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.

Public Health Relevance

Built on two recent advances we made, multicolor multicycle in situ imaging of biomarkers with quantum dots and DNA strand-mediated displacement, we propose to develop an innovative molecular analysis technology for truly multiplexed and quantitative analysis of cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA192985-02
Application #
9076630
Study Section
Special Emphasis Panel (ZCA1)
Project Start
2015-06-01
Project End
2018-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Washington
Department
Biomedical Engineering
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Shang, Jing; Zrazhevskiy, Pavel; Postupna, Nadia et al. (2015) Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling. Sci Rep 5:13651