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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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Related in: MedlinePlus

TGFβR2i allows efficient expansion of ERpos cells.(a) Population doublings as a function of passage number calculated by continuous cell number recordings in triplicate cultures before confluency and plating at a fixed number of 4,000 cells per cm2 per flask at each split. TGFβR2i allows proliferation for up to six passages, corresponding to 15 population doublings (open diamond). If RepSox is omitted, the cells cannot be expanded beyond fourth passage (cross). Initial plating on 3T3 feeders with quantification starting in passage three extends proliferation to more than 10 passages, corresponding to more than 25 population doublings (open circle). Cells from a different sorting (albeit followed for a shorter period) exhibit similar extended proliferative capacity (closed circle). hTERT/shp16-transduced CD166high/CD117low cells subsequently passaged at a fixed number of 6,000 cells per cm2 at each split extended the proliferative capacity even further (closed triangle), and the cells have now been growing for more than 12 passages. hTERT/shp16-transduced CD166high/CD117low cells derived from a different biopsy, split at a ratio of up to 1:4, has so far been growing up to passage 9 (open square). (b) Even beyond 20 population doublings (passage 9), ERpos cells with definitive lifespan maintain ER and PR expression as shown by immunoperoxidase and haematoxylin staining (cells in ninth passage seeded at 4,000 cells per cm2 and stained at day 5 with SP1 prediluted and SAN27, respectively). Scale bar, 50 μm.
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f5: TGFβR2i allows efficient expansion of ERpos cells.(a) Population doublings as a function of passage number calculated by continuous cell number recordings in triplicate cultures before confluency and plating at a fixed number of 4,000 cells per cm2 per flask at each split. TGFβR2i allows proliferation for up to six passages, corresponding to 15 population doublings (open diamond). If RepSox is omitted, the cells cannot be expanded beyond fourth passage (cross). Initial plating on 3T3 feeders with quantification starting in passage three extends proliferation to more than 10 passages, corresponding to more than 25 population doublings (open circle). Cells from a different sorting (albeit followed for a shorter period) exhibit similar extended proliferative capacity (closed circle). hTERT/shp16-transduced CD166high/CD117low cells subsequently passaged at a fixed number of 6,000 cells per cm2 at each split extended the proliferative capacity even further (closed triangle), and the cells have now been growing for more than 12 passages. hTERT/shp16-transduced CD166high/CD117low cells derived from a different biopsy, split at a ratio of up to 1:4, has so far been growing up to passage 9 (open square). (b) Even beyond 20 population doublings (passage 9), ERpos cells with definitive lifespan maintain ER and PR expression as shown by immunoperoxidase and haematoxylin staining (cells in ninth passage seeded at 4,000 cells per cm2 and stained at day 5 with SP1 prediluted and SAN27, respectively). Scale bar, 50 μm.

Mentions: TGFβR2i appeared to support growth of ERpos HBECs also after passaging. To test this systematically, we plated CD166high-derived cells at a density of 6,400 cells per cm2 in primary culture, and the cultures were subsequently passaged at a density of 4,000 cells per cm2. On passaging, ER expression was particularly evident in the dense centre of proliferating colonies. Continuous proliferation under these conditions was maintained for up to six passages, corresponding to 15 population doublings (exemplified in Fig. 5a). Importantly, however, in the absence of RepSox, the cells could not be expanded beyond fourth passage (Fig. 5a). The lifespan and ER expression (ranging from 21 to 77% in second- to fourth-passage cultures, Table 2) were somewhat biopsy dependent, and in general proliferation slowed between fourth and sixth passages, and at the same time downregulation of ER expression was observed. While this narrowed the window of experimentation to up to fourth passage, the cells could easily and reproducibly be replaced with new cultures with similar luminal characteristics (Table 2). However, passage number could be increased by increasing seeding density. Thus, ERpos cells derived from three biopsies could be cultured for another two to three passages, that is, up to passage six to nine when passaged at a high density of 8,000–20,000 cells per cm2. Moreover, the lifespan of ERpos HBECs passaged at low seeding density could be further extended by initial plating on 3T3 feeders, which lead to proliferation for more than 10 passages, corresponding to more than 25 population doublings (Fig. 5a). Cells from a different sorting exhibited similar characteristics (Fig. 5a). We subsequently addressed whether TGFβR2i culture would allow for alternative approaches to extend the lifespan of normal breast-derived ERpos cells. ERpos HBECs were successfully transduced with pBABE-neo-hTERT and pLenti X2 hygro/shp16 constructs and have now been cultured for 4 months with weekly passages at 6,000 cells per cm2, exceeding 12 passages (Fig. 5a). Transduction of ERpos cells from a different biopsy and subsequent splitting at up to 1:4 supported the robustness of this approach. Of note, these long-term cultured cells exhibited a phenotype essentially similar to cultures with definitive lifespan, including a relatively high level of ER expression (Supplementary Fig. 11 and Table 2). Thus, while the proliferation of ERpos cells with definite lifespan is somewhat slow as it took more than 100 days to generate more than 25 population doublings, the present protocol nevertheless allows a considerable expansion of the ERpos cell population extending from low-passage cultures with low seeding density through medium-passage cultures with high seeding density to high-passage cultures of hTERT/shp16-tranduced cells, which provides a relatively wide window of experimentation. Of note, until senescence the cells maintain their expression of ER, Ks20.8 reactivity as well as—mainly in densely packed colonies—expression of PR (Fig. 5b). In conclusion, TGFβR2i readily supports serial subculture of a population of ERpos HBECs, that is, ERpos progenitors.


