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.

Keywords: Breast; Differentiation; Epithelial progenitors; Fibroblast.

Fig. 1

Lineage specific markers are maintained in hTERT immortalized HBFCs. a Diagram depicting the cumulative population doublings of CD105⁺ and CD26⁺ HBFCs transduced with empty vector (evHBFC^CD105; open squares and evHBFC^CD26; open triangles) or hTERT (iHBFC^CD105; closed squares and iHBFC^CD26; closed triangles) and recorded between passages 10 (day zero) and 57 (day 412). Whereas iHBFC^CD105 and iHBFC^CD26 continued to proliferate, empty vector controls ceased to expand after around 18 and 24 population doublings, respectively. Also, note that iHBFC^CD26 have an intrinsic growth advantage irrespective of immortalization. b iHBFCs were examined repeatedly for the expression of lineage markers CD105 and CD26 by immunoperoxidase staining (brown), here illustrated for cells in passage 50. Like their primary ancestors, iHBFC^CD105 are CD105^high/CD26^low (left) and iHBFC^CD26 are CD105^low/CD26^high (right). Nuclei are counterstained with hematoxylin (blue) (bar = 50 μm)

Fig. 2

iHBFCs resemble HBFCs by gene expression profiles and CD140b and CD248 are lobular markers in situ. a Venn diagram showing the number of differentially expressed genes (DEGs; p < 0.05 and fold change ≥ 2) based on genome wide RNA-sequencing of CD105⁺ and CD26⁺ HBFCs and iHBFCs, respectively. Bar diagram shows the percent overlap of DEGs between the cells indicated. b Heatmap of expression values of DEGs annotated with a cluster of differentiation (CD) name represented in a for iHBFC^CD105and iHBFC^CD26. c Cryostat sections of normal breast biopsies stained with peroxidase (brown) for CD140b and CD248 selected based on the CD gene expression profile of iHBFCs. Note the relatively intense staining in TDLUs (left) versus ducts (right). Nuclei are counterstained with hematoxylin (blue) (bar = 100 μm)

Fig. 3

iHBFC^CD105 support luminal epithelial growth and TDLU-like branching morphogenesis. Comparison of the capacity of iHBFC^CD105 and iHBFC^CD26 to induce human breast epithelial morphogenesis. a Phase contrast micrographs of luminal breast epithelial cells co-cultured for 16 days on passage 40 iHBFC^CD105 (left) or iHBFC^CD26 (right) (bar = 100 μm). Only iHBFC^CD105 facilitate elaborate TDLU-like branching morphogenesis. b Double immunofluorescence staining of luminal epithelial/iHBFC co-cultures with K19 (red) and MUC1 (green; bar = 100 μm). Note the staining of correctly polarized MUC1 in K19⁺ structures in both co-cultures. c Illustration of difference in induced branching morphogenesis by iHBFC^CD105 and iHBFC^CD26, respectively, by low magnification imaging and segmentation in ImageJ of branching morphogenesis in luminal epithelial/iHBFC co-cultures stained by peroxidase for K19 (brown). Segmented images show epithelial structures projected in black pixels (bar = 1000 μm). d Dot plot depicting the inductive capacity of seven pairs of iHBFC^CD105 (left) and iHBFC^CD26 (right) measured as the number of luminal epithelial structures per square unit area using luminal epithelial cells from five different biopsies. Consistently, iHBFC^CD105 have higher inductive capacity (asterisk indicates significance at p < 0.05 by Wilcoxon signed-rank test)

