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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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CD24+CD29+ cells maintain their ability to differentiate. CD24+CD29+ cells were sorted out from the W0069 cell line and they were cultured as monolayers. After 3 passages, flow cytometry analysis was performed using anti CD24-PE and anti CD29-FITC (A) or anti CD24-PE and anti 49f-FITC (B). Same strategy was followed in the case of CD24-CD29- sorted out cells (C, D). Representative data after flow cytometry are shown. (E) morphology of double positive or double negative cell in culture under microscope.
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Figure 4: CD24+CD29+ cells maintain their ability to differentiate. CD24+CD29+ cells were sorted out from the W0069 cell line and they were cultured as monolayers. After 3 passages, flow cytometry analysis was performed using anti CD24-PE and anti CD29-FITC (A) or anti CD24-PE and anti 49f-FITC (B). Same strategy was followed in the case of CD24-CD29- sorted out cells (C, D). Representative data after flow cytometry are shown. (E) morphology of double positive or double negative cell in culture under microscope.

Mentions: To provide further evidence to our hypothesis, we sorted out CD24 and CD29 double positive or negative cells and compared their ability for growth, differentiation, and tumorigenesis. After 2-3 passages in conventional tissue culture dishes, the sorted CD24+CD29+ cells were able to differentiate and reconstitute the heterogeneity found in the parental cell line, as revealed by FACS using markers for either CD24/CD29 (Fig. 4A) or CD24/CD49f (Fig. 4B). In contrast, the sorted CD24-CD29- cells could not differentiate and reconstitute the heterogeneity found in the W0069 cell line (Fig 4C, D). Notably, the heterogeneity of the double positive cells was again reflected in the morphology of the cells in culture (Fig. 4E), which was similar to the parental cell line. On the other hand, double negative cells in culture were homogeneous and looked very similar to the W0069-229 subclone. Thus, expansion of double positive cells in a monolayer leads to reconstitution of all different subpopulations found in parental cells, whereas cells depleted of double positive cells were unable to repopulate the CD24+CD29+ fraction.


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)

CD24+CD29+ cells maintain their ability to differentiate. CD24+CD29+ cells were sorted out from the W0069 cell line and they were cultured as monolayers. After 3 passages, flow cytometry analysis was performed using anti CD24-PE and anti CD29-FITC (A) or anti CD24-PE and anti 49f-FITC (B). Same strategy was followed in the case of CD24-CD29- sorted out cells (C, D). Representative data after flow cytometry are shown. (E) morphology of double positive or double negative cell in culture under microscope.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: CD24+CD29+ cells maintain their ability to differentiate. CD24+CD29+ cells were sorted out from the W0069 cell line and they were cultured as monolayers. After 3 passages, flow cytometry analysis was performed using anti CD24-PE and anti CD29-FITC (A) or anti CD24-PE and anti 49f-FITC (B). Same strategy was followed in the case of CD24-CD29- sorted out cells (C, D). Representative data after flow cytometry are shown. (E) morphology of double positive or double negative cell in culture under microscope.
Mentions: To provide further evidence to our hypothesis, we sorted out CD24 and CD29 double positive or negative cells and compared their ability for growth, differentiation, and tumorigenesis. After 2-3 passages in conventional tissue culture dishes, the sorted CD24+CD29+ cells were able to differentiate and reconstitute the heterogeneity found in the parental cell line, as revealed by FACS using markers for either CD24/CD29 (Fig. 4A) or CD24/CD49f (Fig. 4B). In contrast, the sorted CD24-CD29- cells could not differentiate and reconstitute the heterogeneity found in the W0069 cell line (Fig 4C, D). Notably, the heterogeneity of the double positive cells was again reflected in the morphology of the cells in culture (Fig. 4E), which was similar to the parental cell line. On the other hand, double negative cells in culture were homogeneous and looked very similar to the W0069-229 subclone. Thus, expansion of double positive cells in a monolayer leads to reconstitution of all different subpopulations found in parental cells, whereas cells depleted of double positive cells were unable to repopulate the CD24+CD29+ fraction.

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