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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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Multiparametric flow cytometry analysis of mammary epithelial and stem cell markers in matched adherent and sphere cultures of breast cancer cell lines.(A) Experiment design. Matched adherent and sphere cultures of luminal-like and basal-like/claudin-low cell lines were generated in triplicate from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates. Panel 1: MUC1-FITC, HER2-PE and the LIVE/DEAD® red cell viability stain; Panel 2: CD49f-Pacific Blue, Aldefluor® reagent (for ALDH1 activity), CD24-PE, LIVE/DEAD® red, AC133-APC, EpCAM-PerCP Cy5.5 and CD44-APC-Cy7. (B) Gating strategy for simultaneous detection of cell surface markers. Acellular particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity (live cells designated ‘LDR live’). For each experiment, gates were placed based on autofluorescence of unstained adherent or sphere control samples to account for differences in the autofluorescence of cells grown in the different formats. For Aldefluor® (ALDH1 activity), gates were placed based on the fluorescence of the DEAB negative control (refer to supp methods). Population frequencies were determined for individual parameters (MUC1+, HER2+, AC133+, EpCAM+, ALDH1+, CD44+, CD24+, CD49f+). For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (see Fig. 3).
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pone-0064388-g002: Multiparametric flow cytometry analysis of mammary epithelial and stem cell markers in matched adherent and sphere cultures of breast cancer cell lines.(A) Experiment design. Matched adherent and sphere cultures of luminal-like and basal-like/claudin-low cell lines were generated in triplicate from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates. Panel 1: MUC1-FITC, HER2-PE and the LIVE/DEAD® red cell viability stain; Panel 2: CD49f-Pacific Blue, Aldefluor® reagent (for ALDH1 activity), CD24-PE, LIVE/DEAD® red, AC133-APC, EpCAM-PerCP Cy5.5 and CD44-APC-Cy7. (B) Gating strategy for simultaneous detection of cell surface markers. Acellular particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity (live cells designated ‘LDR live’). For each experiment, gates were placed based on autofluorescence of unstained adherent or sphere control samples to account for differences in the autofluorescence of cells grown in the different formats. For Aldefluor® (ALDH1 activity), gates were placed based on the fluorescence of the DEAB negative control (refer to supp methods). Population frequencies were determined for individual parameters (MUC1+, HER2+, AC133+, EpCAM+, ALDH1+, CD44+, CD24+, CD49f+). For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (see Fig. 3).

Mentions: Matched adherent and sphere cultures of luminal and Claudin-low cell lines were generated in triplicate (technical replicates) from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates for simultaneous detection of cell surface markers (Fig. 2). Panel 1: MUC1-FITC (BD), HER2-PE (BD) and the LIVE/DEAD® red cell viability stain (Invitrogen); Panel 2: CD49f-Pacific Blue (Biolegend), Aldefluor® assay reagent (StemCell Technologies), CD24-PE (BD), LIVE/DEAD® red, AC133-APC (Milltenyi), EpCAM-PerCP Cy5.5 (BD) and CD44-APC-Cy7 (Biolegend). Staining with the Aldefluor® assay for detection of ALDH1 activity was done prior to the addition of fluorescent antibodies according to the maufacturer’s instructions. Each Aldefluor-containing sample was prepared in duplicate, and the second negative control sample was immediately quenched in the ALDH1 inhibitor Diethylaminobenzaldehyde (DEAB) as per the manufacturer’s instructions.


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)

Multiparametric flow cytometry analysis of mammary epithelial and stem cell markers in matched adherent and sphere cultures of breast cancer cell lines.(A) Experiment design. Matched adherent and sphere cultures of luminal-like and basal-like/claudin-low cell lines were generated in triplicate from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates. Panel 1: MUC1-FITC, HER2-PE and the LIVE/DEAD® red cell viability stain; Panel 2: CD49f-Pacific Blue, Aldefluor® reagent (for ALDH1 activity), CD24-PE, LIVE/DEAD® red, AC133-APC, EpCAM-PerCP Cy5.5 and CD44-APC-Cy7. (B) Gating strategy for simultaneous detection of cell surface markers. Acellular particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity (live cells designated ‘LDR live’). For each experiment, gates were placed based on autofluorescence of unstained adherent or sphere control samples to account for differences in the autofluorescence of cells grown in the different formats. For Aldefluor® (ALDH1 activity), gates were placed based on the fluorescence of the DEAB negative control (refer to supp methods). Population frequencies were determined for individual parameters (MUC1+, HER2+, AC133+, EpCAM+, ALDH1+, CD44+, CD24+, CD49f+). For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (see Fig. 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672101&req=5

pone-0064388-g002: Multiparametric flow cytometry analysis of mammary epithelial and stem cell markers in matched adherent and sphere cultures of breast cancer cell lines.(A) Experiment design. Matched adherent and sphere cultures of luminal-like and basal-like/claudin-low cell lines were generated in triplicate from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates. Panel 1: MUC1-FITC, HER2-PE and the LIVE/DEAD® red cell viability stain; Panel 2: CD49f-Pacific Blue, Aldefluor® reagent (for ALDH1 activity), CD24-PE, LIVE/DEAD® red, AC133-APC, EpCAM-PerCP Cy5.5 and CD44-APC-Cy7. (B) Gating strategy for simultaneous detection of cell surface markers. Acellular particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity (live cells designated ‘LDR live’). For each experiment, gates were placed based on autofluorescence of unstained adherent or sphere control samples to account for differences in the autofluorescence of cells grown in the different formats. For Aldefluor® (ALDH1 activity), gates were placed based on the fluorescence of the DEAB negative control (refer to supp methods). Population frequencies were determined for individual parameters (MUC1+, HER2+, AC133+, EpCAM+, ALDH1+, CD44+, CD24+, CD49f+). For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (see Fig. 3).
Mentions: Matched adherent and sphere cultures of luminal and Claudin-low cell lines were generated in triplicate (technical replicates) from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates for simultaneous detection of cell surface markers (Fig. 2). Panel 1: MUC1-FITC (BD), HER2-PE (BD) and the LIVE/DEAD® red cell viability stain (Invitrogen); Panel 2: CD49f-Pacific Blue (Biolegend), Aldefluor® assay reagent (StemCell Technologies), CD24-PE (BD), LIVE/DEAD® red, AC133-APC (Milltenyi), EpCAM-PerCP Cy5.5 (BD) and CD44-APC-Cy7 (Biolegend). Staining with the Aldefluor® assay for detection of ALDH1 activity was done prior to the addition of fluorescent antibodies according to the maufacturer’s instructions. Each Aldefluor-containing sample was prepared in duplicate, and the second negative control sample was immediately quenched in the ALDH1 inhibitor Diethylaminobenzaldehyde (DEAB) as per the manufacturer’s instructions.

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