Single-Cell Mass Cytometry of Archived Human Epithelial Tissue for Decoding Cancer Signaling Pathways

  • Cherie’ R. Scurrah
  • Alan J. Simmons
  • Ken S. Lau
Part of the Methods in Molecular Biology book series (MIMB, volume 1884)


The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.

Key words

Single-cell CyTOF FFPE Cryopreserved Mass cytometry Epithelial cells Dissociation Disaggregation Signaling 



This work was supported by NIH grants R01DK103831 and P50CA095103 (K.S.L., C.R.S.), U01CA215798 (A.J.S.), and T32AI007281 (C.R.S.).


  1. 1.
    Shukla S, Meeran SM (2014) Epigenetics of cancer stem cells: pathways and therapeutics. Biochim Biophys Acta 1840:3494–3502CrossRefGoogle Scholar
  2. 2.
    Shimokawa M, Ohta Y, Nishikori S, Matano M, Takano A, Fujii M et al (2017) Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545:187–192CrossRefGoogle Scholar
  3. 3.
    de Sousa e Melo F, Kurtova AV, Harnoss JM, Kljavin N, Hoeck JD, Hung J et al (2017) A distinct role for Lgr5(+) stem cells in primary and metastatic colon cancer. Nature 543:676–680CrossRefGoogle Scholar
  4. 4.
    Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29:4741–4751CrossRefGoogle Scholar
  5. 5.
    Simmons AJ, Lau KS (2017) Deciphering tumor heterogeneity from FFPE tissues: Its promise and challenges. Mol Cell Oncol 4:e1260191CrossRefGoogle Scholar
  6. 6.
    Abrahamsen I, Lorens JB (2013) Evaluating extracellular matrix influence on adherent cell signaling by cold trypsin phosphorylation-specific flow cytometry. BMC Cell Biol 14:36CrossRefGoogle Scholar
  7. 7.
    Zeng J, Mohammadreza A, Gao W, Merza S, Smith D, Kelbauskas L et al (2014) A minimally invasive method for retrieving single adherent cells of different types from cultures. Sci Rep 4:5424CrossRefGoogle Scholar
  8. 8.
    Simmons AJ, Banerjee A, McKinley ET, Scurrah CR, Herring CA, Gewin LS et al (2015) Cytometry-based single-cell analysis of intact epithelial signaling reveals divergent MAPK activation during TNF-α-induced apoptosis. Mol Syst Biol 11:835–835CrossRefGoogle Scholar
  9. 9.
    Bendall SC, Davis KL, Amir e-AD, Tadmor MD, Simonds EF, Chen TJ et al (2014) Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development. Cell 157:714–725CrossRefGoogle Scholar
  10. 10.
    Simmons AJ, Scurrah CR, McKinley ET, Herring CA, Irish JM, Washington MK et al (2016) Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks. Sci Signal 9:rs11CrossRefGoogle Scholar
  11. 11.
    Kokkat TJ, Patel MS, McGarvey D, LiVolsi VA, Baloch ZW (2013) Archived formalin-fixed paraffin-embedded (FFPE) blocks: A valuable underexploited resource for extraction of DNA, RNA, and protein. Biopreserv Biobank 11:101–106CrossRefGoogle Scholar
  12. 12.
    Finck R, Simonds EF, Jager A, Krishnaswamy S, Sachs K, Fantl W et al (2013) Normalization of mass cytometry data with bead standards. Cytometry A 83:483–494CrossRefGoogle Scholar
  13. 13.
    Corver WE, ter Haar NT (2011) High-resolution multiparameter DNA flow cytometry for the detection and sorting of tumor and stromal subpopulations from paraffin-embedded tissues. Curr Protoc Cytom 55:7.37.1–7.37.21Google Scholar
  14. 14.
    Koley D, Bard AJ (2010) Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM). Proc Natl Acad Sci U S A 107:16783–16787CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Cherie’ R. Scurrah
    • 1
  • Alan J. Simmons
    • 1
  • Ken S. Lau
    • 1
  1. 1.Epithelial Biology Center and the Department of Cell and Developmental BiologyVanderbilt University School of MedicineNashvilleUSA

Personalised recommendations