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Propagation of oestrogen receptor-positive and oestrogen-responsive normal human breast cells in culture.

Fridriksdottir AJ, Kim J, Villadsen R, Klitgaard MC, Hopkinson BM, Petersen OW, Rønnov-Jessen L - Nat Commun (2015)

Bottom Line: Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures.We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFβ signalling, and that growth is augmented further in response to oestrogen.These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.

ABSTRACT
Investigating the susceptibility of oestrogen receptor-positive (ER(pos)) normal human breast epithelial cells (HBECs) for clinical purposes or basic research awaits a proficient cell-based assay. Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures. We report a robust technique for isolating ER(pos) HBECs from reduction mammoplasties by FACS using two cell surface markers, CD166 and CD117, and an intracellular cytokeratin marker, Ks20.8, for further tracking single cells in culture. We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFβ signalling, and that growth is augmented further in response to oestrogen. Importantly, ER signalling is functionally active in ER(pos) cells in extended culture. These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer.

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ERpos cells are purified and tracked by sequential CD326/CD271–CD166/CD117 FACS followed by multicolour staining and qRT–PCR.(a) Multicolour flow cytometry of uncultured HBECs incubated with CD326/CD271/CD166/CD117 and visualized pairwise (left diagrams) to recover luminal cells (CD326high) and basal cells (CD271high) and from the luminal gate CD166high and CD117high cells. Smears of sorted cells were stained (right panel) with either of the markers against basal cells, cytokeratin K14; luminal cells, cytokeratin K18; luminal progenitors, cytokeratin K15; Ks20.8 or ER–PR and counterstained with DAPI nuclear stain. Hormone receptor-positive cells are observed primarily among CD166high cells. Scale bar, 50 μm. (b) Purity of sorted cells as determined by staining of smears followed by quantification of the percentage of cells stained with either of the markers cytokeratin K14, K18, K15, Ks20.8 or hormone receptors (ER–PR; 3 × 100 cells per slide, error bars indicate s.d.'s). (c) Heatmap representing qRT–PCR analysis of the relative gene expression of lineage markers in sorted basal cells (basal), CD117high luminal cells (CD117) and CD166high luminal cells (CD166) from six different biopsies. Data confirm lineage-specific transcriptional profiles of the three cell populations and restricts ER expression (ESR1) primarily to CD166high luminal cells. Colour bar indicates the fold difference of the relative gene expression in log2 scale.
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f2: ERpos cells are purified and tracked by sequential CD326/CD271–CD166/CD117 FACS followed by multicolour staining and qRT–PCR.(a) Multicolour flow cytometry of uncultured HBECs incubated with CD326/CD271/CD166/CD117 and visualized pairwise (left diagrams) to recover luminal cells (CD326high) and basal cells (CD271high) and from the luminal gate CD166high and CD117high cells. Smears of sorted cells were stained (right panel) with either of the markers against basal cells, cytokeratin K14; luminal cells, cytokeratin K18; luminal progenitors, cytokeratin K15; Ks20.8 or ER–PR and counterstained with DAPI nuclear stain. Hormone receptor-positive cells are observed primarily among CD166high cells. Scale bar, 50 μm. (b) Purity of sorted cells as determined by staining of smears followed by quantification of the percentage of cells stained with either of the markers cytokeratin K14, K18, K15, Ks20.8 or hormone receptors (ER–PR; 3 × 100 cells per slide, error bars indicate s.d.'s). (c) Heatmap representing qRT–PCR analysis of the relative gene expression of lineage markers in sorted basal cells (basal), CD117high luminal cells (CD117) and CD166high luminal cells (CD166) from six different biopsies. Data confirm lineage-specific transcriptional profiles of the three cell populations and restricts ER expression (ESR1) primarily to CD166high luminal cells. Colour bar indicates the fold difference of the relative gene expression in log2 scale.

