Digital Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples

血浆微样品中肿瘤源性细胞外囊泡的数字纳米等离子体定量

• 批准号: 10037327
• 负责人: Hua Lu
• 金额: $ 63.1万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-25 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10037327
• 关键词: Address; Affect; Antibodies; Benign; Binding; Biological Assay; Biological Markers; Blood Circulation; Blood specimen; CA-19-9 Antigen; CD44 gene; CLIA certified; Cancer Detection; Cells; Clinical; Complex; Detection; Development; Diagnosis; Diagnostic; Disease; Early Diagnosis; Early treatment; Excision; Exhibits; Family history of; Guidelines; Image; Individual; Inherited; Institutes; Laboratories; Lesion; Longitudinal Studies; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Manuals; Methods; Microfabrication; Modeling; Monitor; Mus; Neoadjuvant Therapy; Neoplasm Metastasis; Non-Malignant; Operative Surgical Procedures; Pancreas; Pancreatitis; Patients; Performance; Pilot Projects; Plasma; Procedures; Proteins; Protocols documentation; Reader; Reagent; Regimen; Reporting; Reproducibility; Research; Resectable; Resistance; Resolution; Sampling; Sensitivity and Specificity; Serum; Source; Specificity; Surface; Syndrome; TACSTD1 gene; Therapeutic; Time; Treatment outcome; Tumor stage; Tumor-Derived; Urine; Validation; Variant; Vesicle; Work; advanced disease; base; cancer diagnosis; cancer stem cell; cancer therapy; clinical translation; cohort; design; digital; disorder control; early screening; extracellular vesicles; human disease; human model; improved; improved outcome; longitudinal analysis; minimally invasive; mouse model; nanoparticle; nanoplasmonic; novel; novel marker; pancreatic cancer model; pancreatic cancer patients; pre-clinical; predictive modeling; premalignant; response; sample collection; success; translational impact; treatment response; tumor; tumor initiation

ABSTRACT Extracellular vesicles (EVs) hold great potential as novel biomarkers for minimally invasive detection of early stage tumors, since tumors abundantly secrete EVs that accumulate in the circulation and these EVs can transport factors that regulate tumor initiation, progression, and metastasis. However, most current EV analysis methods require pre-isolation of EVs prior to analysis and are low-throughput and impractical for clinical use. We recently developed a rapid, robust, isolation-free and inexpensive assay that directly quantifies tumor-derived EVs in small volumes (1~5 µL) of serum or plasma. In this assay, EVs that bind probes specific for two different EV target proteins produce a distance-regulated nanoplasmon-enhanced scattering (nPES) effect that allows sensitive detection of specific EVs. In a pilot study we used a nPES assay for a pancreatic cancer (PC)- associated EV marker to distinguish PC cases from non-malignant controls (patients with pancreatitis and healthy individuals) with high reproducibility, specificity, and sensitivity. This assay also differentiated PC tumor stages and tumor responses to neoadjuvant, outperforming CA19-9, a biomarker widely used for PC therapy assessment. Our nPES assay platform has multiple features required for research and clinical translation: 1) It is rapid, high-throughput, and inexpensive; 2) it does not employ EV isolation, avoiding a major source of EV assay variation; 3) it robustly and reproducibly quantifies EV biomarkers from small volumes of serum, plasma or urine, allowing its use in longitudinal analysis of mouse models of human disease; and 4) it can be readily adapted to diagnose and monitor cancers that express other EV biomarkers. Based on the success of our pilot study, we propose to develop and validate an automated and highly reproducible nPES EV assay to allow rapid and accurate PC diagnosis in clinical settings. We hypothesize that a nPES-based digital EV reader will equal or outperform the analytical performance of our current manual assay. We will build a diagnostic EV assay model for early PC detection by examining the ability of proteins reported to be enriched on the surface of EVs derived from PC stem cells or PC-initiating cells (e.g., CD44, CD133 and EpCAM) to diagnose patients with early stage PC and to differentiate them from patients with pre-malignant pancreatic lesions, hereditary syndromes or family history of PC, and individuals with normal pancreases. Specifically, we propose to: 1) Development and fabrication of Chip-nPES platform to achieve single EV resolution; 2) automate and refine our nPES-based digital EV reader to enhance assay reliability and reproducibility. We will also select and validate candidate EV capture and detection antibodies for PC diagnosis; 3) establish and evaluate a diagnostic model that integrates EV biomarkers with known cancer-associations; and 4) perform a pre-clinical validation of this assay in a third-party laboratory. The successful results of this work would have a significant translational impact in cancer management, through reliable and accessible screens for early detection of pancreatic cancer.

摘要 细胞外小泡(EVS)作为一种新型的微创检测早期肿瘤的生物标志物具有很大的潜力 分期肿瘤，因为肿瘤大量分泌在循环中积聚的EV，这些EV可以 调节肿瘤起始、进展和转移的运输因子。然而，大多数当前的电动汽车分析 这种方法需要在分析前预先隔离EVS，而且吞吐量低，不适合临床使用。我们 最近开发了一种快速、可靠、无隔离和廉价的直接定量检测肿瘤来源的方法 小剂量(1~5微克L)血清或血浆。在这项检测中，结合两个不同基因的探针的EVS EV靶蛋白产生距离调节的纳米等离子增强散射(NPES)效应，从而允许 对特定电动汽车的灵敏检测。在一项初步研究中，我们对胰腺癌(PC)进行了NPEs检测- 相关EV标记物用于区分PC病例和非恶性对照(胰腺炎和 健康人)具有高度的重复性、特异性和敏感性。这项检测也能区分PC瘤 新辅助治疗的分期和肿瘤反应优于广泛用于PC治疗的生物标记物CA19-9 评估。我们的NPEs分析平台具有研究和临床翻译所需的多种功能：1)它 快速、高吞吐量和廉价；2)它不采用EV隔离，避免了EV的主要来源 检测变异；3)从少量血清、血浆中可靠地、可重复性地定量肠道病毒生物标志物。 或尿液，允许将其用于对人类疾病的小鼠模型的纵向分析；以及4)它可以很容易地 适用于诊断和监测表达其他EV生物标志物的癌症。基于我们试点的成功 研究中，我们建议开发和验证一种自动化和高度重复性的NPEs EV检测方法，以允许快速 和临床环境中准确的PC诊断。我们假设，基于NPE的数字电动汽车阅读器将等于 或者比我们目前手工化验的分析性能更好。我们将建立一个诊断EV检测模型 通过检测EVS表面报告的浓缩蛋白的能力来检测早期PC 从PC干细胞或PC启动细胞(如CD44、CD133和EpCAM)诊断早期患者 并与胰腺癌前病变、遗传性综合征或家族性疾病的患者进行鉴别。 胰腺癌病史和胰腺正常个体。具体地说，我们建议：1)发展和 制造芯片-NPE平台以实现单一电动汽车的分辨率；2)自动化和提炼我们基于NPE的 数字电动汽车阅读器，以提高检测的可靠性和重复性。我们还将选择和验证候选人 用于PC诊断的EV捕获和检测抗体；3)建立和评估诊断模型 将EV生物标记物与已知的癌症相关性相结合；以及4)进行该检测的临床前验证 在第三方实验室里。这项工作的成功结果将对翻译产生重大影响 癌症管理，通过可靠和可访问的筛查早期发现胰腺癌。