title: Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks.
abstract: Cellular heterogeneity poses a substantial challenge to understanding tissue-level phenotypes and confounds conventional bulk analyses. To analyze signaling at the single-cell level in human tissues, we applied mass cytometry using cytometry time of flight to formalin-fixed, paraffin-embedded (FFPE) normal and diseased intestinal specimens. This technique, called FFPE-DISSECT (disaggregation for intracellular signaling in single epithelial cells from tissue), is a single-cell approach to characterizing signaling states in embedded tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor-α in intestinal enterocytes, goblet cells, and enteroendocrine cells, implicating the downstream RAS-RAF-MEK pathway in determining goblet cell identity. Application of this technique and computational analyses to human colon specimens confirmed the reduced differentiation in colorectal cancer (CRC) compared to normal colon and revealed increased intratissue and intertissue heterogeneity in CRC with quantitative changes in the regulation of signaling pathways. Specifically, coregulation of the kinases p38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in proliferating normal colon cells was lost in CRC. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, enables the rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. This technique can be used to derive cellular landscapes from archived patient samples (beyond CRC) and as a high-resolution tool for disease characterization and subtyping.
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Simmons
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Cherié R
authors.initials: CR
authors.lastname: Scurrah
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Eliot T
authors.initials: ET
authors.lastname: McKinley
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Herring
authors.affiliation: Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Irish
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: M Kay
authors.initials: MK
authors.lastname: Washington
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Veterans Affairs Medical Center, Tennessee Valley Healthcare System, Nashville, TN 37232, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Coffey
authors.affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, TN 37232, USA. ken.s.lau@vanderbilt.edu.
authors.forename: Ken S
authors.initials: KS
authors.lastname: Lau
mesh_terms: D000818:Animals; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology*; D005260:Female; D006801:Humans; D019044:Image Cytometry; D020935:MAP Kinase Signaling System; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D016612:Paraffin Embedding
chemical_list: D009363:Neoplasm Proteins