The online version of this article (doi:10.1186/s12943-017-0642-7) contains supplementary material, which is available to authorized users.
The role of cancer-associated fibroblasts (CAFs) during tumour progression is obscured by the inherently complex, heterotypic nature of fibroblast cells and behaviours in various subtypes of malignancies. Therefore, we sought to identify distinct fibroblast subpopulations at the single-cell level.
Using single-cell quantitative PCR as a powerful tool to study heterogeneity and rare cell events, in a high-throughput manner a panel of gene targets are run simultaneously on transcripts isolated from single cells obtained by fluorescence-activated cell sort. Assessment of cells with stem-like characteristics was attained by anchorage-independent, anoikis-resistant culture.
Single-cell analysis of fibroblasts and their tumour-activated counterparts demonstrated molecularly distinct cell types defined by differential expression of characteristic mesenchymal and fibroblast activation markers. Identified subpopulations presented overlapping gene expression patterns indicating transitional molecular states during fibroblast differentiation. Using single-cell resolution data we generated a molecular differentiation model which enabled the classification of patient-derived fibroblasts, validating our modelling approach. Remarkably, a subset of fibroblasts displayed expression of pluripotency markers, which was enriched for in non-adherent conditions. Yet the ability to form single-cell derived spheres was generally reduced in CAFs and upon fibroblast activation through TGFβ1 ligand and cancer cell-secreted factors. Hence, our data imply the existence of putative stem/progenitor cells as a physiological feature of undifferentiated fibroblasts.
Within this comprehensive study we have identified distinct and intersecting molecular profiles defining fibroblast activation states and propose that underlying cellular heterogeneity, fibroblasts are hierarchically organized. Understanding the molecular make-up of cellular organization and differentiation routes will facilitate the discovery of more specific markers for stromal subtypes and targets for anti-stromal therapies.
Additional file 1: Characterization of patient-derived fibroblasts and clinical information of used tumours. Fibroblast morphology and SMAα expression levels of isolated normal and cancer-associated fibroblasts. Clinical information of all analysed tumours. (PDF 1033 kb)12943_2017_642_MOESM1_ESM.pdf
Additional file 2: Gene correlation analysis. Heatmaps depicting gene correlation analyses according to fibroblast subgroups (normal versus cancer-activated) for CAF cell line model and primary fibroblasts. (PDF 2716 kb)12943_2017_642_MOESM2_ESM.pdf
Tchou J, Kossenkov AV, Chang L, et al. Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles. BMC Med Genet. 2012;5:39.
Melton DA, Cowen C. “Stemness”: Definitions, Criteria, and Standards. Essentials of Stem Cell Biology. 2nd ed. 2009. p. Xxiii–Xxix. CrossRef
- Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts
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