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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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A unique staining signature Ks20.8pos/CD166high/CD117low is eligible for ERpos cell sorting and tracking.(a) Serial cryostat sections of a normal human breast terminal duct lobular unit (TDLU) stained with immunoperoxidase against Ks20.8 (left) and K18 (right), and counterstained with haematoxylin. Note the characteristic scattered staining pattern with Ks20.8 against the uniform lineage-related staining of luminal cells with K18. Scale bar, 50 μm. (b) Multicolour imaging of normal human breast cryostat sections including acini and ducts from four different biopsies stained for Ks20.8 (red), hormone receptors ER–PR (SP1–SP2, green) and DAPI nuclear stain (blue). The Ks20.8pos compartment encompasses the ER–PR-expressing cells. Scale bar, 50 μm. (c) Multicolour imaging of normal human breast cryostat sections stained for CD166 (3A6, red), CD117 (K44.2, red), hormone receptors ER–PR (green) and DAPI stain (blue). While CD166 is highly expressed in hormone receptor-positive cells, CD117 shows the complementary pattern. Scale bar, 50 μm.
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f1: A unique staining signature Ks20.8pos/CD166high/CD117low is eligible for ERpos cell sorting and tracking.(a) Serial cryostat sections of a normal human breast terminal duct lobular unit (TDLU) stained with immunoperoxidase against Ks20.8 (left) and K18 (right), and counterstained with haematoxylin. Note the characteristic scattered staining pattern with Ks20.8 against the uniform lineage-related staining of luminal cells with K18. Scale bar, 50 μm. (b) Multicolour imaging of normal human breast cryostat sections including acini and ducts from four different biopsies stained for Ks20.8 (red), hormone receptors ER–PR (SP1–SP2, green) and DAPI nuclear stain (blue). The Ks20.8pos compartment encompasses the ER–PR-expressing cells. Scale bar, 50 μm. (c) Multicolour imaging of normal human breast cryostat sections stained for CD166 (3A6, red), CD117 (K44.2, red), hormone receptors ER–PR (green) and DAPI stain (blue). While CD166 is highly expressed in hormone receptor-positive cells, CD117 shows the complementary pattern. Scale bar, 50 μm.

Mentions: To answer the long-standing question of whether loss of hormone receptors in culture is due to the loss of cells or the loss of receptor protein expression, it was necessary first to provide tools for cell tracking and sorting of the relevant cells. To unequivocally track ERpos HBECs at the single-cell level we screened our antibody library for surrogate markers with a long half-life, for example, cytokeratins13, in culture. In situ staining of more than 30 reduction mammoplasties revealed a surprising pattern with a monoclonal antibody (clone Ks.20.8) originally raised against cytokeratin 20, a simple epithelial cytokeratin with a very restricted expression pattern and not expressed in normal human breast14. The lack of true cytokeratin 20 expression in normal breast was here confirmed with two specific antibodies (listed in Table 1). Instead, Ks20.8 stained a subpopulation of luminal cells in a unique scattered pattern (Fig. 1a). Ks20.8 antibodies from four different suppliers (Table 1) revealed similar staining patterns (Supplementary Fig. 1a). The characteristic staining pattern led us to speculate that it indeed represented ERpos cells. While ER and Ks20.8 apparently co-localized in acini as well as in ducts (Supplementary Fig. 1b), the immunofluorescence staining was not sufficiently strong to allow quantification. For this purpose, it was necessary to apply dual antibody immunofluorescence. Since ER and progesterone receptor (PR) are expressed coordinately in essentially the same cells in the normal human breast15, we decided to enhance immunofluorescence staining of uncultured cells by mixing antibodies for ER and PR. This approach revealed that the scattered staining with Ks20.8 encompassed almost the entire population of hormone receptor-positive HBECs in acini as well as in ducts (Fig. 1b) with the most evident co-localization in foci with the strongest receptor expression (in four out of eight biopsies tested). The antibody screen unravelled a number of additional markers of Ks20.8pos HBECs some of which are well-known markers of ERpos cells, including activating enhancer-binding protein 2 beta (AP2β), a marker of luminal differentiation, GATA3 (ref. 16), a marker of cell survival/longevity, Bcl2, two TGFβ-mediated, epithelial–mesenchymal transition-related markers, N-glycan/CDw75 antigen17 and N-cadherin (reviewed in ref. 18), as well as the stem cell markers, ALCAM (CD166) (ref. 19) and the laminin receptor, 67LR (ref. 20; Supplementary Fig. 2).


