Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts

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

Heterogeneity of fibroblasts from radicular cyst influenced osteoclastogenesis and bone destruction

“…On the other hand, among the three groups (Cluster from control, Cluster 1 from cysts, and Cluster 2), the differentially expressed osteoclastogenesis‐related genes were COX‐2, IL‐6, IL‐1α, TNF‐α, VEGF‐A, RANKL/OPG, and EDA + FN, which is similar to the profile of genes in the groups of radicular cysts (perilesional sclerotic, unsclerotized, and control), as mentioned above. This result further suggested that in radicular cysts, the activity of bone resorption was influenced by the proportion of different fibroblast subsets, which were categorized here as Cluster 1 and 2 (Busch et al, 2017). Therefore, Cluster 2 may be important in osteoclastogenesis, and the ablation of this subset may have therapeutic applications in bone destruction (Lv et al, 2017; Philippeos et al, 2018).…”

“…Under pathological conditions, the interactions with microenvironment components further facilitate fibroblast differentiation into distinct subsets, by which the development and fate of tumor (Ostman, 2014), fibrosis (Xie et al, 2018), inflammation (Mizoguchi et al, 2018), and wound healing (Guerrero‐Juarez et al, 2019) are influenced. For instance, in lung cancers, cancer‐secreted factors induced the differentiation of cancer‐associated fibroblasts (CAFs) subsets (Busch et al, 2017), expressing different markers, or collagen sets (Lambrechts et al, 2018). Further, the fibroblasts surrounding microvessels became subsets with lower tumor‐promoting function (Suda et al, 2016) relative to other CAFs, while in osteoarthritis, fibroblast subsets localized to the perivascular zone exhibiting proinflammatory potential (Mizoguchi et al, 2018), suggesting the functional differentiation of fibroblast subsets in lesions.…”

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

“…Since the initial report on different subpopulations of αSMA+ myofibroblasts in breast cancer (Lazard et al, 1993), it is now becoming clear that CAFs come with different flavours and that a heterogeneous population exists within a single breast tumour (Bartoschek et al, 2018;Busch et al, 2017;Calvo et al, 2013;Costa et al, 2018;Cremasco et al, 2018;Raz et al, 2018;Su et al, 2018). Here, we have presented data showing the existence of numerous CAF subpopulations in murine models of TNBC, expanding on previous findings within breast cancer that have proposed the existence of only 2 to 4 different CAF subpopulations (Bartoschek et al, 2018;Costa et al, 2018;Cremasco et al, 2018;Raz et al, 2018).…”

“…CAFs have been implicated in many of these tumour-promoting processes (Kalluri, 2016;LeBleu and Kalluri, 2018), yet reports studying the temporal co-evolution of CAFs as tumours progress are very limited. By comparing transcriptomic profiles from breast cancer CAFs and normal mammary fibroblasts Busch et al suggest that CAFs gradually arise from normal fibroblasts via a primed state, as they find that a part of the fibroblasts isolated from histologically normal lumpectomy resection areas cluster together with CAFs (Busch et al, 2017). A study in the MMTV-PyMT breast cancer model briefly touched upon the concept of CAF co-evolution, and found that fibroblasts isolated from progressive stages of tumour development showed increasing extracellular matrix (ECM) remodelling abilities (Calvo et al, 2013).…”

“…Intra-tumoural heterogeneity of cancer cells in breast cancer is well recognised, and emerging evidence points toward breast cancer CAFs being equally heterogeneous (Bartoschek et al, 2018;Busch et al, 2017;Calvo et al, 2013;Costa et al, 2018;Cremasco et al, 2018;Raz et al, 2018;Su et al, 2018). While cancer cell heterogeneity is believed to arise through clonal evolution, the origins of CAFs, and how this influences the CAF heterogeneity, are still being intensely studied.…”