Development of non-invasive biomarker discovery and diagnostics approach for bladder cancer based on urine proteome and phosphoproteome

项目摘要 细胞外囊泡（EV）的分析已经成为发现细胞外囊泡（EV）的非常有前途的领域。 来自生物流体（例如血液、尿液、CSF或其他）的肿瘤相关生物标志物。生物流体的使用 EV在临床环境中提供了许多优点，包括非侵入性收集、合适的 纵向疾病监测的样本来源，更好的肿瘤异质性屏幕截图，更高的 稳定性和样品量，更快的处理时间，更低的废品率和成本， 它们的组织对应物。尽管有巨大的潜力，EV磷酸蛋白组的数据是 几乎不存在在这项NIH SBIR快速通道研究中，我们将进一步开发一种新的尿液 基于EVtrap微珠的分析平台，可完全捕获细胞外囊泡， 将蛋白质组提取和分析方法发展为高效和可重复的方法 用于发现和检测癌症生物标志物。我们已经利用这种方法生成了 一组新的蛋白质/磷蛋白标记物，来自尿EV，用于敏感和非侵入性 检测和监测膀胱癌。在这个项目中，发现的生物标志物小组将 进行验证和完善，以证明临床实用性。以下目标将在 提案的第一阶段：目标1：开发和优化EVtrap，以实现大规模和高通量 EV隔离。目标#2：确定当前膀胱癌生物标志物的可行性并完善 小组。以下目标将在提案的第二阶段完成：目标1：适应EVtrap 用于高通量EV捕获和货物分析的现有自动化设备。目标#2： 并将最终的面板标准化以用于临床应用。通过这个项目的完成，生物标志物 将开发尿液的发现平台，并将开发膀胱癌监测检测试剂盒。 验证，可以克服目前的方法的局限性，因此可以有巨大的 公共卫生影响和市场潜力。

PROJECT SUMMARY Profiling of Extracellular Vesicles (EV) has emerged as a highly promising field for discovery of tumor-relevant biomarkers from biofluids, such as blood, urine, CSF or others. The use of biofluid EVs offers numerous advantages in clinical setting, including non-invasive collection, a suitable sample source for longitudinal disease monitoring, better screenshot of tumor heterogeneity, higher stability and sample volumes, faster processing times, lower rejection rates and costs compared to their tissue counterpart. Despite the immense potential, the data on EV phosphoproteomes are virtually non-existent. In this NIH SBIR Fast-Track study, we will further develop a novel urine analysis platform based on EVtrap beads for complete capture of extracellular vesicles and in-house developed proteome extraction and analysis approach into highly efficient and reproducible method for discovery and detection of cancer biomarkers. We have already utilized this approach to generate a new panel of protein/phosphoprotein markers from urinary EVs for sensitive and non-invasive detection and monitoring of bladder cancer. During this project, the discovered biomarkers panel will be validated and refined to demonstrate clinical utility. The following aims will be completed in the Phase I of the proposal: Aim #1: Develop and optimize EVtrap for large-scale and high-throughput EV isolation. Aim #2: Determine the feasibility of the current bladder cancer biomarkers and refine the panel. The following aims will be completed in the Phase II of the proposal: Aim #1: Adapt EVtrap to an existing automated device for high-throughput EV capture and cargo analysis. Aim #2: Validate and standardize the final panel for clinical utility. By the completion of this project, a biomarker discovery platform from urine will be developed, and bladder cancer monitoring assay will be validated that can overcome the limitations of current approaches, and thus could have an enormous public health impact and market potential.