title: Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts.
abstract: 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.
authors.affiliation: Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Susann.Busch@gu.se.
authors.forename: Susann
authors.identifier: 0000-0001-8119-086X
authors.initials: S
authors.lastname: Busch
authors.affiliation: Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Andersson
authors.affiliation: Department of Surgery, Institute of Clinical Sciences, Transplantation and Regenerative Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Bom
authors.affiliation: Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Claire
authors.initials: C
authors.lastname: Walsh
authors.affiliation: Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Ståhlberg
authors.affiliation: Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Göran
authors.initials: G
authors.lastname: Landberg
mesh_terms: D023102:Anoikis / Q000235:genetics; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D000072645:Cancer-Associated Fibroblasts / Q000378:metabolism* / Q000473:pathology*; D002460:Cell Line; D016000:Cluster Analysis; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D059010:Single-Cell Analysis; D059016:Tumor Microenvironment
chemical_list: D014408:Biomarkers, Tumor
keywords: Breast cancer; Cancer-associated fibroblast; Single-cell analysis; Tumour stroma