Click Chemistry-Mediated Surface Protein Assay for Quantifying Subpopulations of Hepatocellular Carcinoma-associated Extracellular Vesicles

点击化学介导的表面蛋白测定法定量肝细胞癌相关细胞外囊泡亚群

• 批准号: 10737497
• 负责人: Vatche Agopian
• 金额: $ 67.41万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737497
• 关键词: ANXA5 gene; Address; Alcoholic Liver Diseases; Antibodies; Bar Codes; Bioinformatics; Biological Assay; Biology; Biometry; CD81 gene; Cancer Etiology; Case/Control Studies; Cells; Cessation of life; Chemistry; Chronic Hepatitis B; Circulation; Cirrhosis; Clinical; Clinical Practice Guideline; Couples; Coupling; DNA; Development; Diagnosis; Diagnostic; Disease; Early Diagnosis; Etiology; Exhibits; Filtration; GPC3 gene; Goals; Guidelines; Hepatitis B; Hepatitis C; Joints; Liver; Liver Cirrhosis; Liver diseases; Logistic Regressions; Mediating; Medical center; Membrane Proteins; Methods; Modality; Motivation; Oncology; Operative Surgical Procedures; Pathology; Patients; Performance; Phospholipids; Plasma; Precipitation; Primary Malignant Neoplasm of Liver; Primary carcinoma of the liver cells; Prognosis; Protein Analysis; Quantitative Evaluations; Recommendation; Regression Analysis; Reproducibility; Research; Risk; Sampling; Sensitivity and Specificity; Serum; Site; Solid Neoplasm; Specificity; Specimen; Surface; TACSTD1 gene; Techniques; Technology; Time; Training; Tumor-Derived; Ultracentrifugation; Ultrasonography; Validation; Viral hepatitis; alpha-Fetoproteins; assay development; cell type; circulating biomarkers; cohort; detection assay; diagnostic value; early detection biomarkers; extracellular vesicles; hepatocellular carcinoma cell line; in-vitro diagnostics; innovation; lipid nanoparticle; molecular pathology; neoplastic cell; non-alcoholic fatty liver disease; novel marker; particle; predictive modeling; protein biomarkers; technology platform; tumor; tumor heterogeneity; tumor microenvironment; ultrasound

PROJECT SUMMARY Hepatocellular carcinoma (HCC) accounts for 80-85% of primary liver cancers, and mainly occurs in patients with liver cirrhosis or chronic hepatitis B virus (HBV) infection. Prognosis of HCC is dismal primarily due to advanced stage of disease at diagnosis. Current clinical practice guidelines recommend HCC surveillance by biannual liver ultrasound with/without serum alpha-fetoprotein (AFP) for at-risk patients to achieve the goal of detecting HCC at a curative stage. However, their accuracy remains relevantly low with sensitivity between 60- 70% at a specificity of 90%. As such, novel biomarkers for early detection of HCC are still desperately needed. Extracellular vesicles (EVs) are a heterogeneous group of lipid nanoparticles that are released by all types of cells, and even more so by tumor cells and those cells within tumor microenvironment. Tumor-associated EVs are present in circulation at relatively early stages of disease and are readily accessible across all disease stages. Since the surface proteins of tumor-associated EVs could mirror those of the parental tumor cells and those cells within tumor microenvironment, exploiting the diagnostic potential of HCC-associated EVs’ surface protein signatures as a novel biomarker for early detection of HCC holds great promise to significantly augment the ability of current diagnostic modalities. We propose an HCC EV Surface Protein Assay (SPA) to quantify subpopulations of HCC-associated EVs for detecting early-stage HCC. The proposed HCC EV SPA couples two powerful technologies: Click Chemistry- mediated EV Click Beads for isolating different subsets of HCC-associated EVs, and downstream 4-plex real- time immuno-PCR for quantification of the isolated subsets of HCC EVs. 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 subpopulations of tumor-associated EVs. Conventional methods for isolating EVs, such as ultracentrifugation, filtration, and precipitation, are incapable of isolating subpopulation of tumor-associated EVs from total EVs. New research efforts have been devoted to exploring immunoaffinity-based capture techniques for enriching tumor- associated EVs from the plasma samples of patients with 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. In order to address these concerns, our research team developed HCC EV SPA, which combines a click chemistry- mediated tumor-associated EV isolation, and downstream 4-plex real-time immuno-PCR. HCC EV SPA is capable of highly sensitive and specific quantification of 32 subpopulations of HCC EVs in patients’ plasma samples, based on the combined use of 8 different HCC-associated surface protein markers and four EV surface markers. The long-term goal of this R01 proposal is to develop, refine, and validate the HCC EV SPA for detecting early-stage HCC from at-risk liver cirrhotic patients by quantifying subpopulations of HCC EVs.

项目摘要 肝细胞癌（HCC）占原发性肝癌的80-85％，主要发生在患者中 肝硬化或慢性丙型肝炎病毒（HBV）感染。 HCC的预后由于 诊断时疾病的晚期阶段。当前的临床实践指南建议HCC监视 双肝超声检查，具有/没有血清α-毒素蛋白（AFP），以实现危险患者的目标 在治愈阶段检测HCC。但是，它们的准确性与60-之间的灵敏度保持较低 70％的特异性为90％。因此，仍然需要新颖的生物标志物来早日检测HCC。 细胞外蔬菜（EV）是一组异质的脂质纳米颗粒，所有类型释放 细胞，甚至是肿瘤细胞和肿瘤微环境中的细胞。肿瘤相关的电动汽车 在相对较早的疾病阶段出现在循环中，并且在所有疾病阶段都可以轻松获得。 由于肿瘤相关的EV的表面蛋白可以反映出亲本肿瘤细胞的表面蛋白 在肿瘤微环境中，利用了与HCC相关的EVS表面蛋白的诊断潜力 签名作为早期发现HCC的新型生物标志物具有很大的希望，可以显着增加 当前诊断方式的能力。 我们提出了HCC EV表面蛋白测定（SPA），以量化与HCC相关的EV的亚群 检测早期HCC。拟议的HCC EV Spa夫妇伴侣两种强大的技术：点击化学 - 介导的EV点击珠，用于隔离与HCC相关的电动汽车的不同子集和下游4-plex Real- 用于定量HCC EV的分离亚集的时间免疫PCR。出现的主要挑战之一 在用于临床用途的EV利用领域中，缺乏可隔离的可重复和可再现方法 肿瘤相关电动汽车的亚群。隔离电动汽车的常规方法，例如超速离心， 过滤和降水无法从总电动汽车中分离与肿瘤相关的电动汽车的亚群。新的 研究工作已致力于探索基于免疫亲和力的捕获技术，以丰富肿瘤 - 来自不同实体瘤患者血浆样品的相关电动汽车。但是，有挑战 针对单抗体介导的肿瘤衍生的EV富集方法确定，例如有限 灵敏度/特异性以及需要多种捕获抗体克服肿瘤异质性的需求。为了 为了解决这些问题，我们的研究团队开发了HCC EV Spa，该水疗中心结合了点击化学 - 介导的肿瘤相关EV分离，下游4-PLEX实时免疫PCR。 HCC EV水疗中心是 能够对患者血浆中HCC EV的32个亚群进行高度敏感和特异性定量 样品，基于8种不同HCC相关的表面蛋白标记和四个EV表面的共同使用 标记。该R01提案的长期目标是开发，完善和验证HCC EV SPA 通过量化HCC EV的亚群来检测来自高危肝肝硬化患者的早期HCC。