Fig. 4

iHBFC^CD26 convey a ductal-like differentiation of myoepithelial cells. a Images showing FACS sorted CD271^high/MUC1^low breast primary myoepithelial cells in co-culture with iHBFC^CD105 (left) and iHBFC^CD26 (right), fluorescently labeled for K17 (white) and K14 (not shown) by immunocytochemistry. K14 staining was used as a guide in image analysis to identify K14⁺ myoepithelial cells prior to measuring myoepithelial K17 staining intensity. Box plot shows interquartile range and median of K17 mean fluorescence in arbitrary units (AU) of three biopsies (whiskers indicate upper and lower quartiles; asterisk indicates significance at p < 0.05 by Kruskal-Wallis rank-sum test). b Primary myoepithelial/fibroblast co-cultures (iHBFC^CD105 (red), iHBFC^CD26 (gray)), were single cell suspended and stained for CD271 before analysis by FACS. Histogram shows cell count normalized to mode versus myoepithelial CD271 staining intensity in arbitrary units (AU) of a single biopsy (left) and box plot shows the interquartile range and median of the mean of CD271 fluorescence intensity relative to iHBFC^CD105 in arbitrary units of three biopsies (right; whiskers indicate upper and lower quartile, asterisk indicates significance at p < 0.05 by Kruskal-Wallis rank-sum test). c Schematic showing the experimental outline (left): primary CD271⁺ myoepithelial cells are plated onto confluent fibroblast feeders (passage 1 co-culture, Ps1), from which myoepithelial cells are isolated and then re-plated onto new fibroblast feeders (passage 2 co-culture, Ps2). Dot plot (right) shows normalized myoepithelial CD271 fluorescence in arbitrary units (AU) with mean values and standard deviations indicated by vertical bars as measured by FACS of 2250 cells in passage 1 and 2 co-cultures grouped according to feeder (iHBFC^CD105 (red) or iHBFC^CD26 (gray)). Note that the myoepithelial phenotype shifts as a consequence of a switch between fibroblasts (asterisk indicates significance at p < 0.05 by nested t test)

Fig. 5

The luminal differentiation repertoire of myoepithelial progenitors is directed by interaction with specialized fibroblasts. a Comparison of capacity of fibroblasts to direct epithelial progenitor capacity. Myoepithelial cells co-cultured with iHBFC^CD105 or iHBFC^CD26 were passaged and subjected to luminal differentiation conditions at clonal density and peroxidase stained for K19. While the induced K19 appeared mainly scattered when derived from iHBFC^CD105 co-culture (left), additional rather homogenous islets presented from iHBFC^CD26 co-cultures (right). The distinct phenotypes were observed in five out of seven tests with absence of homogeneous islets from iHBFC^CD26 in two tests (bar = 500 μm). b Representative multicolor confocal images (K19, red; K14, green; nuclei, blue) of cryostat sections of xenografted NOG mice 8 weeks after orthotopic injection of myoepithelial cells from primary co-culture with iHBFC^CD105 or iHBFC^CD26. Bilayered epithelial structures were obtained in 6/10 and 5/8 injections from iHBFC^CD105 and iHBFC^CD26, respectively, although at limited numbers, down to a few per transplant. Whereas iHBFC^CD105 co-culture derived myoepithelial cells readily differentiated into luminal K14^−/low/K19⁺ cells, co-culture with iHBFC^CD26 resulted mainly in K14⁺/K19⁺ luminal cells (bar = 50 μm)

Fig. 6

HBFC^CD105 TGF-β signaling supports parenchymal morphogenesis. a Overview of experimental design in which HBFCs are plated and exposed to 10 μM SB431542 or vehicle (DMSO) from day 7 to day 10 at which SB431542 or vehicle are removed and primary CD271^low/MUC1^high luminal breast epithelial cells are added and co-cultured for 10 days prior to assessment of epithelial structure formation. b 15 tests representing recombinations of four fibroblast biopsies and five epithelial biopsies are presented in a paired dot plot and a representative set of micrographs, showing a significant reduction in epithelial structure formation per square unit area in response to SB431542 versus vehicle in HBFC^CD105 co-cultures only (asterisk indicates significance at p < 0.05 by Wilcoxon signed-rank test, ns = not significant) (bar = 1000 μm)