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Identification and characterization of cancer initiating cells from BRCA1 related mammary tumors using markers for normal mammary stem cells.

Vassilopoulos A, Wang RH, Petrovas C, Ambrozak D, Koup R, Deng CX - Int. J. Biol. Sci. (2008)

Bottom Line: The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer.Under low-attachment conditions, we detected "tumorspheres" only in the presence of double positive cells, which maintained their ability to self-renew.These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Genetics of Development, Disease Branch, National Institute of Diabetes, Digestive, Kidney Diseases, National Institutes of Health, Bethesda, Maryland, MD 20892, USA.

ABSTRACT
It is hypothesized that cancer stem cells arise either from normal stem cells or from progenitor cells that have gained the ability to self-renew. Here we determine whether mammary cancer stem cells can be isolated by using antibodies that have been used for the isolation of normal mammary stem cells. We show that BRCA1 mutant cancer cell lines contained a subpopulation of CD24+CD29+ or CD24+CD49f+ cells that exhibited increased proliferation and colony forming ability in vitro, and enhanced tumor-forming ability in vivo. The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer. Under low-attachment conditions, we detected "tumorspheres" only in the presence of double positive cells, which maintained their ability to self-renew. Furthermore, CD24+CD29+ cells could form tubular structures reminiscent of the mammary ductal tree when grown in three-dimensional cultures, implying that these cancer cells maintain some of the characteristics of the normal stem cells. Nevertheless, they could still drive tumor formation since as low as 500 double positive cells immediately after sorting from BRCA1 mutant primary tumors were able to form tumors with the same heterogeneity found in the original tumors. These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.

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Both BRCA1 cancer cell lines and BRCA1 primary tumors contain a distinct subpopulation of CD24+CD29+ cells. (A, B) representative flow cytometry analysis of stained (A) and unstained (B) W0069 cells with CD24-PE and CD29-FITC. (C) representative flow cytometry analysis of W0069 cells stained with CD24-PE and CD49f-FITC. (D) representative flow cytometry analysis of CD31-, CD45-, Ter119- (Lin-) mammary epithelial cells purified from BRCA1 mammary tumors using anti CD24-PE and anti CD29-FITC. (E, F) representative flow cytometry analysis of two different subclones of W0069 cell line after staining with antibodies against CD24 and CD29 [(subclone 228 (E), and subclone 229 (F)]. Numbers represent the percentage of each cell population. G) morphology of different cell lines under microscope.
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Figure 1: Both BRCA1 cancer cell lines and BRCA1 primary tumors contain a distinct subpopulation of CD24+CD29+ cells. (A, B) representative flow cytometry analysis of stained (A) and unstained (B) W0069 cells with CD24-PE and CD29-FITC. (C) representative flow cytometry analysis of W0069 cells stained with CD24-PE and CD49f-FITC. (D) representative flow cytometry analysis of CD31-, CD45-, Ter119- (Lin-) mammary epithelial cells purified from BRCA1 mammary tumors using anti CD24-PE and anti CD29-FITC. (E, F) representative flow cytometry analysis of two different subclones of W0069 cell line after staining with antibodies against CD24 and CD29 [(subclone 228 (E), and subclone 229 (F)]. Numbers represent the percentage of each cell population. G) morphology of different cell lines under microscope.

Mentions: We first tested whether CD24 and CD29, markers for normal mammary stem cells 15, 16, could be used to isolate CD24+CD29+ double positive populations from BRCA1 mutant tumor cells. We first performed flow cytometry analysis of two tumor cell lines derived from mammary tumors of Brca1Ko/Co;p53+/-;WAP-Cre mice 30. We identified a subpopulation of cells in both cancer cell lines that expressed cell surface markers CD24 and CD29 (Fig. 1A). The profile was similar when cells were stained with CD49f instead of CD29 (Fig. 1C). CD49f stains for α6-integrin, which forms heterodimers with β1-integrin (CD29) and it has also been shown before to enrich for mammary stem cells 16. We have also isolated a discrete subpopulation of CD24+CD29+ cells from BRCA1 mutant primary mammary tumors after using antibodies against well-characterized endothelial (CD31) and hematopoietic (CD45 and TER119) antigens to deplete these lineage positive cells followed by fluorescence-activated cell sorting (FACS) (Fig. 1D). The percentage of double positive cells varied among different BRCA1 cancer cell lines and primary tumors (12.8±2.5% in W0069, 16.7±5.3% in W780, and from 15-25% in the primary tumors).


