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

项目摘要 肝细胞癌（HCC）占原发性肝癌的80-85%，主要发生在 伴有肝硬化或慢性B型肝炎病毒（HBV）感染。HCC的预后很差，主要是由于 诊断时疾病处于晚期。目前的临床实践指南建议通过以下方式监测HCC： 对高危患者进行一年两次的肝脏超声检查（有/无血清甲胎蛋白），以实现以下目标： 在治愈阶段检测HCC。然而，它们的准确性仍然相对较低，灵敏度在60- 100之间。 70%，特异性90%。因此，仍然迫切需要用于早期检测HCC的新生物标志物。 细胞外囊泡（EV）是由所有类型的脂质纳米颗粒释放的异质组。 细胞，甚至肿瘤细胞和肿瘤微环境中的那些细胞。肿瘤相关EV 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的表面蛋白， 在肿瘤微环境中，开发HCC相关EV表面蛋白的诊断潜力 作为早期检测HCC的新生物标志物， 目前的诊断方法。 我们提出了一种HCC EV表面蛋白测定（SPA）来定量HCC相关EV的亚群， 检测早期HCC。拟议的HCC EV SPA结合了两项强大的技术：点击化学- 介导的EV点击珠，用于分离HCC相关EV的不同亚群，和下游4重真实的- 时间免疫PCR用于定量HCC EV的分离亚组。新出现的主要挑战之一 在EV临床应用领域，缺乏用于分离 肿瘤相关EV的亚群。用于分离EV的常规方法，例如超离心， 过滤和沉淀不能从总EV中分离肿瘤相关EV亚群。新 研究工作致力于探索基于免疫亲和性的捕获技术， 来自患有不同实体瘤的患者的血浆样品的相关EV。然而， 鉴定了单一抗体介导的肿瘤来源的EV富集方法，例如有限的 因此，这需要更高的灵敏度/特异性，并且需要多种捕获抗体来克服肿瘤异质性。为了 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。HCC EV SPA是 能够对患者血浆中32个HCC EV亚群进行高灵敏度和特异性定量 基于8种不同的HCC相关表面蛋白标志物和4种EV表面标志物的组合使用， 标记。本R 01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硬化患者的早期HCC。

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.