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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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Double positive cells grow in three-dimensional cultures and form well differentiated structures. (A) Immediately after sorting, CD24+CD29+ and CD24-CD29- cells were grown in matrigel with varying number of cells per well. Numbers in table represent the number of colonies in one out of three independent experiments. (B) Pictures showing colonies formed when 1000 cells were inoculated in matrigel. Note, only one colony (arrow) was formed from 1000 double negative cells. (C) Characteristic colonies are shown in higher magnification. Arrows show differentiated structures as well as complex tubular structures formed from double positive cells grown in three dimensions.
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Figure 5: Double positive cells grow in three-dimensional cultures and form well differentiated structures. (A) Immediately after sorting, CD24+CD29+ and CD24-CD29- cells were grown in matrigel with varying number of cells per well. Numbers in table represent the number of colonies in one out of three independent experiments. (B) Pictures showing colonies formed when 1000 cells were inoculated in matrigel. Note, only one colony (arrow) was formed from 1000 double negative cells. (C) Characteristic colonies are shown in higher magnification. Arrows show differentiated structures as well as complex tubular structures formed from double positive cells grown in three dimensions.

Mentions: As mentioned earlier, one of the hallmarks of cancer stem cells is their ability to grow and form colonies under anchorage-independent conditions. Thus, in order to check whether double positive cells are enriched for cancer initiating cells, sorted CD24+CD29+ cells were grown in matrigel. As shown in figure 5A, and 5B, double positive cells exhibited enhanced colony-forming ability, whereas no colonies were formed when 500, 200, or 100 double negative cells were grown under the same experimental conditions. It was shown that epithelial cells grown in three-dimensional cultures recapitulate numerous features of breast epithelium in vivo, including the formation of acini-like spheroids, apicobasal polarization of cells, basal deposition of basement membrane components (collagen IV and laminin V), and in some cases, the production of milk proteins 32, 33. Since the cancer cell lines used were from mammary epithelial cancers, we wanted to investigate whether these cells may differentiate when cultured in three-dimensional cultures. Indeed we found that CD24+CD29+ cells exhibited an expanded differentiative repertoire compared to the double negative cells. While no differentiated structures were found in colonies formed by double negative cells, double positive cells could differentiate into complex tubular and branched structures reminiscent of the mammary ductal tree (Fig. 5C). Previous studies reported that normal mammary stem cells have the ability to recapitulate the architecture of the mammary gland 15, 34. Since this subpopulation of cancer cells maintains the ability to differentiate in three-dimensional culture systems, it is possible that CD24+CD29+ cells share some of the characteristics of normal mammary stem cells.


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)

Double positive cells grow in three-dimensional cultures and form well differentiated structures. (A) Immediately after sorting, CD24+CD29+ and CD24-CD29- cells were grown in matrigel with varying number of cells per well. Numbers in table represent the number of colonies in one out of three independent experiments. (B) Pictures showing colonies formed when 1000 cells were inoculated in matrigel. Note, only one colony (arrow) was formed from 1000 double negative cells. (C) Characteristic colonies are shown in higher magnification. Arrows show differentiated structures as well as complex tubular structures formed from double positive cells grown in three dimensions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2367429&req=5

Figure 5: Double positive cells grow in three-dimensional cultures and form well differentiated structures. (A) Immediately after sorting, CD24+CD29+ and CD24-CD29- cells were grown in matrigel with varying number of cells per well. Numbers in table represent the number of colonies in one out of three independent experiments. (B) Pictures showing colonies formed when 1000 cells were inoculated in matrigel. Note, only one colony (arrow) was formed from 1000 double negative cells. (C) Characteristic colonies are shown in higher magnification. Arrows show differentiated structures as well as complex tubular structures formed from double positive cells grown in three dimensions.
Mentions: As mentioned earlier, one of the hallmarks of cancer stem cells is their ability to grow and form colonies under anchorage-independent conditions. Thus, in order to check whether double positive cells are enriched for cancer initiating cells, sorted CD24+CD29+ cells were grown in matrigel. As shown in figure 5A, and 5B, double positive cells exhibited enhanced colony-forming ability, whereas no colonies were formed when 500, 200, or 100 double negative cells were grown under the same experimental conditions. It was shown that epithelial cells grown in three-dimensional cultures recapitulate numerous features of breast epithelium in vivo, including the formation of acini-like spheroids, apicobasal polarization of cells, basal deposition of basement membrane components (collagen IV and laminin V), and in some cases, the production of milk proteins 32, 33. Since the cancer cell lines used were from mammary epithelial cancers, we wanted to investigate whether these cells may differentiate when cultured in three-dimensional cultures. Indeed we found that CD24+CD29+ cells exhibited an expanded differentiative repertoire compared to the double negative cells. While no differentiated structures were found in colonies formed by double negative cells, double positive cells could differentiate into complex tubular and branched structures reminiscent of the mammary ductal tree (Fig. 5C). Previous studies reported that normal mammary stem cells have the ability to recapitulate the architecture of the mammary gland 15, 34. Since this subpopulation of cancer cells maintains the ability to differentiate in three-dimensional culture systems, it is possible that CD24+CD29+ cells share some of the characteristics of normal mammary stem cells.

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