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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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Differences in the frequencies of functional mammary epithelial subpopulations in parallel sphere and adherent cultures of breast cancer cell lines assessed by multiparametric flow cytometry analysis.Adherent and sphere cultures were dissociated, stained with fluorescent antibody conjugates and analysed as described in Fig. 2. Heat maps show changes in subpopulation frequencies within the live cell population. Red shades = higher frequency in spheres compared to matched adherent cultures; blue shades = lower frequency in spheres; empty = not consistent and/or not statistically significant across biological replicates; diagonal line = not determined. Statistical significance was determined using two-way ANOVA and 2-tailed, paired t-tests. Results are depicted only where the trend was directionally consistent and statistically significant over biological replicates (refer to methods). Significance levels are colour-coded: light pink/blue: P = 0.05–0.001; mid-pink/blue: P = 0.001–0.0001; dark red/blue: P<0.0001). Dotted boxes indicate changes of interest: significant enrichment with AC133+ cells in 3/6 cell lines (A), and frequent shifts in CD49f+/CD24+ phenotypes with sphere culture (B).
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pone-0064388-g003: Differences in the frequencies of functional mammary epithelial subpopulations in parallel sphere and adherent cultures of breast cancer cell lines assessed by multiparametric flow cytometry analysis.Adherent and sphere cultures were dissociated, stained with fluorescent antibody conjugates and analysed as described in Fig. 2. Heat maps show changes in subpopulation frequencies within the live cell population. Red shades = higher frequency in spheres compared to matched adherent cultures; blue shades = lower frequency in spheres; empty = not consistent and/or not statistically significant across biological replicates; diagonal line = not determined. Statistical significance was determined using two-way ANOVA and 2-tailed, paired t-tests. Results are depicted only where the trend was directionally consistent and statistically significant over biological replicates (refer to methods). Significance levels are colour-coded: light pink/blue: P = 0.05–0.001; mid-pink/blue: P = 0.001–0.0001; dark red/blue: P<0.0001). Dotted boxes indicate changes of interest: significant enrichment with AC133+ cells in 3/6 cell lines (A), and frequent shifts in CD49f+/CD24+ phenotypes with sphere culture (B).

Mentions: Raw fluorescence data was collected on a FACSAria I flow cytometer (Becton Dickinson) using FACSDiva acquisition software (v6.1.3; BD). Particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity. 1×104 Events that fell within the live cell gate were collected for each sample. Manual fluorescence compensation was performed on each occasion, then retrospectively checked and modified if necessary using FCS Express analysis software (v4.0; DeNovo Software). For each experiment, gates were placed based on unstained adherent or sphere control samples to account for variations in the autofluorescence of cells grown in the different formats. For Aldefluor®-stained samples, gates were placed based on the fluorescence of parallel samples stained with the ALDH1 inhibitor, DEAB. Population frequencies were determined for individual parameters. For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (Figure 3/4). Samples stained with the relevant isotype controls were checked to ensure best gate placement, and ‘fluorescence minus one’ (FMO) controls were checked to ensure the fluorescent panels chosen could be accurately compensated.


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)

Differences in the frequencies of functional mammary epithelial subpopulations in parallel sphere and adherent cultures of breast cancer cell lines assessed by multiparametric flow cytometry analysis.Adherent and sphere cultures were dissociated, stained with fluorescent antibody conjugates and analysed as described in Fig. 2. Heat maps show changes in subpopulation frequencies within the live cell population. Red shades = higher frequency in spheres compared to matched adherent cultures; blue shades = lower frequency in spheres; empty = not consistent and/or not statistically significant across biological replicates; diagonal line = not determined. Statistical significance was determined using two-way ANOVA and 2-tailed, paired t-tests. Results are depicted only where the trend was directionally consistent and statistically significant over biological replicates (refer to methods). Significance levels are colour-coded: light pink/blue: P = 0.05–0.001; mid-pink/blue: P = 0.001–0.0001; dark red/blue: P<0.0001). Dotted boxes indicate changes of interest: significant enrichment with AC133+ cells in 3/6 cell lines (A), and frequent shifts in CD49f+/CD24+ phenotypes with sphere culture (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672101&req=5

pone-0064388-g003: Differences in the frequencies of functional mammary epithelial subpopulations in parallel sphere and adherent cultures of breast cancer cell lines assessed by multiparametric flow cytometry analysis.Adherent and sphere cultures were dissociated, stained with fluorescent antibody conjugates and analysed as described in Fig. 2. Heat maps show changes in subpopulation frequencies within the live cell population. Red shades = higher frequency in spheres compared to matched adherent cultures; blue shades = lower frequency in spheres; empty = not consistent and/or not statistically significant across biological replicates; diagonal line = not determined. Statistical significance was determined using two-way ANOVA and 2-tailed, paired t-tests. Results are depicted only where the trend was directionally consistent and statistically significant over biological replicates (refer to methods). Significance levels are colour-coded: light pink/blue: P = 0.05–0.001; mid-pink/blue: P = 0.001–0.0001; dark red/blue: P<0.0001). Dotted boxes indicate changes of interest: significant enrichment with AC133+ cells in 3/6 cell lines (A), and frequent shifts in CD49f+/CD24+ phenotypes with sphere culture (B).
Mentions: Raw fluorescence data was collected on a FACSAria I flow cytometer (Becton Dickinson) using FACSDiva acquisition software (v6.1.3; BD). Particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity. 1×104 Events that fell within the live cell gate were collected for each sample. Manual fluorescence compensation was performed on each occasion, then retrospectively checked and modified if necessary using FCS Express analysis software (v4.0; DeNovo Software). For each experiment, gates were placed based on unstained adherent or sphere control samples to account for variations in the autofluorescence of cells grown in the different formats. For Aldefluor®-stained samples, gates were placed based on the fluorescence of parallel samples stained with the ALDH1 inhibitor, DEAB. Population frequencies were determined for individual parameters. For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (Figure 3/4). Samples stained with the relevant isotype controls were checked to ensure best gate placement, and ‘fluorescence minus one’ (FMO) controls were checked to ensure the fluorescent panels chosen could be accurately compensated.

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