Clinical platform for high-throughput analyses of extracellular vesicles

细胞外小泡(EVS)已成为组织活检的一种有希望的替代物，具有潜在的应用前景 实现非侵入性、实时的癌症监测。大多数癌细胞将大量的EV释放到 携带亲本肿瘤分子成分的循环。因此，分析电动汽车可以提供新的途径 评估肿瘤负荷和肿瘤异质性。然而，将EV分析应用到临床工作中的fl现在是 由于缺乏标准化、实用的方法而受阻。常规化验工具(例如，超速离心法， Western blotting，ELISA)需要大量的样本和广泛的处理；它们对于 临床应用。样品处理和测试方案的变化常常导致不一致和可变 fi结束符。该提案的目标是：i)通过为以下方面提供一个强大的平台来应对这些技术挑战 EV蛋白分析，以及ii)严格评估EV作为癌症生物标志物的临床价值。我们组成了一个 实现这一目标的战略学术-行业合作伙伴关系：Exosome Diagnostics，行业领先者 基于EV的癌症诊断，提供开发和制造体外诊断医疗的现成能力 以及马萨诸塞州总医院的系统生物学中心，该中心是开发新技术的先驱 电动汽车分析的分析技术。这些团队将带来他们的多学科专业知识，创新 技术和互补资源，以开展以下翻译项目：第一，我们将建立 一个灵敏、高通量的EV蛋白筛选平台。该系统将采用我们最新开发的 基于新型表面等离子体共振(SPR)的NPLEx(纳米等离子体外切体)技术 通过纳米孔结构。我们的初步研究表明，NPLEx的敏感度比 常规方法和消耗的样本量仍然很少(0.1μL)。新的NPLEx系统将拥有 扩大了分析能力和商业生产的可扩展性。我们将设计一款新的SPR芯片和一个 大规模并行EV筛查的检测仪器，并建立标准化的检测方案。 利用Exosome诊断公司的开发和监管专业知识，最终的平台将是 准备好转化为临床诊断实验室。第二，我们将进行有针对性的临床研究， 特别是检测EV蛋白信号是否可以用作癌症检测的生物标志物 治疗监测。我们将收集正在接受治疗的卵巢癌患者的循环EV，以及 跟踪EV蛋白水平的连续变化。我们将确保检测的可靠性和重复性 临床可翻译的EV诊断。我们将严格执行设备设计和样品的质量控制 处理，收集注解良好的患者和对照样本，并进行多点测试。技术上的 而这项研究的科学fic结果可能会对癌症产生显著的翻译影响，建立 一种可靠的、高度特异的fi-c检测方法，用于指导治疗决策和评估治疗效果fi。外切体 诊断将提供监管指导以及营销新测试和设备的渠道。

Extracellular vesicles (EVs) have emerged as a promising surrogate for the tissue biopsy, potentially enabling non-invasive, real-time cancer monitoring. Most cancer cells release large numbers of EVs into circulation that carry molecular constituents of the parent tumor. Analyzing EVs could thus offer new avenues to assess tumor burden and tumor heterogeneity. Adoption of EV analyses into a clinical workflow, however, is impeded by the lack of standardized, practical methods. Conventional assay tools (e.g., ultracentrifugation, Western blotting, ELISA) require large sample volumes and extensive processing; they are impractical for clinical applications. Variations in sample handling and testing protocols often lead to inconsistent and variable findings. The goal of this proposal is i) to address such technical challenges by advancing a robust platform for EV protein analyses, and ii) to rigorously evaluate EVs' clinical value as cancer biomarkers. We formed a strategic academic-industry partnership to achieve this goal: Exosome Diagnostics, an industry leader in EV-based cancer diagnostics, offering ready capacity to develop and manufacture in-vitro diagnostic medical devices; and the Center for Systems Biology at Massachusetts General Hospital, a pioneer in developing novel analytical technologies for EV analyses. These teams will bring in their multidisciplinary expertise, innovative technologies and complementary resources to carry out the following translational projects: First, we will build a sensitive, high-throughput platform for EV protein screening. The system will adopt our recently developed nPLEX (nano-plasmonic exosome) technology that is based on novel surface plasmon resonance (SPR) through nanohole structures. Our initial study showed that nPLEX achieved >1000-fold higher sensitivity than conventional methods and yet consumed scant sample volumes (0.1 μL). The new nPLEX system will have expanded analytical capacity and scalability for commercial production. We will design a new SPR chip and a detection instrument for massively parallel EV screening, and also establish standardized assay protocols. Leveraging the developmental and regulatory expertise of Exosome Diagnostics, the resulting platform will be ready for translation into clinical diagnostic laboratories. Second, we will perform a targeted clinical study, particularly testing whether EV protein signatures can be used as a biomarker for cancer detection and treatment monitoring. We will collect circulating EVs from ovarian cancer patients undergoing therapies, and track serial changes of EV protein levels. We will ensure assay reliability and reproducibility to deliver clinically translatable EV diagnostics. We will follow stringent quality control on device design and sample processing, accrue well-annotated patient and control samples, and perform multisite testing. The technical and scientific outcomes of this research could have a significant translational impact in cancer, establishing a robust, highly specific assay to guide treatment decision and assess therapeutic efficacy. Exosome Diagnostics will provide regulatory guidance as well as channels to market new tests and devices.