Extracellular Vesicle-Based Digital Scoring Assay for Detecting Early-stage Hepatocellular Carcinoma

项目总结 细胞外小泡(EVS)是一组磷脂双层包裹的异质颗粒 由所有类型的细胞释放，尤其是肿瘤细胞。因为肿瘤的生物分子货物- 衍生的EVS反映了亲代肿瘤细胞的EVS，表征了肿瘤衍生的EVS并描绘了它们的货物 预计将具有实质性的诊断价值。肝细胞癌(HCC)，第四常见 全世界与癌症相关的死亡原因，最常发生在潜在的肝硬变患者 继发于酒精性肝病(ALD)、非酒精性脂肪性肝病(NAFLD)或乙肝/丙型肝炎 感染。任何原因引起的肝硬变都是公认的肝细胞癌的危险因素；然而，目前的监测 腹部影像和血清生物标志物(如AFP)方案对诊断肝细胞癌的敏感性较差。 在早期阶段，当它有可能治愈的时候。因此，敏感区分早期阶段的生物标志物 高危肝硬变的肝细胞癌是迫切需要的。探讨EVS和EV在肝癌诊断中的潜力 用于检测早期肝癌的货物剖面图具有显著增强Current的能力的巨大希望 诊断模式。 我们提出了一种用于检测早期肝细胞癌的肝细胞癌EV数字评分方法，它结合了两个非常强大的 技术：用于肝癌EV纯化的EV点击芯片和逆转录-液滴数字聚合酶链式反应(RT-RT-DELP) DdPCR)，用于电动汽车货物概况分析。电动汽车临床应用领域出现的主要挑战之一 用途是缺乏可靠和可重复的方法来分离纯肿瘤来源的EV群体。 传统的分离肠道病毒的方法，如超速离心法、过滤法和沉淀法是不能实现的。 区分肿瘤来源的电动汽车和非肿瘤来源的电动汽车。新的研究努力致力于 探索基于免疫亲和力的捕获技术在不同实体肿瘤中丰富肿瘤来源的EVS。 然而，单一抗体介导的肿瘤来源的EV的富集性存在挑战。 方法，如有限的敏感度/特异度和需要多种捕获抗体来克服 肿瘤异质性。EV Click芯片可以通过基于两步共价化学的 肿瘤来源的EV纯化(点击化学介导的EV捕获/二硫化物裂解驱动的EV释放) 而不是抗体介导的EV捕获。纯化的肝癌EV然后可以通过定量a 通过结合RT-ddPCR技术，筛选出20个肝细胞癌特异的mRNA标记物。拟议的研究将 实施：i)两个功能组件(即电动汽车点击芯片)的探索性开发和优化 和RT-ddPCR)和分析验证所提出的肝细胞癌EV数字评分试验，以及ii)对 肝细胞癌EV数字评分方法对早期肝细胞癌的诊断性能 培训和验证队列。此R01提案的长期目标是开发、优化和验证 建议的肝细胞癌EV数字评分方法用于检测高危肝硬变患者的早期肝细胞癌。

PROJECT SUMMARY Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer-enclosed particles that are released by all types of cells, and even more so by tumor cells. Since the biomolecular cargoes of tumor- derived EVs mirror those of the parental tumor cells, characterizing tumor-derived EVs and profiling their cargo are expected to be of substantial diagnostic value. Hepatocellular carcinoma (HCC), the fourth most common cause of cancer-related deaths worldwide, most often develops in patients with underlying liver cirrhosis secondary to alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), or hepatitis B/C infections. Cirrhosis from any cause is a well-established risk factor for HCC; however, current surveillance regimens with abdominal imaging and serum biomarkers (e.g., AFP) have poor sensitivity for diagnosing HCC at an early stage, when it is potentially curable. Therefore, biomarkers that sensitively distinguish early-stage HCC from at-risk liver cirrhosis are desperately needed. Exploring the diagnostic potential of HCC EVs and EV cargo profiling for detecting early-stage HCC holds great promise to significantly augment the ability of current diagnostic modalities. We propose an HCC EV digital scoring assay for detecting early-stage HCC, which couples two very powerful technologies: EV Click Chip for purification of HCC EVs and reverse-transcription droplet digital PCR (RT- ddPCR) for EV cargo profiling. One of the major challenges emerging in the field of EV utilization for clinical use is the lack of robust and reproducible methods for the isolation of a pure tumor-derived EV population. Conventional methods for isolating EVs, such as ultracentrifugation, filtration, and precipitation, are incapable of discriminating tumor-derived EVs from non-tumor-derived EVs. New research efforts have been devoted to exploring immunoaffinity-based capture techniques for enriching tumor-derived EVs in different solid tumors. However, there are challenges identified for the single antibody-mediated tumor-derived EV enriching approaches, such as limited sensitivity/specificity and a need for multiple capture antibodies to overcome the tumor heterogeneity. The EV Click Chips can address these concerns with a 2-step covalent chemistry-based tumor-derived EV purification (click chemistry-mediated EV capture/disulfide cleavage-driven EV release) instead of antibody-mediated EV capture. The purified HCC EVs can then be characterized by quantifying a panel of 20 HCC-specific mRNA markers by incorporating RT-ddPCR technology. The proposed research will conduct: i) an exploratory development and optimization of the two functional components (i.e., EV Click Chip and RT-ddPCR) and analytically validate the proposed HCC EV digital scoring assay, and ii) an evaluation of the diagnostic performance of the proposed HCC EV digital scoring assay for detecting early-stage HCC using training and validation cohorts. The long-term goal of this R01 proposal is to develop, optimize, and validate the proposed HCC EV digital scoring assay for detecting early-stage HCC from at-risk liver cirrhotic patients.