Cancer molecular analysis using quantum dots

 描述（由申请人提供）：单个细胞的全面分子表征有助于理解细胞病理生理学和我们诊断的能力 并控制复杂病理过程的进展，例如癌症。原则上，用独特的报告探针标记每个生物分子并以亚细胞分辨率高灵敏度检测其定位可以实现这一目标。然而，没有一种传统的生物医学技术能够经受住挑战，受到可以同时分析的分子靶标数量的限制，提供有限的单细胞信息，并且通常利用定性而不是定量分析。将基于纳米颗粒的新型成像技术转化为生物医学研究和临床诊断，有望为解决肿瘤的表型异质性提供强大的工具，打开研究低丰度事件的大门，这些事件通常被批量处理掩盖或完全消除，并阐明癌症生物标志物特征，以进行准确诊断和靶向治疗。为了实现这一承诺，我们最近发明了一种基于光学成像分辨率的单细胞中数十至数百种肿瘤生物标志物的循环成像的多细胞分子谱分析技术（Nat.Commun. 2013和Nat.Protocols2013）。基于这项工作和我们在DNA链介导的快速和多克隆分析中取得的另一项进展（JACS 2011），我们建议探索和开发新一代快速但温和脱色条件的多克隆蛋白质组学技术。我们的方法利用简单的生物缀合和自组装机制用于直接的探针制备，用于编码分子靶标的杂交抗体-DNA探针，用于定量和荧光成像的量子点的独特光学性质，以及DNA支点介导的置换的温和和快速动力学。这些特征的独特组合（得到我们最近初步结果的支持）产生了简单但强大的技术，该技术应能够原位对单个癌细胞进行分子表征，同时不需要专门的技术技能或对常见染色方法和成像仪器进行重大修改。因此，如果成功的话，所提出的QD技术应该很容易适用于广泛的应用，特别是检查癌细胞中的分子信号传导途径，评估对治疗的反应，以及开发多重诊断和预后面板，从而有望在癌症研究和临床实践中产生重大影响。

 DESCRIPTION (provided by applicant): Comprehensive molecular characterization of individual cells is instrumental for understanding cell pathophysiology and our ability to diagnose and control the progression of complex pathological processes, such as cancer. In principle, tagging each biomolecule with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution can achieve this goal. Yet, none of the conventional biomedical techniques can stand up to the challenge, suffering from a limitation in the number of molecular targets that can be analyzed simultaneously, providing limited single cell information, and often utilizing qualitative rather than quantitative analysis. Translation of novel nanoparticl-based imaging technologies into biomedical research and clinical diagnostics promises to provide powerful tools for addressing phenotypic heterogeneity of tumors, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating cancer biomarker signatures for accurate diagnosis and targeted therapy. To deliver on this promise, we recently invented a multicolor multicycle molecular profiling technology based on cyclic imaging of tens to hundreds of tumor biomarkers in single cells with optical imaging resolution (Nat. Commun. 2013 & Nat. Protocols 2013). Built on this work and another advance we made on DNA strand mediated displacement for fast and multicycle analysis (JACS 2011), we propose to explore and develop a new generation of multicolor multicycle proteomic technology with fast yet mild destaining condition. Our approach takes advantage of simple bioconjugation and self-assembly mechanisms for straightforward probe preparation, hybrid antibody-DNA probes for encoding of molecular targets, the unique optical properties of quantum dots for quantitative and multicolor imaging, and the mildness and fast kinetics of DNA toehold- mediated displacement. Unique combination of these features (which are supported by our recent preliminary results) yields simple, but powerful technology that should enable molecular characterization of individual cancer cells in situ, while requiring no specialized technical skills or significant modifications to common staining methodology and imaging instrumentation. As a result, if successful, the proposed QD technology should be readily applicable for a wide range of applications, in particular examination of molecular signaling pathways in cancer cells, evaluation of response to therapy, and development of multiplexed diagnostic and prognostic panels, thus promising to produce a substantial impact in cancer research and clinical practice.