NAFLD, distinguish NAFL from NASH and help risk stratify patients. Such a test will replace highly invasive liver biopsy currently used for diagnosis and will revolutionize the care of patients with NAFLD. It will also allow early diagnosis of NASH in at risk populations.

抽象的 非酒精性脂肪肝病（NAFLD）是美国最常见的肝病。 NAFLD 是一种 相对非进行性非酒精性脂肪肝 (NAFL) 和进行性非酒精性脂肪肝的综合体 脂肪性肝炎（NASH）。虽然肝脂肪变性是 NAFL 和 NASH 的保守特征，但后者是 其特点是肝损伤、炎症、纤维化，以及肝癌和肝硬化的风险。目前， 没有有效的非侵入性和可扩展的筛查策略来区分 NAFL 和 NASH 也不监测 NASH 进展。因此，对相对容易的生物标志物的需求存在显着的未满足的需求。 获得，可以随着时间的推移反复测试，可以区分 NAFL 和 NASH，在病理生理学上是 - 形成，并帮助对 NASH 患者进行风险分层。细胞外囊泡 (EV) 携带来自患病细胞的信号 和器官，因此代表了 NAFL 和 NASH 的理想生物标志物。然而，电动汽车的发展 基于生物标志物的方法受到了一些挑战的困扰。第一个挑战涉及大样本 传统上用于 EV 分析的质谱方法（例如 LC-MS/MS）的体积要求。这 限制了它们在分析稀缺患者样本方面的效用。第二个挑战在于电动汽车的发布 来自患病肝脏的细胞被循环稀释，仅占总 EV 的一小部分 (~1%) 存在于外周血中。这使得识别疾病进展或治疗重新的生物标志物变得困难。 响应。在这个项目中，我们将通过开发新技术来解决上述挑战：1） 纳米弹丸 (NP) 二次离子质谱 (SIMS) 将使用以下技术对 EV 进行多重分析 微升样品和 2) 微流体器官型肝脏培养物，使我们能够收获未稀释的肝脏 EV 来自健康或患病的组织。 该项目完成后，我们将拥有强大的蛋白质组质谱技术 和 EV 的脂质组学分析。该项目中电动汽车生物标志物的识别未来将得到充分利用 进行基于血液的测试，可诊断 NAFLD、区分 NAFL 和 NASH 并帮助风险分层 患者。这种测试将取代目前用于诊断的高侵入性肝活检，并将彻底改变 NAFLD 患者的护理。它还将有助于对高危人群进行 NASH 早期诊断。