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

基于细胞外囊泡的数字评分法检测早期肝细胞癌

• 批准号: 10097789
• 负责人: Yazhen Zhu
• 金额: $ 65.97万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-18 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10097789
• 关键词: AFP gene; Abdomen; Address; Alcoholic Liver Diseases; Antibodies; Bioinformatics; Biological; Biological Assay; Biological Markers; Biology; Biometry; Blood Circulation; Cancer Etiology; Cessation of life; Chemistry; Cirrhosis; Clinical; Couples; Development; Devices; Diagnosis; Diagnostic; Disulfides; Early Diagnosis; Ensure; Etiology; Evaluation; Exhibits; Filtration; Goals; Hepatitis B; Image; Infection; Joints; Liver; Liver Cirrhosis; Mediating; Medical center; Messenger RNA; Methods; Microfluidics; Minor; Modality; Modeling; Motivation; Nanostructures; Oncology; Operative Surgical Procedures; Patients; Performance; Phospholipids; Plasma; Population; Precipitation; Primary carcinoma of the liver cells; Quantitative Evaluations; Regimen; Reporting; Reproducibility; Research; Research Personnel; Reverse Transcription; Risk; Risk Factors; Sampling; Secondary to; Sensitivity and Specificity; Serum; Site; Solid Neoplasm; Specificity; Specimen; Surface; Techniques; Technology; Training; Tumor-Derived; Ultracentrifugation; Ultrasonography; Validation; Yang; assay development; base; blind; cell type; cohort; digital; extracellular vesicles; hepatocellular carcinoma cell line; in-vitro diagnostics; innovation; molecular pathology; nanomaterials; neoplastic cell; non-alcoholic fatty liver disease; particle; standard of care; tumor heterogeneity; vesicular release

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.

项目概要 细胞外囊泡 (EV) 是一组异质的磷脂双层封闭颗粒， 由所有类型的细胞释放，尤其是肿瘤细胞释放。由于肿瘤的生物分子货物 衍生的 EV 与亲本肿瘤细胞相似，表征肿瘤衍生的 EV 并分析其货物 预计具有重要的诊断价值。肝细胞癌 (HCC)，第四常见的癌症 全世界癌症相关死亡的原因，最常见于患有潜在肝硬化的患者 继发于酒精性肝病 (ALD)、非酒精性脂肪肝病 (NAFLD) 或乙型/丙型肝炎 感染。任何原因引起的肝硬化都是 HCC 的一个明确的危险因素；然而，目前的监控 采用腹部影像学和血清生物标志物（例如 AFP）的治疗方案诊断 HCC 的敏感性较差 在早期阶段，当它有可能治愈时。因此，敏感区分早期阶段的生物标志物 迫切需要来自高危肝硬化的 HCC。探索 HCC EV 和 EV 的诊断潜力 用于检测早期 HCC 的货物分析有望显着增强当前的能力 诊断方式。 我们提出了一种用于检测早期 HCC 的 HCC EV 数字评分测定，它将两种非常强大的功能结合起来 技术：用于纯化 HCC EV 的 EV Click 芯片和逆转录液滴数字 PCR (RT- ddPCR）用于电动汽车货物分析。 EV临床应用领域出现的主要挑战之一 使用的原因是缺乏可靠且可重复的方法来分离纯肿瘤来源的 EV 群体。 超速离心、过滤和沉淀等传统方法无法分离 EV 区分肿瘤来源的 EV 和非肿瘤来源的 EV 的方法。新的研究工作致力于 探索基于免疫亲和力的捕获技术来富集不同实体瘤中肿瘤来源的 EV。 然而，单一抗体介导的肿瘤源性 EV 富集存在一些挑战。 方法，例如有限的灵敏度/特异性以及需要多种捕获抗体来克服 肿瘤异质性。 EV Click 芯片可以通过基于共价化学的两步法解决这些问题 肿瘤源性 EV 纯化（点击化学介导的 EV 捕获/二硫键裂解驱动的 EV 释放） 而不是抗体介导的 EV 捕获。然后可以通过量化纯化的 HCC EV 来表征 通过结合 RT-ddPCR 技术，一组 20 个 HCC 特异性 mRNA 标记物。拟议的研究将 进行：i) 两个功能组件（即 EV Click 芯片）的探索性开发和优化 和 RT-ddPCR）并分析验证拟议的 HCC EV 数字评分测定，以及 ii）评估 所提出的 HCC EV 数字评分测定法用于检测早期 HCC 的诊断性能 培训和验证队列。该 R01 提案的长期目标是开发、优化和验证 拟议的 HCC EV 数字评分测定用于检测高危肝硬化患者的早期 HCC。