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)

A unique staining signature Ks20.8pos/CD166high/CD117low is eligible for ERpos cell sorting and tracking.(a) Serial cryostat sections of a normal human breast terminal duct lobular unit (TDLU) stained with immunoperoxidase against Ks20.8 (left) and K18 (right), and counterstained with haematoxylin. Note the characteristic scattered staining pattern with Ks20.8 against the uniform lineage-related staining of luminal cells with K18. Scale bar, 50 μm. (b) Multicolour imaging of normal human breast cryostat sections including acini and ducts from four different biopsies stained for Ks20.8 (red), hormone receptors ER–PR (SP1–SP2, green) and DAPI nuclear stain (blue). The Ks20.8pos compartment encompasses the ER–PR-expressing cells. Scale bar, 50 μm. (c) Multicolour imaging of normal human breast cryostat sections stained for CD166 (3A6, red), CD117 (K44.2, red), hormone receptors ER–PR (green) and DAPI stain (blue). While CD166 is highly expressed in hormone receptor-positive cells, CD117 shows the complementary pattern. Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: A unique staining signature Ks20.8pos/CD166high/CD117low is eligible for ERpos cell sorting and tracking.(a) Serial cryostat sections of a normal human breast terminal duct lobular unit (TDLU) stained with immunoperoxidase against Ks20.8 (left) and K18 (right), and counterstained with haematoxylin. Note the characteristic scattered staining pattern with Ks20.8 against the uniform lineage-related staining of luminal cells with K18. Scale bar, 50 μm. (b) Multicolour imaging of normal human breast cryostat sections including acini and ducts from four different biopsies stained for Ks20.8 (red), hormone receptors ER–PR (SP1–SP2, green) and DAPI nuclear stain (blue). The Ks20.8pos compartment encompasses the ER–PR-expressing cells. Scale bar, 50 μm. (c) Multicolour imaging of normal human breast cryostat sections stained for CD166 (3A6, red), CD117 (K44.2, red), hormone receptors ER–PR (green) and DAPI stain (blue). While CD166 is highly expressed in hormone receptor-positive cells, CD117 shows the complementary pattern. Scale bar, 50 μm.
Mentions: To answer the long-standing question of whether loss of hormone receptors in culture is due to the loss of cells or the loss of receptor protein expression, it was necessary first to provide tools for cell tracking and sorting of the relevant cells. To unequivocally track ERpos HBECs at the single-cell level we screened our antibody library for surrogate markers with a long half-life, for example, cytokeratins13, in culture. In situ staining of more than 30 reduction mammoplasties revealed a surprising pattern with a monoclonal antibody (clone Ks.20.8) originally raised against cytokeratin 20, a simple epithelial cytokeratin with a very restricted expression pattern and not expressed in normal human breast14. The lack of true cytokeratin 20 expression in normal breast was here confirmed with two specific antibodies (listed in Table 1). Instead, Ks20.8 stained a subpopulation of luminal cells in a unique scattered pattern (Fig. 1a). Ks20.8 antibodies from four different suppliers (Table 1) revealed similar staining patterns (Supplementary Fig. 1a). The characteristic staining pattern led us to speculate that it indeed represented ERpos cells. While ER and Ks20.8 apparently co-localized in acini as well as in ducts (Supplementary Fig. 1b), the immunofluorescence staining was not sufficiently strong to allow quantification. For this purpose, it was necessary to apply dual antibody immunofluorescence. Since ER and progesterone receptor (PR) are expressed coordinately in essentially the same cells in the normal human breast15, we decided to enhance immunofluorescence staining of uncultured cells by mixing antibodies for ER and PR. This approach revealed that the scattered staining with Ks20.8 encompassed almost the entire population of hormone receptor-positive HBECs in acini as well as in ducts (Fig. 1b) with the most evident co-localization in foci with the strongest receptor expression (in four out of eight biopsies tested). The antibody screen unravelled a number of additional markers of Ks20.8pos HBECs some of which are well-known markers of ERpos cells, including activating enhancer-binding protein 2 beta (AP2β), a marker of luminal differentiation, GATA3 (ref. 16), a marker of cell survival/longevity, Bcl2, two TGFβ-mediated, epithelial–mesenchymal transition-related markers, N-glycan/CDw75 antigen17 and N-cadherin (reviewed in ref. 18), as well as the stem cell markers, ALCAM (CD166) (ref. 19) and the laminin receptor, 67LR (ref. 20; Supplementary Fig. 2).

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