Fibroblasts direct differentiation of human breast epithelial progenitors

Abstract

Background Breast cancer arises within specific regions in the human breast referred to as the terminal duct lobular units (TDLUs). These are relatively dynamic structures characterized by sex hormone driven cyclic epithelial turnover. TDLUs consist of unique parenchymal entities embedded within a fibroblast-rich lobular stroma. Here, we established and characterized a new human breast lobular fibroblast cell line against its interlobular counterpart with a view to assessing the role of region-specific stromal cues in the control of TDLU dynamics. Methods Primary lobular and interlobular fibroblasts were transduced to express human telomerase reverse transcriptase (hTERT). Differentiation of the established cell lines along lobular and interlobular pathways was determined by immunocytochemical staining and genome-wide RNA sequencing. Their functional properties were further characterized by analysis of mesenchymal stem cell (MSC) differentiation repertoire in culture and in vivo. The cells’ physiological relevance for parenchymal differentiation was examined in heterotypic co-culture with fluorescence-activated cell sorting (FACS)-purified normal breast primary luminal or myoepithelial progenitors. The co-cultures were immunostained for quantitative assessment of epithelial branching morphogenesis, polarization, growth, and luminal epithelial maturation. In extension, myoepithelial progenitors were tested for luminal differentiation capacity in culture and in mouse xenografts. To unravel the significance of transforming growth factor-beta (TGF-β)-mediated crosstalk in TDLU-like morphogenesis and differentiation, fibroblasts were incubated with the TGF-β signaling inhibitor, SB431542, prior to heterotypic co-culture with luminal cells. Results hTERT immortalized fibroblast cell lines retained critical phenotypic traits in culture and linked to primary fibroblasts. Cell culture assays and transplantation to mice showed that the origin of fibroblasts determines TDLU-like and ductal-like differentiation of epithelial progenitors. Whereas lobular fibroblasts supported a high level of branching morphogenesis by luminal cells, interlobular fibroblasts supported ductal-like myoepithelial characteristics. TDLU-like morphogenesis, at least in part, relied on intact TGF-β signaling. Conclusions The significance of the most prominent cell type in normal breast stroma, the fibroblast, in directing epithelial differentiation is largely unknown. Through establishment of lobular and interlobular fibroblast cell lines, we here demonstrate that epithelial progenitors are submitted to stromal cues for site-specific differentiation. Our findings lend credence to considering stromal subtleties of crucial importance in the development of normal breast and, in turn, breast cancer.

A single‐cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast

“…Moreover, close inspection of the fibroblast population (cluster 1) indicated that a lower proportion of cells expressed key marker genes such as PDGFRA, PDGFRB, CD34, and the matrix‐associated genes POSTN and COL3A1 (also SPARC, COL5A2, COL14A1 ) in post‐menopausal tissue (Fig 3A). High‐resolution confocal imaging of breast tissue to address expression and spatial distribution indicated a marked decrease in PDGFRβ+ fibroblasts in post‐menopausal tissue (Fig 3B), including the interlobular fibroblasts (Morsing et al, 2020). Some variation between post‐menopausal tissue sections was apparent, in part reflecting the regression of TDLUs.…”

A single-cell atlas of the healthy breast tissues reveals clinically relevant clusters of breast epithelial cells

“…23 Similarly, functionally heterogeneous fibroblasts in the normal breast have been described. 24 Epithelial cell types were dominant. Using CD49f/EpCAM expression pattern as well as TP63/NFIB, ELF5/EHF, and FOXA1/ESR1 as functional markers of basal/stem, luminal progenitors, and mature luminal cells, we performed subcluster analyses of epithelial cells, which revealed 13 different epithelial cells (Figures 1B and 1C).…”

“…Because our study involved breast biopsies of healthy women, tissue is very limited in quantity, and breast-region-specific differences in the cell types could not be ascertained. Recent studies have identified different biology and marker profiles in Article ll fibroblasts of the ductal and lobular regions of the breast, 24 and it will not be a surprise if transcriptome of epithelial cells in the ductal and lobular regions are different, which the current approaches cannot address. Lastly, it is difficult to account for inter-individual differences in transcriptome due to genetic diversity in the human population.…”

Transition to invasive breast cancer is associated with progressive changes in the structure and composition of tumor stroma

“…Notably, this analysis 274 also revealed a transition from a more luminal-like state in normal samples to a more 275 mesenchymal-like state in synchronous DCIS that aligned with analogous shifts in tumor 276 cell differentiation and function ( Figure 4I). In light of previous studies revealing a functional and structural interdependence between myoepithelium and the surrounding stroma (Jones et al, 2003;Morsing et al, 2020), we next sought to determine if the progressive loss of myoepithelial integrity observed here correlated with changes in fibroblast function and extracellular matrix remodeling (ECM). Single cell clustering revealed four fibroblast populations that included normal (CD36 high), resting (VIM-only), myofibroblast (SMA + ), and CAF (FAP + ) subsets ( Figure 5A).…”