Identification and characterization of cancer initiating cells from BRCA1 related mammary tumors using markers for normal mammary stem cells.

Vassilopoulos A, Wang RH, Petrovas C, Ambrozak D, Koup R, Deng CX - Int. J. Biol. Sci. (2008)

Both BRCA1 cancer cell lines and BRCA1 primary tumors contain a distinct subpopulation of CD24+CD29+ cells. (A, B) representative flow cytometry analysis of stained (A) and unstained (B) W0069 cells with CD24-PE and CD29-FITC. (C) representative flow cytometry analysis of W0069 cells stained with CD24-PE and CD49f-FITC. (D) representative flow cytometry analysis of CD31-, CD45-, Ter119- (Lin-) mammary epithelial cells purified from BRCA1 mammary tumors using anti CD24-PE and anti CD29-FITC. (E, F) representative flow cytometry analysis of two different subclones of W0069 cell line after staining with antibodies against CD24 and CD29 [(subclone 228 (E), and subclone 229 (F)]. Numbers represent the percentage of each cell population. G) morphology of different cell lines under microscope.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2367429&req=5

Figure 1: Both BRCA1 cancer cell lines and BRCA1 primary tumors contain a distinct subpopulation of CD24+CD29+ cells. (A, B) representative flow cytometry analysis of stained (A) and unstained (B) W0069 cells with CD24-PE and CD29-FITC. (C) representative flow cytometry analysis of W0069 cells stained with CD24-PE and CD49f-FITC. (D) representative flow cytometry analysis of CD31-, CD45-, Ter119- (Lin-) mammary epithelial cells purified from BRCA1 mammary tumors using anti CD24-PE and anti CD29-FITC. (E, F) representative flow cytometry analysis of two different subclones of W0069 cell line after staining with antibodies against CD24 and CD29 [(subclone 228 (E), and subclone 229 (F)]. Numbers represent the percentage of each cell population. G) morphology of different cell lines under microscope.
Mentions: We first tested whether CD24 and CD29, markers for normal mammary stem cells 15, 16, could be used to isolate CD24+CD29+ double positive populations from BRCA1 mutant tumor cells. We first performed flow cytometry analysis of two tumor cell lines derived from mammary tumors of Brca1Ko/Co;p53+/-;WAP-Cre mice 30. We identified a subpopulation of cells in both cancer cell lines that expressed cell surface markers CD24 and CD29 (Fig. 1A). The profile was similar when cells were stained with CD49f instead of CD29 (Fig. 1C). CD49f stains for α6-integrin, which forms heterodimers with β1-integrin (CD29) and it has also been shown before to enrich for mammary stem cells 16. We have also isolated a discrete subpopulation of CD24+CD29+ cells from BRCA1 mutant primary mammary tumors after using antibodies against well-characterized endothelial (CD31) and hematopoietic (CD45 and TER119) antigens to deplete these lineage positive cells followed by fluorescence-activated cell sorting (FACS) (Fig. 1D). The percentage of double positive cells varied among different BRCA1 cancer cell lines and primary tumors (12.8±2.5% in W0069, 16.7±5.3% in W780, and from 15-25% in the primary tumors).

Bottom Line: The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer.Under low-attachment conditions, we detected "tumorspheres" only in the presence of double positive cells, which maintained their ability to self-renew.These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Genetics of Development, Disease Branch, National Institute of Diabetes, Digestive, Kidney Diseases, National Institutes of Health, Bethesda, Maryland, MD 20892, USA.

ABSTRACT
It is hypothesized that cancer stem cells arise either from normal stem cells or from progenitor cells that have gained the ability to self-renew. Here we determine whether mammary cancer stem cells can be isolated by using antibodies that have been used for the isolation of normal mammary stem cells. We show that BRCA1 mutant cancer cell lines contained a subpopulation of CD24+CD29+ or CD24+CD49f+ cells that exhibited increased proliferation and colony forming ability in vitro, and enhanced tumor-forming ability in vivo. The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer. Under low-attachment conditions, we detected "tumorspheres" only in the presence of double positive cells, which maintained their ability to self-renew. Furthermore, CD24+CD29+ cells could form tubular structures reminiscent of the mammary ductal tree when grown in three-dimensional cultures, implying that these cancer cells maintain some of the characteristics of the normal stem cells. Nevertheless, they could still drive tumor formation since as low as 500 double positive cells immediately after sorting from BRCA1 mutant primary tumors were able to form tumors with the same heterogeneity found in the original tumors. These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.

Show MeSH
Related in: MedlinePlus