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)

TGFβR2i allows efficient expansion of ERpos cells.(a) Population doublings as a function of passage number calculated by continuous cell number recordings in triplicate cultures before confluency and plating at a fixed number of 4,000 cells per cm2 per flask at each split. TGFβR2i allows proliferation for up to six passages, corresponding to 15 population doublings (open diamond). If RepSox is omitted, the cells cannot be expanded beyond fourth passage (cross). Initial plating on 3T3 feeders with quantification starting in passage three extends proliferation to more than 10 passages, corresponding to more than 25 population doublings (open circle). Cells from a different sorting (albeit followed for a shorter period) exhibit similar extended proliferative capacity (closed circle). hTERT/shp16-transduced CD166high/CD117low cells subsequently passaged at a fixed number of 6,000 cells per cm2 at each split extended the proliferative capacity even further (closed triangle), and the cells have now been growing for more than 12 passages. hTERT/shp16-transduced CD166high/CD117low cells derived from a different biopsy, split at a ratio of up to 1:4, has so far been growing up to passage 9 (open square). (b) Even beyond 20 population doublings (passage 9), ERpos cells with definitive lifespan maintain ER and PR expression as shown by immunoperoxidase and haematoxylin staining (cells in ninth passage seeded at 4,000 cells per cm2 and stained at day 5 with SP1 prediluted and SAN27, respectively). 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

f5: TGFβR2i allows efficient expansion of ERpos cells.(a) Population doublings as a function of passage number calculated by continuous cell number recordings in triplicate cultures before confluency and plating at a fixed number of 4,000 cells per cm2 per flask at each split. TGFβR2i allows proliferation for up to six passages, corresponding to 15 population doublings (open diamond). If RepSox is omitted, the cells cannot be expanded beyond fourth passage (cross). Initial plating on 3T3 feeders with quantification starting in passage three extends proliferation to more than 10 passages, corresponding to more than 25 population doublings (open circle). Cells from a different sorting (albeit followed for a shorter period) exhibit similar extended proliferative capacity (closed circle). hTERT/shp16-transduced CD166high/CD117low cells subsequently passaged at a fixed number of 6,000 cells per cm2 at each split extended the proliferative capacity even further (closed triangle), and the cells have now been growing for more than 12 passages. hTERT/shp16-transduced CD166high/CD117low cells derived from a different biopsy, split at a ratio of up to 1:4, has so far been growing up to passage 9 (open square). (b) Even beyond 20 population doublings (passage 9), ERpos cells with definitive lifespan maintain ER and PR expression as shown by immunoperoxidase and haematoxylin staining (cells in ninth passage seeded at 4,000 cells per cm2 and stained at day 5 with SP1 prediluted and SAN27, respectively). Scale bar, 50 μm.
Mentions: TGFβR2i appeared to support growth of ERpos HBECs also after passaging. To test this systematically, we plated CD166high-derived cells at a density of 6,400 cells per cm2 in primary culture, and the cultures were subsequently passaged at a density of 4,000 cells per cm2. On passaging, ER expression was particularly evident in the dense centre of proliferating colonies. Continuous proliferation under these conditions was maintained for up to six passages, corresponding to 15 population doublings (exemplified in Fig. 5a). Importantly, however, in the absence of RepSox, the cells could not be expanded beyond fourth passage (Fig. 5a). The lifespan and ER expression (ranging from 21 to 77% in second- to fourth-passage cultures, Table 2) were somewhat biopsy dependent, and in general proliferation slowed between fourth and sixth passages, and at the same time downregulation of ER expression was observed. While this narrowed the window of experimentation to up to fourth passage, the cells could easily and reproducibly be replaced with new cultures with similar luminal characteristics (Table 2). However, passage number could be increased by increasing seeding density. Thus, ERpos cells derived from three biopsies could be cultured for another two to three passages, that is, up to passage six to nine when passaged at a high density of 8,000–20,000 cells per cm2. Moreover, the lifespan of ERpos HBECs passaged at low seeding density could be further extended by initial plating on 3T3 feeders, which lead to proliferation for more than 10 passages, corresponding to more than 25 population doublings (Fig. 5a). Cells from a different sorting exhibited similar characteristics (Fig. 5a). We subsequently addressed whether TGFβR2i culture would allow for alternative approaches to extend the lifespan of normal breast-derived ERpos cells. ERpos HBECs were successfully transduced with pBABE-neo-hTERT and pLenti X2 hygro/shp16 constructs and have now been cultured for 4 months with weekly passages at 6,000 cells per cm2, exceeding 12 passages (Fig. 5a). Transduction of ERpos cells from a different biopsy and subsequent splitting at up to 1:4 supported the robustness of this approach. Of note, these long-term cultured cells exhibited a phenotype essentially similar to cultures with definitive lifespan, including a relatively high level of ER expression (Supplementary Fig. 11 and Table 2). Thus, while the proliferation of ERpos cells with definite lifespan is somewhat slow as it took more than 100 days to generate more than 25 population doublings, the present protocol nevertheless allows a considerable expansion of the ERpos cell population extending from low-passage cultures with low seeding density through medium-passage cultures with high seeding density to high-passage cultures of hTERT/shp16-tranduced cells, which provides a relatively wide window of experimentation. Of note, until senescence the cells maintain their expression of ER, Ks20.8 reactivity as well as—mainly in densely packed colonies—expression of PR (Fig. 5b). In conclusion, TGFβR2i readily supports serial subculture of a population of ERpos HBECs, that is, ERpos progenitors.

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