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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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Enhanced self-renewal capacity and tumor growth ability of CD24+CD29+ cells compared to double negative cells. (A) morphology of tumorspheres grown in ultra-low attachment plates from W0069 cells, CD24+CD29+, and CD24-CD29- cells in culture. CD24-CD29- cells can form small cell aggregates but they cannot form tumorspheres. (B) Quantification of tumorsphere initiating cells was done by performing limiting dilution assay after plating 1 cell/well in 96 well plates. (C) CD24+CD29+ and CD24-CD29- cells were sorted out from primary BRCA1 tumors. Immediately after sorting, 500 cells from each group were injected into the fourth mammary fat pad of nude mice. Denominators in the table represent the number of injections, and numerators represent the number of resultant tumors. Tumor growth rates were monitored as described in Materials and Methods and average tumor size is shown.
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Figure 6: Enhanced self-renewal capacity and tumor growth ability of CD24+CD29+ cells compared to double negative cells. (A) morphology of tumorspheres grown in ultra-low attachment plates from W0069 cells, CD24+CD29+, and CD24-CD29- cells in culture. CD24-CD29- cells can form small cell aggregates but they cannot form tumorspheres. (B) Quantification of tumorsphere initiating cells was done by performing limiting dilution assay after plating 1 cell/well in 96 well plates. (C) CD24+CD29+ and CD24-CD29- cells were sorted out from primary BRCA1 tumors. Immediately after sorting, 500 cells from each group were injected into the fourth mammary fat pad of nude mice. Denominators in the table represent the number of injections, and numerators represent the number of resultant tumors. Tumor growth rates were monitored as described in Materials and Methods and average tumor size is shown.

Mentions: Most stem cell enrichment protocols rely on immunosorting according to the cell surface expression of some proteins. Alternatively, it has been shown that mammospheres, which are spherical colonies formed by epithelial cells when cultured on non-adherent surfaces are enriched in stem/progenitor cells 35. Since cancer initiating cells resemble stem cells in their ability to grow as “tumorspheres” in culture, we wanted to check whether CD24+CD29+ cells maintain the potential to grow under conditions in which they cannot attach to a solid substratum. Whereas many cells underwent anoikis as expected, both parental and double positive cells were able to form “tumorspheres” after 3-4 days in culture (Fig. 6A). After enzymatic dissociation of these spheres, we could still get secondary “tumorspheres”, indicating that they are enriched for cancer initiating cells with self-renewal capacity. In contrast, the majority of double negative cells grown in suspension were non-viable floating cells with some cells growing as aggregates, which eventually deteriorated. To further confirm that these tumorspheres represented the progeny of an individual cell, we plated cells in limiting dilution with one cell, on the average, in each well of 96-well plates. Of note, we found that both parental and double positive cells had almost the same number of cells capable of giving rise to “tumorspheres” (419±96 vs. 391±128 per 10000 cells), while no tumorsphere was found in the double negative cells (Fig. 6B).


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)

Enhanced self-renewal capacity and tumor growth ability of CD24+CD29+ cells compared to double negative cells. (A) morphology of tumorspheres grown in ultra-low attachment plates from W0069 cells, CD24+CD29+, and CD24-CD29- cells in culture. CD24-CD29- cells can form small cell aggregates but they cannot form tumorspheres. (B) Quantification of tumorsphere initiating cells was done by performing limiting dilution assay after plating 1 cell/well in 96 well plates. (C) CD24+CD29+ and CD24-CD29- cells were sorted out from primary BRCA1 tumors. Immediately after sorting, 500 cells from each group were injected into the fourth mammary fat pad of nude mice. Denominators in the table represent the number of injections, and numerators represent the number of resultant tumors. Tumor growth rates were monitored as described in Materials and Methods and average tumor size is shown.
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Related In: Results  -  Collection

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

Figure 6: Enhanced self-renewal capacity and tumor growth ability of CD24+CD29+ cells compared to double negative cells. (A) morphology of tumorspheres grown in ultra-low attachment plates from W0069 cells, CD24+CD29+, and CD24-CD29- cells in culture. CD24-CD29- cells can form small cell aggregates but they cannot form tumorspheres. (B) Quantification of tumorsphere initiating cells was done by performing limiting dilution assay after plating 1 cell/well in 96 well plates. (C) CD24+CD29+ and CD24-CD29- cells were sorted out from primary BRCA1 tumors. Immediately after sorting, 500 cells from each group were injected into the fourth mammary fat pad of nude mice. Denominators in the table represent the number of injections, and numerators represent the number of resultant tumors. Tumor growth rates were monitored as described in Materials and Methods and average tumor size is shown.
Mentions: Most stem cell enrichment protocols rely on immunosorting according to the cell surface expression of some proteins. Alternatively, it has been shown that mammospheres, which are spherical colonies formed by epithelial cells when cultured on non-adherent surfaces are enriched in stem/progenitor cells 35. Since cancer initiating cells resemble stem cells in their ability to grow as “tumorspheres” in culture, we wanted to check whether CD24+CD29+ cells maintain the potential to grow under conditions in which they cannot attach to a solid substratum. Whereas many cells underwent anoikis as expected, both parental and double positive cells were able to form “tumorspheres” after 3-4 days in culture (Fig. 6A). After enzymatic dissociation of these spheres, we could still get secondary “tumorspheres”, indicating that they are enriched for cancer initiating cells with self-renewal capacity. In contrast, the majority of double negative cells grown in suspension were non-viable floating cells with some cells growing as aggregates, which eventually deteriorated. To further confirm that these tumorspheres represented the progeny of an individual cell, we plated cells in limiting dilution with one cell, on the average, in each well of 96-well plates. Of note, we found that both parental and double positive cells had almost the same number of cells capable of giving rise to “tumorspheres” (419±96 vs. 391±128 per 10000 cells), while no tumorsphere was found in the double negative cells (Fig. 6B).

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