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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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Metastatic cells from pleural effusions isolated from breast cancer patients can form mammospheres. (a) Cells were isolated from pleural effusions (PEs) and placed in non-differentiating medium in non-adherent culture flasks (see Materials and methods). PE14, PE6, PE21, and PE8 show four representative cultures. (b) PE mammospheres were disrupted and the cells plated onto glass coverslips in medium supplemented with 1% foetal calf serum. After 5 days of adherent culture, the cells were stained with antibodies to MUC1 (HMFG2), CK5 (D5/6), CK14 (LL002), and CK19 (BA17). (c) Breast cancer cell lines were also placed in non-differentiating medium in non-adherent culture flasks. Mammospheres could be seen developing in MCF7 and SKBR3 cultures, while MDAMB231 produced loosely adhered clumps of cells.
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Figure 1: Metastatic cells from pleural effusions isolated from breast cancer patients can form mammospheres. (a) Cells were isolated from pleural effusions (PEs) and placed in non-differentiating medium in non-adherent culture flasks (see Materials and methods). PE14, PE6, PE21, and PE8 show four representative cultures. (b) PE mammospheres were disrupted and the cells plated onto glass coverslips in medium supplemented with 1% foetal calf serum. After 5 days of adherent culture, the cells were stained with antibodies to MUC1 (HMFG2), CK5 (D5/6), CK14 (LL002), and CK19 (BA17). (c) Breast cancer cell lines were also placed in non-differentiating medium in non-adherent culture flasks. Mammospheres could be seen developing in MCF7 and SKBR3 cultures, while MDAMB231 produced loosely adhered clumps of cells.

Mentions: Single-cell suspensions derived from pleural effusions from 27 breast cancer patients or ascites from 5 breast cancer patients were placed in mammosphere culture. Most of the samples had been frozen for more than 20 years, although five cultures were from fresh specimens of pleural effusions collected in 2006 (designated 06 followed by the PE number in Table 1). Twenty out of 27 pleural effusion samples (74%) produced viable mammospheres (20 to 100 μm) that could be cultured past passage 2 (Table 1). In many cases, these could be passaged further. However, no ascites samples produced viable mammospheres beyond a second passage (0/5). Figure 1a shows mammospheres cultured from four samples, and for comparison, the mammospheres produced by three breast cancer cell lines are shown in Figure 1c. Only the two epithelial cell lines produced discrete relatively large mammospheres, which could be passaged. The fibroblastic-like cell line MDA-MB-231 formed small loosely adherent structures, which, however, did survive passaging.


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)

Metastatic cells from pleural effusions isolated from breast cancer patients can form mammospheres. (a) Cells were isolated from pleural effusions (PEs) and placed in non-differentiating medium in non-adherent culture flasks (see Materials and methods). PE14, PE6, PE21, and PE8 show four representative cultures. (b) PE mammospheres were disrupted and the cells plated onto glass coverslips in medium supplemented with 1% foetal calf serum. After 5 days of adherent culture, the cells were stained with antibodies to MUC1 (HMFG2), CK5 (D5/6), CK14 (LL002), and CK19 (BA17). (c) Breast cancer cell lines were also placed in non-differentiating medium in non-adherent culture flasks. Mammospheres could be seen developing in MCF7 and SKBR3 cultures, while MDAMB231 produced loosely adhered clumps of cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Metastatic cells from pleural effusions isolated from breast cancer patients can form mammospheres. (a) Cells were isolated from pleural effusions (PEs) and placed in non-differentiating medium in non-adherent culture flasks (see Materials and methods). PE14, PE6, PE21, and PE8 show four representative cultures. (b) PE mammospheres were disrupted and the cells plated onto glass coverslips in medium supplemented with 1% foetal calf serum. After 5 days of adherent culture, the cells were stained with antibodies to MUC1 (HMFG2), CK5 (D5/6), CK14 (LL002), and CK19 (BA17). (c) Breast cancer cell lines were also placed in non-differentiating medium in non-adherent culture flasks. Mammospheres could be seen developing in MCF7 and SKBR3 cultures, while MDAMB231 produced loosely adhered clumps of cells.
Mentions: Single-cell suspensions derived from pleural effusions from 27 breast cancer patients or ascites from 5 breast cancer patients were placed in mammosphere culture. Most of the samples had been frozen for more than 20 years, although five cultures were from fresh specimens of pleural effusions collected in 2006 (designated 06 followed by the PE number in Table 1). Twenty out of 27 pleural effusion samples (74%) produced viable mammospheres (20 to 100 μm) that could be cultured past passage 2 (Table 1). In many cases, these could be passaged further. However, no ascites samples produced viable mammospheres beyond a second passage (0/5). Figure 1a shows mammospheres cultured from four samples, and for comparison, the mammospheres produced by three breast cancer cell lines are shown in Figure 1c. Only the two epithelial cell lines produced discrete relatively large mammospheres, which could be passaged. The fibroblastic-like cell line MDA-MB-231 formed small loosely adherent structures, which, however, did survive passaging.

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