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An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models.

Donatello S, Hudson L, Cottell DC, Blanco A, Aurrekoetxea I, Shelly MJ, Dervan PA, Kell MR, Stokes M, Hill AD, Hopkins AM - J. Exp. Clin. Cancer Res. (2011)

Bottom Line: Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells.Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Royal College of Surgeons in Ireland; Dublin, Ireland.

ABSTRACT

Background: Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.

Methods: Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.

Results: Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells.

Conclusions: Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

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Related in: MedlinePlus

Characterization of tumour and non-tumour primary cultures. A. Organoid-derived cultures (A, top panels, 10X magnification) from both tumour and non-tumour specimens had large polygonal cells (lower panels, lpc) surrounded by small polygonal cells (lower panels, spc, 20X magnification). B. Representative tumour and non-tumour cultures (passages 1-3) were analyzed for expression of the epithelial markers K19, K18 and ESA and the myoepithelial markers SMA, K14 and vimentin (scale bar 50 μm). C. Representative cultures were immunoblotted for expression of epithelial (K19, K18) and myoepithelial (vimentin, p63) markers.
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Figure 1: Characterization of tumour and non-tumour primary cultures. A. Organoid-derived cultures (A, top panels, 10X magnification) from both tumour and non-tumour specimens had large polygonal cells (lower panels, lpc) surrounded by small polygonal cells (lower panels, spc, 20X magnification). B. Representative tumour and non-tumour cultures (passages 1-3) were analyzed for expression of the epithelial markers K19, K18 and ESA and the myoepithelial markers SMA, K14 and vimentin (scale bar 50 μm). C. Representative cultures were immunoblotted for expression of epithelial (K19, K18) and myoepithelial (vimentin, p63) markers.

Mentions: Primary cultures of both non-tumour (NT) and tumour (T) human breast tissue yielded adherent organoids with outwardly-proliferating colonies (Figure 1A, left). Two cellular populations were observed - large polygonal cells in colony centres (lpc; Figure 1A, right), and small polygonal cells (spc) at the peripheries. Since spc and lpc resembled respectively myoepithelial and luminal epithelial cells, expression of epithelial and myoepithelial markers was examined by immunofluorescence microscopy (Figure 1B). In comparison to the negative control (-ve), cultures were mostly dual-positive for epithelial markers such as K18, K19 or epithelial-specific antigen (ESA) and myoepithelial markers such as K14, vimentin or smooth muscle actin (SMA). Western blot (Figure 1C) detection of K18 was not as sensitive as immufluorescence analysis, since only some of the cultures expressed K18. Interestingly our analysis (Figure 1C) also revealed that 3 out of 4 non-tumour cultures expressed high levels of the epithelial marker K19 and low levels of the myoepithelial marker p63. In contrast, 3 out of 4 tumour cultures expressed low levels of K19 but high levels of p63. Western blotting analysis also confirmed high expression of the myoepithelial marker vimentin.


An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models.

Donatello S, Hudson L, Cottell DC, Blanco A, Aurrekoetxea I, Shelly MJ, Dervan PA, Kell MR, Stokes M, Hill AD, Hopkins AM - J. Exp. Clin. Cancer Res. (2011)

Characterization of tumour and non-tumour primary cultures. A. Organoid-derived cultures (A, top panels, 10X magnification) from both tumour and non-tumour specimens had large polygonal cells (lower panels, lpc) surrounded by small polygonal cells (lower panels, spc, 20X magnification). B. Representative tumour and non-tumour cultures (passages 1-3) were analyzed for expression of the epithelial markers K19, K18 and ESA and the myoepithelial markers SMA, K14 and vimentin (scale bar 50 μm). C. Representative cultures were immunoblotted for expression of epithelial (K19, K18) and myoepithelial (vimentin, p63) markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094256&req=5

Figure 1: Characterization of tumour and non-tumour primary cultures. A. Organoid-derived cultures (A, top panels, 10X magnification) from both tumour and non-tumour specimens had large polygonal cells (lower panels, lpc) surrounded by small polygonal cells (lower panels, spc, 20X magnification). B. Representative tumour and non-tumour cultures (passages 1-3) were analyzed for expression of the epithelial markers K19, K18 and ESA and the myoepithelial markers SMA, K14 and vimentin (scale bar 50 μm). C. Representative cultures were immunoblotted for expression of epithelial (K19, K18) and myoepithelial (vimentin, p63) markers.
Mentions: Primary cultures of both non-tumour (NT) and tumour (T) human breast tissue yielded adherent organoids with outwardly-proliferating colonies (Figure 1A, left). Two cellular populations were observed - large polygonal cells in colony centres (lpc; Figure 1A, right), and small polygonal cells (spc) at the peripheries. Since spc and lpc resembled respectively myoepithelial and luminal epithelial cells, expression of epithelial and myoepithelial markers was examined by immunofluorescence microscopy (Figure 1B). In comparison to the negative control (-ve), cultures were mostly dual-positive for epithelial markers such as K18, K19 or epithelial-specific antigen (ESA) and myoepithelial markers such as K14, vimentin or smooth muscle actin (SMA). Western blot (Figure 1C) detection of K18 was not as sensitive as immufluorescence analysis, since only some of the cultures expressed K18. Interestingly our analysis (Figure 1C) also revealed that 3 out of 4 non-tumour cultures expressed high levels of the epithelial marker K19 and low levels of the myoepithelial marker p63. In contrast, 3 out of 4 tumour cultures expressed low levels of K19 but high levels of p63. Western blotting analysis also confirmed high expression of the myoepithelial marker vimentin.

Bottom Line: Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells.Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Royal College of Surgeons in Ireland; Dublin, Ireland.

ABSTRACT

Background: Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.

Methods: Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.

Results: Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells.

Conclusions: Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

Show MeSH
Related in: MedlinePlus