title: Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.
abstract: 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.
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Simmons
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Amrita
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Eliot T
authors.initials: ET
authors.lastname: McKinley
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Cherie' R
authors.initials: CR
authors.lastname: Scurrah
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Chemical and Physical Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Herring
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Veterans Affairs Medical Center, Tennessee Valley Healthcare System, Nashville, TN, USA.
authors.forename: Leslie S
authors.initials: LS
authors.lastname: Gewin
authors.affiliation: The Transplant Center and Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Ryota
authors.initials: R
authors.lastname: Masuzaki
authors.affiliation: The Transplant Center and Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Karp
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Franklin
authors.affiliation: Life Sciences Division, GE Global Research, Niskayuna, NY, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gerdes
authors.affiliation: Departments of Cancer Biology, and Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Irish
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Veterans Affairs Medical Center, Tennessee Valley Healthcare System, Nashville, TN, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Coffey
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA Department of Chemical and Physical Biology, Vanderbilt University Medical Center, Nashville, TN, USA ken.s.lau@vanderbilt.edu.
authors.forename: Ken S
authors.initials: KS
authors.lastname: Lau
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D015398:Signal Transduction; D059010:Single-Cell Analysis; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D014409:Tumor Necrosis Factor-alpha
keywords: CyTOF; TNF; apoptosis; epithelial signaling; single‐cell biology