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In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.

Smart CE, Morrison BJ, Saunus JM, Vargas AC, Keith P, Reid L, Wockner L, Askarian-Amiri M, Amiri MA, Sarkar D, Simpson PT, Clarke C, Schmidt CW, Reynolds BA, Lakhani SR, Lopez JA - PLoS ONE (2013)

Bottom Line: Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats.Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage.We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them.

View Article: PubMed Central - PubMed

Affiliation: UQ Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia. c.smart@uq.edu.au

ABSTRACT
Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

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Relative frequencies of luminal and myoepithelial/bipotent human mammary epithelial cells in MUC1+-derived mammosphere cultures.MUC1+ sphere cultures were prepared as described in Fig. 5A, in parallel with matched MUC1+ adherent cultures, then dissociated and used in a colony forming cell (CFC) assay to determine the clonogenicity of luminal and myoepithelial/bipotent progenitor compartments (which give rise to colonies with luminal and mixed/myo morphologies respectively). (A) Light micrographs of Geimsa-stained colonies grown from dissociated spheres and parallel adherent cultures. Representative colonies are shown for adherent- (i-iii) and sphere-derived (iv-vi) cells. This includes examples of luminal (i, iv and v), mixed (ii, iii) and myoepithelial (vi) colony morphologies. (B) Overall clonogenicity (colonies formed as a percentage of total cells seeded) of spheres and parallel adherent cultures. (C) Comparison of luminal and myo/bipotent progenitor cell clonogenicity of spheres and parallel adherent cultures. Data are from four biological replicates (specimens from 4 patients), each performed in triplicate. Statistical tests used were paired, two-tailed students t-tests (P values indicated; ns = not significant).
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pone-0064388-g006: Relative frequencies of luminal and myoepithelial/bipotent human mammary epithelial cells in MUC1+-derived mammosphere cultures.MUC1+ sphere cultures were prepared as described in Fig. 5A, in parallel with matched MUC1+ adherent cultures, then dissociated and used in a colony forming cell (CFC) assay to determine the clonogenicity of luminal and myoepithelial/bipotent progenitor compartments (which give rise to colonies with luminal and mixed/myo morphologies respectively). (A) Light micrographs of Geimsa-stained colonies grown from dissociated spheres and parallel adherent cultures. Representative colonies are shown for adherent- (i-iii) and sphere-derived (iv-vi) cells. This includes examples of luminal (i, iv and v), mixed (ii, iii) and myoepithelial (vi) colony morphologies. (B) Overall clonogenicity (colonies formed as a percentage of total cells seeded) of spheres and parallel adherent cultures. (C) Comparison of luminal and myo/bipotent progenitor cell clonogenicity of spheres and parallel adherent cultures. Data are from four biological replicates (specimens from 4 patients), each performed in triplicate. Statistical tests used were paired, two-tailed students t-tests (P values indicated; ns = not significant).

Mentions: If primary mammospheres are enriched for progenitor cells, they might be expected to show increased in vitro clonogenicity, and produce a high frequency of colonies with mixed luminal and myoepithelial features, reflecting bipotency [9]. Consistent with this idea, the immuno-phenotype of MUC1+-derived hollow spheres (Fig. 5D) suggested enrichment with luminal progenitors. We tested this hypothesis using the colony forming cell (CFC) assay [28], which determines the relative frequencies of major mammary epithelial progenitor types (luminal- and myoepithelial-restricted, and bipotent progenitors) based on the morphologies of colonies that emerge from cells seeded at clonal density (luminal, myoepithelial or mixed respectively; Fig. 6A).


In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.

Smart CE, Morrison BJ, Saunus JM, Vargas AC, Keith P, Reid L, Wockner L, Askarian-Amiri M, Amiri MA, Sarkar D, Simpson PT, Clarke C, Schmidt CW, Reynolds BA, Lakhani SR, Lopez JA - PLoS ONE (2013)

Relative frequencies of luminal and myoepithelial/bipotent human mammary epithelial cells in MUC1+-derived mammosphere cultures.MUC1+ sphere cultures were prepared as described in Fig. 5A, in parallel with matched MUC1+ adherent cultures, then dissociated and used in a colony forming cell (CFC) assay to determine the clonogenicity of luminal and myoepithelial/bipotent progenitor compartments (which give rise to colonies with luminal and mixed/myo morphologies respectively). (A) Light micrographs of Geimsa-stained colonies grown from dissociated spheres and parallel adherent cultures. Representative colonies are shown for adherent- (i-iii) and sphere-derived (iv-vi) cells. This includes examples of luminal (i, iv and v), mixed (ii, iii) and myoepithelial (vi) colony morphologies. (B) Overall clonogenicity (colonies formed as a percentage of total cells seeded) of spheres and parallel adherent cultures. (C) Comparison of luminal and myo/bipotent progenitor cell clonogenicity of spheres and parallel adherent cultures. Data are from four biological replicates (specimens from 4 patients), each performed in triplicate. Statistical tests used were paired, two-tailed students t-tests (P values indicated; ns = not significant).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672101&req=5

pone-0064388-g006: Relative frequencies of luminal and myoepithelial/bipotent human mammary epithelial cells in MUC1+-derived mammosphere cultures.MUC1+ sphere cultures were prepared as described in Fig. 5A, in parallel with matched MUC1+ adherent cultures, then dissociated and used in a colony forming cell (CFC) assay to determine the clonogenicity of luminal and myoepithelial/bipotent progenitor compartments (which give rise to colonies with luminal and mixed/myo morphologies respectively). (A) Light micrographs of Geimsa-stained colonies grown from dissociated spheres and parallel adherent cultures. Representative colonies are shown for adherent- (i-iii) and sphere-derived (iv-vi) cells. This includes examples of luminal (i, iv and v), mixed (ii, iii) and myoepithelial (vi) colony morphologies. (B) Overall clonogenicity (colonies formed as a percentage of total cells seeded) of spheres and parallel adherent cultures. (C) Comparison of luminal and myo/bipotent progenitor cell clonogenicity of spheres and parallel adherent cultures. Data are from four biological replicates (specimens from 4 patients), each performed in triplicate. Statistical tests used were paired, two-tailed students t-tests (P values indicated; ns = not significant).
Mentions: If primary mammospheres are enriched for progenitor cells, they might be expected to show increased in vitro clonogenicity, and produce a high frequency of colonies with mixed luminal and myoepithelial features, reflecting bipotency [9]. Consistent with this idea, the immuno-phenotype of MUC1+-derived hollow spheres (Fig. 5D) suggested enrichment with luminal progenitors. We tested this hypothesis using the colony forming cell (CFC) assay [28], which determines the relative frequencies of major mammary epithelial progenitor types (luminal- and myoepithelial-restricted, and bipotent progenitors) based on the morphologies of colonies that emerge from cells seeded at clonal density (luminal, myoepithelial or mixed respectively; Fig. 6A).

Bottom Line: Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats.Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage.We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them.

View Article: PubMed Central - PubMed

Affiliation: UQ Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia. c.smart@uq.edu.au

ABSTRACT
Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

Show MeSH
Related in: MedlinePlus