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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 by pleural effusion cells as detected by antibodies SWA11 and ML5. Cells isolated from pleural effusions were stained with unconjugated SWA11 and ML5, and antibody binding was detected by fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody (see Materials and methods). Filled histograms, 2° antibody only; thin grey line, SWA11; thick black line, ML5. PE, pleural effusion.
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Figure 2: CD24 expression by pleural effusion cells as detected by antibodies SWA11 and ML5. Cells isolated from pleural effusions were stained with unconjugated SWA11 and ML5, and antibody binding was detected by fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody (see Materials and methods). Filled histograms, 2° antibody only; thin grey line, SWA11; thick black line, ML5. PE, pleural effusion.

Mentions: A CD44+ CD24low/- population was isolated from breast tumours and pleural effusions by Al-Hajj and colleagues [12] and these cells were found to be enriched with tumorigenic cells. In these experiments, cells were sometimes gated for epithelial specific antigen-positive (ESA+) cells, and there was a suggestion that the ESA+ subset of CD44+/CD24low/- cells were more tumorigenic. Using the same CD24 antibody used by Al Hajj and colleagues [12] (ML5) and the same CD44 antibody, we found that only some pleural effusion samples yielded subfractions of cells and these could be detected whether or not the cells were first gated as ESA+ (data not shown). We therefore proceeded to analyse the samples without gating for ESA. We also checked whether there was any difference in staining for CD24 using the SWA11 antibody, which is reactive with all glycoforms of CD24, and ML5, the antibody used by Ponti and colleagues [10] and Al-Hajj and colleagues [12]. Figure 2 shows that, for six pleural effusion samples, we found that SWA11 did not give a stronger signal or detect more CD24+ cells. All the samples were therefore analysed using the phycoerythrin-conjugated ML5 antibody (see Materials and methods).


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 by pleural effusion cells as detected by antibodies SWA11 and ML5. Cells isolated from pleural effusions were stained with unconjugated SWA11 and ML5, and antibody binding was detected by fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody (see Materials and methods). Filled histograms, 2° antibody only; thin grey line, SWA11; thick black line, ML5. PE, pleural effusion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: CD24 expression by pleural effusion cells as detected by antibodies SWA11 and ML5. Cells isolated from pleural effusions were stained with unconjugated SWA11 and ML5, and antibody binding was detected by fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody (see Materials and methods). Filled histograms, 2° antibody only; thin grey line, SWA11; thick black line, ML5. PE, pleural effusion.
Mentions: A CD44+ CD24low/- population was isolated from breast tumours and pleural effusions by Al-Hajj and colleagues [12] and these cells were found to be enriched with tumorigenic cells. In these experiments, cells were sometimes gated for epithelial specific antigen-positive (ESA+) cells, and there was a suggestion that the ESA+ subset of CD44+/CD24low/- cells were more tumorigenic. Using the same CD24 antibody used by Al Hajj and colleagues [12] (ML5) and the same CD44 antibody, we found that only some pleural effusion samples yielded subfractions of cells and these could be detected whether or not the cells were first gated as ESA+ (data not shown). We therefore proceeded to analyse the samples without gating for ESA. We also checked whether there was any difference in staining for CD24 using the SWA11 antibody, which is reactive with all glycoforms of CD24, and ML5, the antibody used by Ponti and colleagues [10] and Al-Hajj and colleagues [12]. Figure 2 shows that, for six pleural effusion samples, we found that SWA11 did not give a stronger signal or detect more CD24+ cells. All the samples were therefore analysed using the phycoerythrin-conjugated ML5 antibody (see Materials and methods).

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