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Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Original publication

DOI

10.1073/pnas.1300136110

Type

Journal article

Journal

Proc Natl Acad Sci U S A

Publication Date

16/07/2013

Volume

110

Pages

11982 - 11987

Keywords

cancer diagnostics, high-content cellular analysis, image analysis, mTOR, multiplexing, 3,3'-Diaminobenzidine, Biomarkers, Tumor, Breast Neoplasms, Cell Line, Tumor, Colonic Neoplasms, Female, Formaldehyde, Humans, Image Processing, Computer-Assisted, Immunohistochemistry, In Situ Hybridization, Fluorescence, Microscopy, Fluorescence, Paraffin Embedding, Receptor, ErbB-2, Receptors, Androgen, Receptors, Estrogen, Statistics, Nonparametric, Tumor Suppressor Protein p53