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Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells.

Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero-Stanke L, Taylor-Papadimitriou J, Burchell JM - Breast Cancer Res. (2008)

Bottom Line: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged.The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres.Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Breast Cancer Biology Group, King's College London School of Medicine, Guy's Hospital Campus, Great Maze Pond, London, UK.

ABSTRACT

Introduction: The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44).

Methods: We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented.

Results: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44.

Conclusion: This paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic 'stem' cells to therapeutic agents and for further characterisation of the tumour-inducing subpopulation of breast cancer cells.

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CD24 expression in mammospheres. (a) Mammospheres from pleural effusion PE14 and (b) cells from disrupted PE14 mammospheres differentiated in the presence of serum were stained for CD24 expression using the SWA11 antibody, and binding was visualised using rabbit anti-mouse Alexa 488-conjugated antibody (left panels). Right panels are the same cells stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride). PE, pleural effusion.
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Figure 4: CD24 expression in mammospheres. (a) Mammospheres from pleural effusion PE14 and (b) cells from disrupted PE14 mammospheres differentiated in the presence of serum were stained for CD24 expression using the SWA11 antibody, and binding was visualised using rabbit anti-mouse Alexa 488-conjugated antibody (left panels). Right panels are the same cells stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride). PE, pleural effusion.

Mentions: The flow profile of the PE14 sample was particularly striking as surface expression of CD24 and CD44 by the uncultured cells was uniformly negative (Figure 3). Moreover, mammospheres from this sample were large and could be extensively passaged. Staining of PE14 mammospheres for CD24 with either the SWA11 or ML5 antibody showed that CD24 was low or absent (Figure 4a), as was also observed by Ponti and colleagues [10] in mammospheres cultured from recurrent breast cancer or MCF7 cells. As with the lineage markers, expression was increased upon differentiation (Figure 4b).


Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells.

Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero-Stanke L, Taylor-Papadimitriou J, Burchell JM - Breast Cancer Res. (2008)

CD24 expression in mammospheres. (a) Mammospheres from pleural effusion PE14 and (b) cells from disrupted PE14 mammospheres differentiated in the presence of serum were stained for CD24 expression using the SWA11 antibody, and binding was visualised using rabbit anti-mouse Alexa 488-conjugated antibody (left panels). Right panels are the same cells stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride). PE, pleural effusion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481500&req=5

Figure 4: CD24 expression in mammospheres. (a) Mammospheres from pleural effusion PE14 and (b) cells from disrupted PE14 mammospheres differentiated in the presence of serum were stained for CD24 expression using the SWA11 antibody, and binding was visualised using rabbit anti-mouse Alexa 488-conjugated antibody (left panels). Right panels are the same cells stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride). PE, pleural effusion.
Mentions: The flow profile of the PE14 sample was particularly striking as surface expression of CD24 and CD44 by the uncultured cells was uniformly negative (Figure 3). Moreover, mammospheres from this sample were large and could be extensively passaged. Staining of PE14 mammospheres for CD24 with either the SWA11 or ML5 antibody showed that CD24 was low or absent (Figure 4a), as was also observed by Ponti and colleagues [10] in mammospheres cultured from recurrent breast cancer or MCF7 cells. As with the lineage markers, expression was increased upon differentiation (Figure 4b).

Bottom Line: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged.The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres.Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Breast Cancer Biology Group, King's College London School of Medicine, Guy's Hospital Campus, Great Maze Pond, London, UK.

ABSTRACT

Introduction: The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44).

Methods: We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented.

Results: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44.

Conclusion: This paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic 'stem' cells to therapeutic agents and for further characterisation of the tumour-inducing subpopulation of breast cancer cells.

Show MeSH
Related in: MedlinePlus