Cytometry‐based single‐cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF‐α‐induced apoptosis in vivo

Molecular Systems Biology - Tập 11 Số 10 - 2015
Alan J. Simmons1,2, Amrita Banerjee1,2, Eliot T. McKinley3,2, Cherie’ R. Scurrah1,2, Charles A. Herring4,2, Leslie Gewin1,3,5, Ryota Masuzaki6, Seth J. Karp6, Jeffrey L. Franklin1,2, Michael J. Gerdes7, Jonathan M. Irish8, Robert J. Coffey1,3,2,5, Ken S. Lau1,4,2
1Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA
2Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA
3Department of Medicine, Vanderbilt University Medical Center, Nashville, TN USA.
4Department of Chemical and Physical Biology Vanderbilt University Medical Center Nashville TN USA
5Veterans Affairs Medical Center, Tennessee Valley Healthcare System, Nashville, TN, USA
6The Transplant Center and Department of Surgery Vanderbilt University Medical Center Nashville TN USA
7Life Sciences Division GE Global Research Niskayuna NY USA
8Departments of Cancer Biology, and Pathology, Microbiology and Immunology Vanderbilt University Medical Center Nashville TN USA

Tóm tắt

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.

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Tài liệu tham khảo

10.1186/1471-2121-14-36

10.1038/nbt.2594

10.1038/nm.3488

10.1053/gast.2002.32975

10.1083/jcb.201009141

10.1126/science.1198704

10.1016/j.cell.2014.04.005

10.1006/exer.2001.1019

10.1074/jbc.M114.590240

10.1097/00042737-200506000-00002

10.1002/aja.1001410403

10.1056/NEJMoa0904492

10.1098/rstb.2013.0014

Gage GJ, 2012, Whole animal perfusion fixation for rodents, J Vis Exp, 65, e3564

10.1083/jcb.201010127

10.1007/s00018-012-0984-7

10.1073/pnas.1300136110

10.1681/ASN.2010020147

10.1038/sj.onc.1209615

10.1038/nmeth.2869

10.1111/j.1365-2613.2011.00767.x

10.1152/ajpgi.00408.2005

10.1016/j.imbio.2008.01.003

10.1038/ncb3084

10.1016/j.cell.2004.06.028

10.1038/ncb2384

10.1016/j.cellimm.2005.10.005

10.1074/jbc.M300725200

10.1016/j.intimp.2004.09.027

10.1016/j.clim.2003.11.009

10.1016/j.cell.2007.01.049

10.1126/scisignal.2001338

10.1371/journal.pbio.1001393

10.1039/c3ib40062j

10.1126/scisignal.2000634

10.1002/cyto.a.20965

10.1152/ajpgi.00481.2012

10.1038/nature14152

10.4110/in.2013.13.2.43

10.1074/jbc.M115.667345

Paulus U, 1993, The differentiation and lineage development of goblet cells in the murine small intestinal crypt: experimental and modelling studies, J Cell Sci, 106, 473, 10.1242/jcs.106.2.473

10.2741/4012

10.1016/j.scr.2014.09.002

10.1016/j.cell.2012.02.042

10.1016/j.cellsig.2014.03.015

Rojanapo W, 1980, The prevalence, metabolism and migration of goblet cells in rat intestine following the induction of rapid, synchronous vitamin A deficiency, J Nutr, 110, 178, 10.1093/jn/110.1.178

10.1016/S0960-9822(01)00587-5

10.1038/msb.2009.87

10.1038/nature09637

10.1038/nrm2430

10.1074/jbc.M300096200

10.1038/nbt.2859

10.1146/annurev.physiol.010908.163145

10.1242/dmm.013284

WrightN AlisonM(1984)The biology of epithelial cell populations.Oxford:Clarendon Press

10.1159/000373965

10.1038/ncomms8058

10.1016/j.proghi.2006.09.001

10.1016/j.bbrc.2014.10.134

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Molecular Systems Biology

Tập 11 Số 10

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