Single-Cell Protein Activity Profiling in Breast Cancer Cells and Tissues

项目摘要 分子图谱技术的进步使人们能够识别出越来越多的 疾病生物标志物，每个都提高了更准确，甚至可能是针对患者的诊断的可能性。 然而，目前量化蛋白质生物标记物的方法速度慢且费力，通常需要大量的 大量的活组织，最终不能提供完整、完整的功能蛋白的量化 活动。为了解决这些局限性，我们开发了一种创新的依赖活性的邻近结扎术 平台(ADPL)，最终目标是获得多路、超灵敏的蛋白质活性量化 直接在复杂的生物样本中。在癌症样本的背景下，我们假设定义一个 基于蛋白质活性的分子图谱-生物信号的最终结果-可以更准确地 准确告知诊断和治疗方案，允许早期干预并改善临床结果。 该提案概述了两个可靠的、基于ADPL的高分辨率 平台，能够交替地同时可视化和量化数十种活性酶 肿瘤组织、细胞和生物液。我们目前在使用这一综合平台方面的知识和专业知识将 用于下列目的：1)开发新的化学蛋白质组探针 包括激酶在内的癌症相关酶的多路、全家庭活动图谱；2)高级 组织上交换-ADPL和芯片上单细胞ADPL平台的开发，用于同时、 高分辨率可视化和原位定量一组活性酶；以及3)应用这些 乳腺癌组织和细胞的技术，以创建准确和精确的活动特征并识别 这些特征与临床结果之间的潜在相关性。为了实现这些目标，我们将发展 新的化学蛋白质组分子探针，验证一组癌症相关酶的ADPL试剂 生物标志物，并在细胞系和肿瘤组织中执行严格的、公制驱动的基准分析。 该项目的完成将验证ADPL平台在各种癌症中的应用，提供第一个单细胞 乳腺肿瘤的活动性分布，并提供乳腺癌肿瘤活动性特征之间的新的关联， 疾病严重程度和对治疗的反应。

Project Summary Advances in molecular profiling technologies have enabled the identification of ever-increasing numbers of disease biomarkers, each raising the potential for more precise, and possibly even patient-specific, diagnoses. Current methods of quantifying protein biomarkers, however, are slow and laborious, often require large quantities of biopsied tissues, and ultimately fail to provide a complete, intact quantification of functional protein activity. To address these limitations, we have developed an innovative activity dependent proximity ligation platform (ADPL), with the ultimate goal of obtaining multiplexed, ultrasensitive quantification of protein activity directly in complex biological samples. In the context of cancer specimens, we hypothesize that defining a molecular profile based on protein activity – the end result of biologic signaling – can more precisely and accurately inform diagnoses and treatment regimes, allowing early intervention and improving clinical outcomes. This proposal outlines the advanced development and validation of two reliable, high-resolution ADPL-based platforms, with the power to alternately visualize and quantify dozens of active enzymes simultaneously from tumor tissue, cells and biofluids. Our current knowledge and expertise in the use of this integrated platform will be leveraged in the service of the following aims: 1) development of novel chemoproteomic probes for multiplexed, family-wide activity mapping of cancer-relevant enzymes, including kinases; 2) advanced development of on-tissue Exchange-ADPL and on-chip single-cell ADPL (scADPL) platforms for simultaneous, high resolution visualization and quantification of a panel of active enzymes in situ; and 3) applying these technologies to breast cancer tissues and cells to create accurate and precise activity signatures and identify potential correlations between these signatures and clinical outcomes. To accomplish these aims we will develop new chemical proteomic molecular probes, validate a panel of ADPL reagents for cancer-associated enzyme biomarkers, and perform rigorous, metric-driven benchmarking analyses in cell lines and tumor tissues. Completion of this project will validate ADPL platforms for use in diverse cancers, provide the first single-cell activity profiles of breast tumors, and provide novel correlations between breast cancer tumor activity signatures, disease severity and response to therapy.