Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing

Michael Bartoschek,Åsa Björklund,Chris D Madsen,David Lindgren,Matteo Bocci,Göran Karlsson,Kristian Pietras,Gyula Pekar,Christer Larsson,Jonas Bergh,John Lövrot,Nikolay Oskolkov,Mikael Sommarin,Markus Ringnér

doi:10.1038/s41467-018-07582-3

Abstract

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.

Highlights

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity
Due to the close correlation of the significantly differentially expressed (SDE) genes in vascular CAFs (vCAFs) with genes involved in vascular development, we investigated the expression of prototypical marker genes for endothelial cells, such as Cdh[5], Pecam[1], Cd34, and Tie[1] to rule out inadvertent contamination of the vCAF population
In order to determine whether the functionally distinct gene programs of vCAFs and matrix CAFs (mCAFs) could be discerned from the analysis of bulk data from the The Cancer Genome Atlas (TCGA) database, we investigated the correlation between the condensed gene profiles for each cellular subtype and metagenes for their inferred functions, i.e., regulation of angiogenesis and extracellular matrix (ECM) production

Summary

Introduction

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. The lack of congruency in marker expression raises the possibility that CAFs comprise a diverse group of cells made up of several subtypes[4] Support for this notion comes from recent studies of e.g., pancreatic ductal adenocarcinoma[5], breast carcinoma[6,7,8], colon carcinoma[9], and lung adenocarcinoma[10], in which functionally distinct subclasses of CAFs were identified by various means based on expression of a limited set of markers. Our work dissects the CAF population within breast tumors at single cell resolution and reveals a previously unappreciated functional diversity within the tumor microenvironment that opens up for further development of tools for precision medicine

Methods

Results

Conclusion