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: 10560611
• 负责人: Yazhen Zhu
• 金额: $ 63.28万
• 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/10560611
• 关键词: AFP gene; Abdomen; Address; Alcoholic Liver Diseases; Antibodies; Bioinformatics; Biological; Biological Assay; Biological Markers; Biology; Biometry; Cancer Etiology; Cessation of life; Chemistry; Circulation; Cirrhosis; Clinical; Couples; Development; Devices; Diagnosis; Diagnostic; Disulfides; Early Diagnosis; Ensure; Etiology; Evaluation; Exhibits; Filtration; Goals; Hepatitis B; Hepatitis C; Image; Infection; Joints; Liver; Liver Cirrhosis; Mediating; Medical center; Messenger RNA; Methods; Microfluidics; Minor; Modality; Modeling; Motivation; Nanostructures; Non-Invasive Detection; 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; Specific qualifier value; Specificity; Specimen; Surface; Techniques; Technology; Training; Tumor-Derived; Ultracentrifugation; Ultrasonography; Validation; assay development; cell type; cohort; detection assay; diagnostic value; 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; AFP gene; Abdomen; Address; Alcoholic Liver Diseases; Antibodies; Bioinformatics; Biological; Biological Assay; Biological Markers; Biology; Biometry; Cancer Etiology; Cessation of life; Chemistry; Circulation; Cirrhosis; Clinical; Couples; Development; Devices; Diagnosis; Diagnostic; Disulfides; Early Diagnosis; Ensure; Etiology; Evaluation; Exhibits; Filtration; Goals; Hepatitis B; Hepatitis C; Image; Infection; Joints; Liver; Liver Cirrhosis; Mediating; Medical center; Messenger RNA; Methods; Microfluidics; Minor; Modality; Modeling; Motivation; Nanostructures; Non-Invasive Detection; 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; Specific qualifier value; Specificity; Specimen; Surface; Techniques; Technology; Training; Tumor-Derived; Ultracentrifugation; Ultrasonography; Validation; assay development; cell type; cohort; detection assay; diagnostic value; 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.

项目摘要 细胞外囊泡（EV）是一组异质的磷脂双层粘合颗粒 由所有类型的细胞释放，甚至由肿瘤细胞释放。由于肿瘤的生物分子货物 派生的电动汽车反映了亲本肿瘤细胞的EV，表征了肿瘤衍生的EV并分析其货物 预计将具有实质性的诊断价值。肝细胞癌（HCC），第四大常见 全世界与癌症相关的死亡的原因，最常见的是潜在肝硬化患者 继发于酒精性肝病（ALD），非酒精脂肪肝病（NAFLD）或乙型肝炎 感染。任何原因引起的肝硬化是HCC的公认危险因素。但是，目前的监视 具有腹部成像和血清生物标志物（例如AFP）的方案对诊断HCC的敏感性差 在早期阶段，它可能可以治愈。因此，敏感区分早期的生物标志物 迫切需要来自高危肝肝硬化的HCC。探索HCC EV和EV的诊断潜力 用于检测早期HCC的货物分析具有很大的希望，可以显着增强当前的能力 诊断方式。 我们提出了一个用于检测早期HCC的HCC EV数字评分测定法，该测定法伴随着两个非常强大的功能 技术：EV点击芯片以纯化HCC EV和反向转录液滴数字PCR（RT-） DDPCR）用于EV货物分析。临床的EV利用领域出现的主要挑战之一 使用是缺乏可重现的方法来隔离纯肿瘤衍生的EV种群。 隔离电动汽车的常规方法，例如超速离心，过滤和沉淀 将肿瘤衍生的电动汽车与非肿瘤衍生的电动汽车区分开。新的研究工作已致力于 探索基于免疫亲和力的捕获技术，以丰富不同实体瘤中肿瘤衍生的电动汽车。 但是，对于单抗体介导的肿瘤衍生的EV富集存在挑战 方法，例如有限的灵敏度/特异性以及需要多种捕获抗体克服的方法 肿瘤异质性。 EV点击芯片可以通过2步共价基于化学的问题来解决这些问题 肿瘤衍生的EV纯化（单击化学介导的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A来表征纯化的HCC EV 通过合并RT-DDPCR技术，由20个HCC特异性mRNA标记的面板组成。拟议的研究将 行为：i）两个功能组件的探索性开发和优化（即，EV点击芯片 和RT-DDPCR）并分析验证提出的HCC EV数字评分测定法，以及II）评估 拟议的HCC EV数字评分测定法的诊断性能用于检测早期HCC 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数字评分测定法，用于检测高危肝肝硬化患者的早期HCC。