Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics

Abstract

Introduction: Whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response is not known.

Methods: We generated and characterized 16 cell lines from five distinct Brca1deficient mouse mammary tumors with respect to their cancer stem cell characteristics.

Results: All cell lines derived from one tumor included increased numbers of CD44+/CD24- cells, which were previously identified as human breast cancer stem cells. All cell lines derived from another mammary tumor exhibited low levels of CD44+/CD24- cells, but they harbored 2% to 5.9% CD133+ cells, which were previously associated with cancer stem cells in other human and murine tumors. When plated in the absence of attachment without presorting, only those cell lines that were enriched in either stem cell marker formed spheroids, which were further enriched in cells expressing the respective cancer stem cell marker. In contrast, cells sorted for CD44+/CD24- or CD133+ markers lost their stem cell phenotype when cultured in monolayers. As few as 50 to 100 CD44+/CD24- or CD133+ sorted cells rapidly formed tumors in nonobese diabetic/severe combined immunodeficient mice, whereas 50-fold to 100-fold higher numbers of parental or stem cell depleted cells were required to form few, slow-growing tumors. Expression of stem cell associated genes, including Oct4, Notch1, Aldh1, Fgfr1, and Sox1, was increased in CD44+/CD24- and CD133+ cells. In addition, cells sorted for cancer stem cell markers and spheroid-forming cells were significantly more resistant to DNA-damaging drugs than were parental or stem cell depleted populations, and they were sensitized to the drugs by the heat shock protein-90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride).

Conclusion: Brca1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44+/CD24- cells represent a population that correlates with human breast cancer stem cells.

Figure 1

Expression of putative stem cell markers from cells derived from five individual Brca1 tumors. (a) Cell surface expression of CD44 is relatively uniform across all cell lines. The open histograms, outlined by a black line, represent positive staining for CD44, and gray filled histograms show negative controls stained with matched isotype antibody. (b) Expression of CD24 is variable, with highest level in B.15 cells. (c) There was substantial expression of CD133⁺ cells, accounting for 5.9% of the total population, among RP.1 cells. (d) Co-expression of CD44 and CD24 markers. The quadrants are gated to separate double positive, single positive, and double negative populations. The percentage of cells with CD44⁺/CD24^- markers is indicated at the upper left of each panel. The highest fraction is in cells derived from A1 tumor, as represented by A1.8 cells. One of more than three independent experiments is shown here.

Figure 2

Cells that express stem cell markers survive and form spheroid structures in the absence of attachment. (a) Frequency of spheroid formation is increased in Brca1 A1.8 cells sorted for SC⁺ (CD44⁺/CD24^-) as compared with SC^- (CD24⁺/CD44^-) populations. The results shown are from long-term spheroid assay performed by limiting dilution. The black bars represent numbers of spheroids formed by CD44⁺/CD24^- sorted cells/well and the white bars represent numbers from CD24⁺/CD44^- cells ± standard deviation from six replicate wells at the end of 2 weeks in culture. The numbers on the ordinate show the number of cells plated/well. One of three independent experiments is shown. (b) Unsorted Brca1 A1.8 cells plated in the absence of attachment form spheroids, which are enriched in cells expressing stem cell markers. A1.8 were plated as single cells in low binding plates for 2 to 3 weeks, and the resulting spheroids were expanded for 4 subsequent passages, dispersed into single cell population, and stained with appropriate antibodies (as described in Materials and methods). The CD44 and CD24 markers are shown as an open histogram on the left, and filled histograms show negative controls stained with matched isotype antibody. The single stain analysis is on the right panel; the double staining is shown on the bottom. Results are presented as percentage of CD44⁺/CD24^- cells from the total population. Note the appearance of a dual population that contains CD24^-/Low cells not evident in parental cells run side-by-side with the spheroid-derived cells and illustrated for the parental A1.8 cell line in Figure 1. One representative experiment from more than three is shown here.

Figure 3

Brca1 cells expressing stem cell markers and cells growing as spheroids are highly resistant to cisplatin. A1.8 parental cells, cells from dispersed spheroids, and cells sorted for Stem Cell + (CD44⁺/CD24^-) and Stem Cell – (CD44^-/CD24⁺) markers were treated simultaneously with increasing concentrations of cisplatin for 48 hours. (a) Parental A1.8 cells (solid symbols) compared with spheroid-derived cells (open symbols). (b) A1.8 cells sorted as Stem Cell + (open symbols) and Stem Cell – (solid symbols) populations. (c) Morphologic appearance of control untreated cells and cells exposed to 8 μmol/l cisplatin, as indicated on each panel. Note the formation of aggregates from spheroid-derived dispersed cells at the end of 72 hours in culture. One of three independent experiments is shown here.

Figure 4

17-DMAG is synergistic with cisplatin for Brca1 parental and cancer stem cells. Cells were treated with increasing concentrations of cisplatin, 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), or a combination of both drugs at a constant 1:3 ratio. (a) Simultaneous treatment for 48 hours. The open circles and dotted lines represent exposure to 17-DMAG, the filled circles and dotted lines represent exposure to cisplatin as single agents, and the filled squares linked by a solid line show combination. (b) Sequential addition of the drugs. The filled circles show exposure for 48 hours to cisplatin, the open circles and dotted lines represent 24 hours pf exposure to 17-DMAG, and the filled squares linked by a solid line show combination. The ordinate indicates concentrations of cisplatin, and the error bars represent ± standatd deviation from sextuplicates wells of one of more than three independent experiments. (c) Visual representation of Combination Index (CI) for each combination calculated using CalcuSyn Software. The closed circles represent simultaneous treatment and the open circles represent sequential addition of cisplatin followed by 17-DMAG. Values of CI below 1 indicate synergy, values above 1 represent antagonism, and CI = 1 corresponds to an additive effect. (d) 17-DMAG sensitizes parental and cancer stem cells to Cisplatin. CI for cisplatin and 17-DMAG added simultaneously (+) or sequentially (→) to parental cells or cells sorted for putative cancer stem cell markers. Data are derived from more than three independent experiments.

Figure 5

Cells sorted for expression of CD44⁺/CD24^- or CD133⁺ markers are enriched in tumor-initiating cells. Tumor initiating capacity was determined by implantation of 50, 100, 500 or 1,000 cells in mouse fat pad (MFP) #4 of nonobese diabetic/severe combined immunodeficient mice. (a) RP.1 CD133⁺ cells compared with RP.1 CD133^- cells, and (b) A1.8 CD44⁺/CD24^- cells compared with parental cells. Tumor growth rates were monitored, as described in Materials and methods, and average tumor size is shown for each cell type and cell number implanted based on triplicate implantations. (c) Total number of mice that grew tumors, as determined from independent experiments performed for each cell line. The percentages of positive tumors are indicated in parentheses.

Figure 6

Cells growing as spheroids express stem cell proteins. Immunofluorescence of spheroids formed by A1.8 and RP.1 cells after four passages were stained for Numb, Oct4, and CD133. Cells growing as spheroids were allowed to attach to an eight-well slide, fixed, and stained with indicated antibodies. Staining for Oct4 is visualized in both cell lines, whereas expression of Numb and CD1333 is evident only in RP.1 cells.