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

Additional file 1:

File providing a list of oligonucleotide primer sequences for the mouse ABC transporters and plasma membrane calcium ATPase 4 (housekeeping gene) for quantitative real-time RT-PCR.

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Additional file 2:

File showing the morphologic appearance of all 16 cell lines developed from five original independent Brca1 mammary tumors (A1, B, P3, P2, and RP) grown in monolayer.

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Additional file 3:

File showing that expression of putative stem cell markers (CD44⁺/CD24^- and CD133⁺) occurs on distinct and non-overlapping cell populations. (A) A1.8 cells stained simultaneously with antibodies for CD44, CD24, and CD133 (upper panel). The lower panel shows compensated dual staining for CD44/CD133, CD44/CD24, and triple staining for all three markers. Only 0.02% of A1.8 cells express all 3 markers. (B) RP.1 cells were stained and analyzed as above. No cells bearing all three markers are detectable. One of two independent analyses is shown here.

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Additional file 4:

File showing that RP.1 cells growing as spheroids in the absence of attachment are enriched in CD133⁺ cells. (A) Parental cells and (B) cells dissociated from spheroids after expanding for four passages in vitro were stained side-by-side for CD133 and examined by fluorescence-activated cell sorting. The percentage of CD133⁺ cells is indicated in each box. Note that a distinct CD133^High population is now evident in spheroid-derived cells. One of three independent experiments is shown here.

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Additional file 5:

File showing the morphologic appearance of unsorted cells plated in the absence of attachment from six cell lines that represent five individual tumors. A1.1, A1.8, B.15, P3.17, P2.1, and RP.1 cells were grown in 96-well low-binding plates for 2 weeks, dispersed into single cells, and expanded in six-well low-binding plates. One of more than three independent experiments is shown here.

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Additional file 6:

File showing differences in frequency of CD44/CD24 cells in A1.8 cell line that were growing in monolayer as compared to spheroids. (A) Fluorescence-activated cell sorting (FACS) analysis of stem cell markers from unsorted A1.8 parental cells is compared with SC⁺ (CD44⁺/CD24^-) and SC^- (CD44^-/CD24⁺) cells sorted by FACS after growing as monolayers in the third passage (P.3). (B) RP.1 parental and CD133⁺ and CD133^- cells sorted and passaged as monolayer twice (P.2) before analysis. One of three independent experiments is shown.

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Additional file 7:

File showing the sensitivity of Brca1 cell lines to doxorubicin, cisplatin, and the HSP90 inhibitor 17-DMAG. Cytotoxicity is determined by MTS assay for four representative Brca1 cell lines: A1.8, P3.17, B.15, and RP.1. Cells were exposed to increasing concentrations of (A) doxorubicin, (B) cisplatin, and (C) the HSP90 inhibitor 17-DMAG. Percentage survival (± standard deviation from six replicate wells) after 24 hours of exposure to drugs is represented by open symbols and dotted lines, and after 48 hours by solid symbols and lines. The ordinate shows concentrations of individual drugs. One of three independent experiments for each cell type is shown here.

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Additional file 8:

File showing the differences in expression of ABC transporters, Abcg2 and Abcb1, detected among the cell lines and parental tumors. (A) Expression of Abcg2 among six Brca1 cell lines. (B) Expression of Abcb1 in five cell lines that represent each one of the five independent tumors. Relative (C) Abcb1 and (D) Abcg2 expression in parental cells, cells sorted for respective stem cell markers, and unsorted cells growing as spheroids. Expression of each transporter is normalized to Pmca4 housekeeping gene, as described in Materials and methods. The bars represent ± standard deviation from triplicate samples. One of three independent experiments is shown.

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Additional file 9:

File showing estrogen receptor (ESR)1 expression in individual cell lines and normal mouse mammary gland from 8-week-old C57BL6 mice, as determined by quantitative RT-PCR. The data were calculated using the ΔΔCT method from duplicate samples, in which the expression in each sample run was compared with expression in mammary gland, averaged, and normalized to cyclophilin, which was used as a housekeeping gene.

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