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

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.

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.

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.

Figure 3

CD24 and CD44 expression by pleural effusion cells. Cells isolated from pleural effusions were analysed for their expression of CD44 and CD24 by flow cytometry using antibody clone G44-26 conjugated to fluorescein isothiocyanate to detect CD44 and using the ML5 antibody conjugated to phycoerythrin to detect CD24 (see Materials and methods). Control analyses were performed with an isotype-matched antibody. This is shown for the sample PE14 (Iso) along with the analysis with CD44 and CD24 antibodies (Test). For the other samples, the lines to form the quadrants were applied based on the analysis with the control antibody. The percentages in the right bottom quadrant refer to the CD44⁺ CD24^low/- population. PE, pleural effusion.

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.

Figure 5

Mammosphere-derived cells from pleural effusions are tumorigenic. Five thousand uncultured or mammosphere-derived cells were injected into severe combined immunodeficiency disease mice, and tumour development was monitored. Survival graphs for PE14 (a) and PE66 (b) are shown. (c) Survival curves of mice injected with different numbers of cells (as indicated) derived from PE14 mammospheres. (d) Tumours derived from cells isolated from PE14 mammosphere cultures were removed, fixed, sectioned, and stained for CD24 (SWA11), MUC1, and CK14 and CK19 expression, as described in Materials and methods. CK, cytokeratin; H&E, haematoxylin and eosin; ms, mammosphere; PE, pleural effusion.