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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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Loss of ERpos cells in culture is due to both lack of growth and down modulation of ER expression.(a) T25 flasks in triplicate of basal cells (basal), CD117 high and CD166high ERpos luminal cells plated at a clonal density of 104 cells per flask (400 cells per cm2) and stained with haematoxylin after 14 days in culture. Note that only CD117high cells are colony forming at clonal density. (b) A higher magnification of cultures started at a higher cell density (3,000 cells per cm2) and immunoperoxidase stained at day 9 for Ks20.8 (upper row) or ER (SP1 prediluted, lower row; inset shows positive staining for ER at day 1 after plating) and counterstained with haematoxylin. Whereas ER expression is rapidly lost, the non-colony-forming cells of the CD166high cultures remain Ks20.8 positive and traceable in culture. Scale bar, 50 μm.
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f3: Loss of ERpos cells in culture is due to both lack of growth and down modulation of ER expression.(a) T25 flasks in triplicate of basal cells (basal), CD117 high and CD166high ERpos luminal cells plated at a clonal density of 104 cells per flask (400 cells per cm2) and stained with haematoxylin after 14 days in culture. Note that only CD117high cells are colony forming at clonal density. (b) A higher magnification of cultures started at a higher cell density (3,000 cells per cm2) and immunoperoxidase stained at day 9 for Ks20.8 (upper row) or ER (SP1 prediluted, lower row; inset shows positive staining for ER at day 1 after plating) and counterstained with haematoxylin. Whereas ER expression is rapidly lost, the non-colony-forming cells of the CD166high cultures remain Ks20.8 positive and traceable in culture. Scale bar, 50 μm.

Mentions: With the surrogate markers in hand we were now able to examine the behaviour of sorted ERpos HBECs in culture, potentially irrespective of ER expression. From the point of view that favourable conditions for luminal epithelial cells also apply to ERpos HBECs, we refined a protocol to permit ample colony formation of luminal cells at clonal density. As cell culture plastic we used Primaria, a substrate with a high content of nitrogen previously shown to promote adhesion of breast cells25. The growth medium ‘FAD2' was modified from previously described media for culturing keratinocytes or basal breast epithelium on mouse fibroblast feeders2627. The basal medium is Ham's F12:DMEM (1:3) similar to that of Tan et al.27, but with less serum, that is, 5%, as in Liu et al.26 Under these conditions, we gauged for colony formation among the three FACS gated populations described above. When plated at a clonal density of 400 cells per cm2, indeed colony forming luminal cells from the CD117high gate were highly favoured over basal cells (Fig. 3a). However, it was also clear that the ERpos HBECs from the CD166high gate entirely failed to form colonies under similar conditions. However, a scrutiny of cultures at a higher magnification revealed that CD166high cells in fact did not disappear from the culture. Rather, they plated, survived and also stained with Ks20.8 as a testimony of their original identity, but they entirely refrained from growth and rapidly lost the ER protein (Fig. 3b). By comparison, other HBEC culture media, that is, M87A (ref. 28), MEGM or WIT-P-NC did not support plating of CD166high cells. Therefore, we conclude that failure of culturing ERpos HBECs is caused by both lack of growth and loss of ER expression under conditions otherwise favouring propagation of luminal epithelial cells.


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)

Loss of ERpos cells in culture is due to both lack of growth and down modulation of ER expression.(a) T25 flasks in triplicate of basal cells (basal), CD117 high and CD166high ERpos luminal cells plated at a clonal density of 104 cells per flask (400 cells per cm2) and stained with haematoxylin after 14 days in culture. Note that only CD117high cells are colony forming at clonal density. (b) A higher magnification of cultures started at a higher cell density (3,000 cells per cm2) and immunoperoxidase stained at day 9 for Ks20.8 (upper row) or ER (SP1 prediluted, lower row; inset shows positive staining for ER at day 1 after plating) and counterstained with haematoxylin. Whereas ER expression is rapidly lost, the non-colony-forming cells of the CD166high cultures remain Ks20.8 positive and traceable in culture. Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660059&req=5

f3: Loss of ERpos cells in culture is due to both lack of growth and down modulation of ER expression.(a) T25 flasks in triplicate of basal cells (basal), CD117 high and CD166high ERpos luminal cells plated at a clonal density of 104 cells per flask (400 cells per cm2) and stained with haematoxylin after 14 days in culture. Note that only CD117high cells are colony forming at clonal density. (b) A higher magnification of cultures started at a higher cell density (3,000 cells per cm2) and immunoperoxidase stained at day 9 for Ks20.8 (upper row) or ER (SP1 prediluted, lower row; inset shows positive staining for ER at day 1 after plating) and counterstained with haematoxylin. Whereas ER expression is rapidly lost, the non-colony-forming cells of the CD166high cultures remain Ks20.8 positive and traceable in culture. Scale bar, 50 μm.
Mentions: With the surrogate markers in hand we were now able to examine the behaviour of sorted ERpos HBECs in culture, potentially irrespective of ER expression. From the point of view that favourable conditions for luminal epithelial cells also apply to ERpos HBECs, we refined a protocol to permit ample colony formation of luminal cells at clonal density. As cell culture plastic we used Primaria, a substrate with a high content of nitrogen previously shown to promote adhesion of breast cells25. The growth medium ‘FAD2' was modified from previously described media for culturing keratinocytes or basal breast epithelium on mouse fibroblast feeders2627. The basal medium is Ham's F12:DMEM (1:3) similar to that of Tan et al.27, but with less serum, that is, 5%, as in Liu et al.26 Under these conditions, we gauged for colony formation among the three FACS gated populations described above. When plated at a clonal density of 400 cells per cm2, indeed colony forming luminal cells from the CD117high gate were highly favoured over basal cells (Fig. 3a). However, it was also clear that the ERpos HBECs from the CD166high gate entirely failed to form colonies under similar conditions. However, a scrutiny of cultures at a higher magnification revealed that CD166high cells in fact did not disappear from the culture. Rather, they plated, survived and also stained with Ks20.8 as a testimony of their original identity, but they entirely refrained from growth and rapidly lost the ER protein (Fig. 3b). By comparison, other HBEC culture media, that is, M87A (ref. 28), MEGM or WIT-P-NC did not support plating of CD166high cells. Therefore, we conclude that failure of culturing ERpos HBECs is caused by both lack of growth and loss of ER expression under conditions otherwise favouring propagation of luminal epithelial cells.

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