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.

项目概要 肝细胞癌（HCC）占原发性肝癌的80-85%，主要发生在患者身上 患有肝硬化或慢性乙型肝炎病毒 (HBV) 感染的 HCC 预后不佳，主要是由于。 目前的临床实践指南建议在诊断时进行 HCC 监测。 对于高危患者，每年两次进行含/不含血清甲胎蛋白 (AFP) 的肝脏超声检查，以实现以下目标： 然而，它们的准确性仍然较低，灵敏度在 60- 之间。 70%，特异性为 90%，因此，仍然迫切需要用于早期检测 HCC 的新型生物标志物。 细胞外囊泡 (EV) 是一组异质脂质纳米颗粒，由所有类型的细胞释放 细胞，尤其是肿瘤细胞和肿瘤微环境中的细胞。 它们在疾病的相对早期阶段就存在于循环中，并且在所有疾病阶段都很容易获得。 由于肿瘤相关 EV 的表面蛋白可以反映亲本肿瘤细胞和这些细胞的表面蛋白 在肿瘤微环境中，利用 HCC 相关 EV 表面蛋白的诊断潜力 签名作为一种用于早期检测 HCC 的新型生物标志物，有望显着增强 HCC 的诊断价值。 当前诊断方式的能力。 我们提出 HCC EV 表面蛋白测定 (SPA) 来量化 HCC 相关 EV 的亚群 拟议的 HCC EV SPA 结合了两种强大的技术：Click Chemistry- 介导的 EV Click Beads 用于分离 HCC 相关 EV 的不同子集，以及下游 4 重实数 时间免疫 PCR 对 HCC EV 的孤立子集进行定量是新出现的主要挑战之一。 在 EV 临床应用领域，缺乏可靠且可重复的方法来分离 分离 EV 的传统方法，例如超速离心， 过滤和沉淀无法从总 EV 中分离出肿瘤相关的 EV 亚群。 研究工作致力于探索基于免疫亲和力的捕获技术来富集肿瘤 然而，从不同实体瘤患者的血浆样本中提取相关的 EV 仍然存在挑战。 确定了单抗体介导的肿瘤源性 EV 富集方法，例如有限的 敏感性/特异性以及需要多种捕获抗体来克服肿瘤异质性。 为了解决这些问题，我们的研究团队开发了 HCC EV SPA，它结合了点击化学- 介导的肿瘤相关 EV 分离和下游 4 重实时免疫 PCR 是 HCC EV SPA 的关键。 能够对患者血浆中 32 个 HCC EV 亚群进行高度灵敏和特异的定量 样本，基于 8 种不同的 HCC 相关表面蛋白标记物和 4 种 EV 表面的组合使用 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 亚群来检测高危肝硬化患者的早期 HCC。