Mentions: For cell sorting purposes we found that CD166 and CD117 made good candidate surface markers of potential ERpos and ERneg cells, respectively—again as revealed by enhanced multicolour immunofluorescence (Fig. 1c). The mutual exclusivity of CD117 and ER (Fig. 1c) confirms what has been reported by others21. Accordingly, we designed a fluorescence-activated cell sorting (FACS) protocol to first separate the basal cell population from the luminal cell population based on EpCAM (CD326) and NGFR (CD271) followed by sorting with CD166 and CD117 to further dissect the luminal compartment (Fig. 2a and Supplementary Fig. 3). This protocol yields three populations, the purity of which was assessed by staining smears with lineage and progenitor markers K14, K18 and K15 (ref. 22), ER–PR as well as the novel ERpos cell surrogate marker Ks20.8 (Fig. 2a). As expected, we found that ERpos/PRpos HBECs were highly enriched in the CD166high/CD117low gate (Fig. 2b). While immunofluorescence staining for ER alone evoked a cytoplasmic background staining, which prevented reliable quantification, the smears turned out to be ideal for accurate assessment of the level of co-localization of ER–PR and Ks20.8. Indeed, up to 90% of Ks20.8pos cells were also ER–PRpos (Supplementary Fig. 4) and up to 87% of ER–PRpos cells were Ks20.8pos. The separation of the three subpopulations was further validated by quantitative reverse transcription–PCR (qRT–PCR), which confirmed a high ER expression (ESR1) in the CD166high cells compared with the other subpopulations (Fig. 2c). An additional panel of markers further distinguished the two luminal subpopulations from the basal cell population (Supplementary Fig. 5). Importantly, we found that known ER signalling-related genes such as trefoil factor family-1 (TFF1; ref. 23) and growth regulation by oestrogen in breast cancer 1 (GREB1; ref. 24) were highly expressed in CD166high cells as compared with other HBECs (Supplementary Fig. 5). The degree of separation in the CD166/CD117 FACS analysis was, however, somewhat biopsy dependent. In a series of six biopsies originating from women between 19 and 44 years old, five exhibited a similar separation with 11–49% of the cells being CD166high/CD117low, while one biopsy apparently did not contain a CD117high population (Supplementary Fig. 6). As an alternative, CD117 could be replaced with the laminin receptor 67LR in the CD166 FACS to obtain enriched ERpos HBECs (80% increase in Ks20.8-positive cells in the 67LRhigh gate versus the 67LRlow gate; Supplementary Fig. 7). In conclusion, the combination of Ks20.8, CD166 and CD117 is a promising marker for ERpos HBEC tracking and sorting.


Propagation of oestrogen receptor-positive and oestrogen-responsive normal human breast cells in culture.

Fridriksdottir AJ, Kim J, Villadsen R, Klitgaard MC, Hopkinson BM, Petersen OW, Rønnov-Jessen L - Nat Commun (2015)

ERpos cells are purified and tracked by sequential CD326/CD271–CD166/CD117 FACS followed by multicolour staining and qRT–PCR.(a) Multicolour flow cytometry of uncultured HBECs incubated with CD326/CD271/CD166/CD117 and visualized pairwise (left diagrams) to recover luminal cells (CD326high) and basal cells (CD271high) and from the luminal gate CD166high and CD117high cells. Smears of sorted cells were stained (right panel) with either of the markers against basal cells, cytokeratin K14; luminal cells, cytokeratin K18; luminal progenitors, cytokeratin K15; Ks20.8 or ER–PR and counterstained with DAPI nuclear stain. Hormone receptor-positive cells are observed primarily among CD166high cells. Scale bar, 50 μm. (b) Purity of sorted cells as determined by staining of smears followed by quantification of the percentage of cells stained with either of the markers cytokeratin K14, K18, K15, Ks20.8 or hormone receptors (ER–PR; 3 × 100 cells per slide, error bars indicate s.d.'s). (c) Heatmap representing qRT–PCR analysis of the relative gene expression of lineage markers in sorted basal cells (basal), CD117high luminal cells (CD117) and CD166high luminal cells (CD166) from six different biopsies. Data confirm lineage-specific transcriptional profiles of the three cell populations and restricts ER expression (ESR1) primarily to CD166high luminal cells. Colour bar indicates the fold difference of the relative gene expression in log2 scale.
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Related In: Results  -  Collection

