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Molecular Cancer

, 16:73

First Online: 03 April 2017Received: 16 November 2016Accepted: 22 March 2017DOI: 10.1186-s12943-017-0642-7

Cite this article as: Busch, S., Andersson, D., Bom, E. et al. Mol Cancer 2017 16: 73. doi:10.1186-s12943-017-0642-7

Abstract

BackgroundThe 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.

MethodsUsing 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.

ResultsSingle-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.

ConclusionsWithin 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.

KeywordsCancer-associated fibroblast Breast cancer Tumour stroma Single-cell analysis Abbreviations7AAD7-amino-actinomycin D

ABCG2Adenosine triphosphate-binding cassette sub-family G member 2

ACTA2Actin, alpha 2, smooth muscle, aorta

ALDH1A3Aldehyde dehydrogenase 1 family member A3

ANOVAAnalysis of variance

BCSCBreast cancer-specific stem cells

BSABovine serum albumin

CAFsCancer-associated fibroblasts

CAV1Caveolin 1

CCL5C-C motif chemokine ligand 5

CCNA2Cyclin A2

CD24Cluster of differentiation 24

CD44Cluster of differentiation 44

CDH1Cadherin 1

CDKN1ACyclin dependent kinase inhibitor 1A

cDNAComplementary deoxyribonucleic acid

CO2Carbon dioxide

COL1A1Collagen, type I, alpha 1

CTGFConnective tissue growth factor

CXCL12C-X-C motif chemokine ligand 12

DNADeoxyribonucleic acid

DNaseDeoxyribonuclease

DNERDelta-notch like epidermal growth factor repeat containing

ECMExtracellular matrix

EPCAMEpithelial cell adhesion molecule

ERαOestrogen receptor alpha

ESR1Oestrogen receptor 1

expCAFsExperimentally generated cancer-associated fibroblasts

FACSFluorescence-activated cell sorter

FAP1Fas-associated phosphatase 1

FBSFoetal bovine serum

FN1Fibronectin 1

FOSL1Fos-like antigen 1

FSP1Fibroblast-specific protein 1

GAPDHGlyceraldehyde-3-phosphate dehydrogenase

HGFHepatocyte growth factor

HIF1AHypoxia-induced factor 1 alpha

ID1Inhibitor of deoxyribonucleic acid binding 1

KRT14Keratin 14

KRT18Keratin 18

KRT4Keratin 4

LOXLysyl oxidase

LOXL2Lysyl oxidase like 2

MKI67Marker of proliferation Ki-67

MMP2Matrix metallopeptidase 2

MSCMesenchymal stem cell

NANOGNanog homeobox

PCAPrinciple component analysis

PCNAProliferating cell nuclear antigen

PCRPolymerase chain reaction

PDGFAPlatelet-derived growth factor

PGRProgesterone receptor

poly-HEMAPoly-2-hydroxyethyl methacrylate

POU5F1POU class 5 homeobox 1

RNARibonucleic acid

RNaseRibonuclease

RT-PCRReverse transcriptase polymerase chain reaction

RUNX1Runt-related transcription factor 1

SEMStandard error of mean

SMAαSmooth muscle actin alpha

SNAI1Snail family transcriptional repressor 1

SNAI2Snail family transcriptional repressor 2

SOMKohonen self-organizing maps

SOX2Sex determining region Y-box 2

TGFB1Transforming growth factor beta 1

TGFβTransforming growth factor beta

TNCTenascin C

TP53Tumor protein p53

TWIST1Twist family basic helix-loop-helix transcription factor 1

VEGFAVascular endothelial growth factor A

VIMVimentin

Electronic supplementary materialThe online version of this article doi:10.1186-s12943-017-0642-7 contains supplementary material, which is available to authorized users.

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Autor: Susann Busch - Daniel Andersson - Eva Bom - Claire Walsh - Anders Ståhlberg - Göran Landberg

Fuente: https://link.springer.com/







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