Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-{alpha}-induced apoptosis in vivo
Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue—DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.
Source: Molecular Systems Biology - Category: Molecular Biology Authors: Simmons, A. J., Banerjee, A., McKinley, E. T., Scurrah, C. R., Herring, C. A., Gewin, L. S., Masuzaki, R., Karp, S. J., Franklin, J. L., Gerdes, M. J., Irish, J. M., Coffey, R. J., Lau, K. S. Tags: Methods & Resources, Signal Transduction Articles Source Type: research
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