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Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

Sci. Signal.  11 Oct 2016:
Vol. 9, Issue 449, pp. rs11
DOI: 10.1126/scisignal.aah4413

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Detecting intratumoral heterogeneity in the clinic

The clinical approach to treating cancer has become more patient-directed, because tumors of the same tissue type can vary considerably between patients. Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue currently provides tissue-level data to inform the therapeutic strategy. However, these techniques miss the heterogeneity between cells within the same tumor; thus, an indicated strategy may kill some portions of the tumor (but not the entire tumor), and clinicians and patients must constantly fight new tumor growth. Simmons et al. developed a technique that provides single-cell–level data from clinical FFPE samples. Using this technique, called FFPE-DISSECT, the authors found distinct signaling profiles in different cells of the proliferative colon crypt and additional differences in the ways in which these profiles were disrupted in malignant crypt cells. This approach may enable more effective early treatment strategies and ultimately better outcomes for patients.

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.

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