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f2: ERpos cells are purified and tracked by sequential CD326/CD271–CD166/CD117 FACS followed by multicolour staining and qRT–PCR.(a) Multicolour flow cytometry of uncultured HBECs incubated with CD326/CD271/CD166/CD117 and visualized pairwise (left diagrams) to recover luminal cells (CD326high) and basal cells (CD271high) and from the luminal gate CD166high and CD117high cells. Smears of sorted cells were stained (right panel) with either of the markers against basal cells, cytokeratin K14; luminal cells, cytokeratin K18; luminal progenitors, cytokeratin K15; Ks20.8 or ER–PR and counterstained with DAPI nuclear stain. Hormone receptor-positive cells are observed primarily among CD166high cells. Scale bar, 50 μm. (b) Purity of sorted cells as determined by staining of smears followed by quantification of the percentage of cells stained with either of the markers cytokeratin K14, K18, K15, Ks20.8 or hormone receptors (ER–PR; 3 × 100 cells per slide, error bars indicate s.d.'s). (c) Heatmap representing qRT–PCR analysis of the relative gene expression of lineage markers in sorted basal cells (basal), CD117high luminal cells (CD117) and CD166high luminal cells (CD166) from six different biopsies. Data confirm lineage-specific transcriptional profiles of the three cell populations and restricts ER expression (ESR1) primarily to CD166high luminal cells. Colour bar indicates the fold difference of the relative gene expression in log2 scale.
Mentions: For cell sorting purposes we found that CD166 and CD117 made good candidate surface markers of potential ERpos and ERneg cells, respectively—again as revealed by enhanced multicolour immunofluorescence (Fig. 1c). The mutual exclusivity of CD117 and ER (Fig. 1c) confirms what has been reported by others21. Accordingly, we designed a fluorescence-activated cell sorting (FACS) protocol to first separate the basal cell population from the luminal cell population based on EpCAM (CD326) and NGFR (CD271) followed by sorting with CD166 and CD117 to further dissect the luminal compartment (Fig. 2a and Supplementary Fig. 3). This protocol yields three populations, the purity of which was assessed by staining smears with lineage and progenitor markers K14, K18 and K15 (ref. 22), ER–PR as well as the novel ERpos cell surrogate marker Ks20.8 (Fig. 2a). As expected, we found that ERpos/PRpos HBECs were highly enriched in the CD166high/CD117low gate (Fig. 2b). While immunofluorescence staining for ER alone evoked a cytoplasmic background staining, which prevented reliable quantification, the smears turned out to be ideal for accurate assessment of the level of co-localization of ER–PR and Ks20.8. Indeed, up to 90% of Ks20.8pos cells were also ER–PRpos (Supplementary Fig. 4) and up to 87% of ER–PRpos cells were Ks20.8pos. The separation of the three subpopulations was further validated by quantitative reverse transcription–PCR (qRT–PCR), which confirmed a high ER expression (ESR1) in the CD166high cells compared with the other subpopulations (Fig. 2c). An additional panel of markers further distinguished the two luminal subpopulations from the basal cell population (Supplementary Fig. 5). Importantly, we found that known ER signalling-related genes such as trefoil factor family-1 (TFF1; ref. 23) and growth regulation by oestrogen in breast cancer 1 (GREB1; ref. 24) were highly expressed in CD166high cells as compared with other HBECs (Supplementary Fig. 5). The degree of separation in the CD166/CD117 FACS analysis was, however, somewhat biopsy dependent. In a series of six biopsies originating from women between 19 and 44 years old, five exhibited a similar separation with 11–49% of the cells being CD166high/CD117low, while one biopsy apparently did not contain a CD117high population (Supplementary Fig. 6). As an alternative, CD117 could be replaced with the laminin receptor 67LR in the CD166 FACS to obtain enriched ERpos HBECs (80% increase in Ks20.8-positive cells in the 67LRhigh gate versus the 67LRlow gate; Supplementary Fig. 7). In conclusion, the combination of Ks20.8, CD166 and CD117 is a promising marker for ERpos HBEC tracking and sorting.

Bottom Line: Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures.We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFβ signalling, and that growth is augmented further in response to oestrogen.These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.

ABSTRACT
Investigating the susceptibility of oestrogen receptor-positive (ER(pos)) normal human breast epithelial cells (HBECs) for clinical purposes or basic research awaits a proficient cell-based assay. Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures. We report a robust technique for isolating ER(pos) HBECs from reduction mammoplasties by FACS using two cell surface markers, CD166 and CD117, and an intracellular cytokeratin marker, Ks20.8, for further tracking single cells in culture. We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFβ signalling, and that growth is augmented further in response to oestrogen. Importantly, ER signalling is functionally active in ER(pos) cells in extended culture. These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer.

Show MeSH
Related in: MedlinePlus