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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 affect characteristics of cancer cell lines including proliferation and anchorage-independent growth. (A) W0069, W0069-228, and W0069-229 cells were grown for 72h and cell proliferation was assessed by MTT assay at 24h, 48h and 72h, respectively. (B) 5x104 cells for each cell line were grown in 6 well plates. Every 24h cells were collected and the number of cells was measured by using Z1 Coulter counter up to 8 days. (C, D) cells were grown either in soft agar (C) or in matrigel (D), respectively. Numbers represent the number of colonies in one out of three independent experiments. Characteristic pictures of colonies in both soft agar and matrigel are shown on the right.
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Figure 2: CD24+CD29+ cells affect characteristics of cancer cell lines including proliferation and anchorage-independent growth. (A) W0069, W0069-228, and W0069-229 cells were grown for 72h and cell proliferation was assessed by MTT assay at 24h, 48h and 72h, respectively. (B) 5x104 cells for each cell line were grown in 6 well plates. Every 24h cells were collected and the number of cells was measured by using Z1 Coulter counter up to 8 days. (C, D) cells were grown either in soft agar (C) or in matrigel (D), respectively. Numbers represent the number of colonies in one out of three independent experiments. Characteristic pictures of colonies in both soft agar and matrigel are shown on the right.

Mentions: The differences in the number of CD24+CD29+ cells in subclones led us to test whether there are any different phenotypes between these clones. Specifically, we were interested in characteristics related to cancer stem cell phenotype such as the ability to proliferate or grow under low attachment conditions either in liquid culture or in matrigel. The proliferation capacity correlated with the amount of double positive cells since both W0069 and W0069-228 cells proliferated much faster than W0069-229 cells (Fig. 2A, B). Furthermore, both W0069 and W0069-228 could form colonies in soft agar and matrigel, respectively, while no colonies were formed when the same number of W0069-229 cells was tested under identical experimental conditions (Fig. 2C, D). To determine the tumor-initiating capacity of the cell lines, we injected subclones containing different percentages of double positive cells to nude mice subcutaneously and monitored tumor formation. W0069-228 cells showed enhanced tumorigenicity compared to W0069-229 cells as indicated by measuring the tumor volume at different time points after injection (Fig. 3A). This was further confirmed by staining tumors with the proliferation marker Ki67, since it was shown that in W0069-228 tumors the majority of cells exhibited positive staining compared to the lower amount of Ki67 positive cells found in W0069-229 tumors (61±4.9% vs. 34±1.1%, respectively), suggesting that indeed W0069-228 cells are more tumorigenic than W0069-229 cells (Fig. 3B). The same results were obtained when the different cell lines were injected directly to the mammary fat pad of nude mice. After 106 cells of either W0069 or W0069-228 cells were injected, all mice formed tumors (6/6) in both cases after one month. On the other hand, when W0069-229 cells were injected, only half of the mice (3/6) formed tumors. In addition, tumors generated from W0069 and W0069-228 cells were much larger (>1cm) than tumors formed from the same number of W0069-229 cells (<0.5cm) when the tumors were measured one month after injection (Fig. 3C). Based on previous studies it is believed that tumors are composed of a heterogeneous population of cells, with differences in their phenotypic characteristics and proliferative potential, including cancer cells with indefinite proliferative potential and those with limited or no proliferative potential 11, 31. Thus, our data indicates that the presence of CD24+CD29+ cells may drive tumorigenicity of these cell lines, implying that this subpopulation shares the characteristics of cancer initiating 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)

CD24+CD29+ cells affect characteristics of cancer cell lines including proliferation and anchorage-independent growth. (A) W0069, W0069-228, and W0069-229 cells were grown for 72h and cell proliferation was assessed by MTT assay at 24h, 48h and 72h, respectively. (B) 5x104 cells for each cell line were grown in 6 well plates. Every 24h cells were collected and the number of cells was measured by using Z1 Coulter counter up to 8 days. (C, D) cells were grown either in soft agar (C) or in matrigel (D), respectively. Numbers represent the number of colonies in one out of three independent experiments. Characteristic pictures of colonies in both soft agar and matrigel are shown on the right.
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Related In: Results  -  Collection

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

Figure 2: CD24+CD29+ cells affect characteristics of cancer cell lines including proliferation and anchorage-independent growth. (A) W0069, W0069-228, and W0069-229 cells were grown for 72h and cell proliferation was assessed by MTT assay at 24h, 48h and 72h, respectively. (B) 5x104 cells for each cell line were grown in 6 well plates. Every 24h cells were collected and the number of cells was measured by using Z1 Coulter counter up to 8 days. (C, D) cells were grown either in soft agar (C) or in matrigel (D), respectively. Numbers represent the number of colonies in one out of three independent experiments. Characteristic pictures of colonies in both soft agar and matrigel are shown on the right.
Mentions: The differences in the number of CD24+CD29+ cells in subclones led us to test whether there are any different phenotypes between these clones. Specifically, we were interested in characteristics related to cancer stem cell phenotype such as the ability to proliferate or grow under low attachment conditions either in liquid culture or in matrigel. The proliferation capacity correlated with the amount of double positive cells since both W0069 and W0069-228 cells proliferated much faster than W0069-229 cells (Fig. 2A, B). Furthermore, both W0069 and W0069-228 could form colonies in soft agar and matrigel, respectively, while no colonies were formed when the same number of W0069-229 cells was tested under identical experimental conditions (Fig. 2C, D). To determine the tumor-initiating capacity of the cell lines, we injected subclones containing different percentages of double positive cells to nude mice subcutaneously and monitored tumor formation. W0069-228 cells showed enhanced tumorigenicity compared to W0069-229 cells as indicated by measuring the tumor volume at different time points after injection (Fig. 3A). This was further confirmed by staining tumors with the proliferation marker Ki67, since it was shown that in W0069-228 tumors the majority of cells exhibited positive staining compared to the lower amount of Ki67 positive cells found in W0069-229 tumors (61±4.9% vs. 34±1.1%, respectively), suggesting that indeed W0069-228 cells are more tumorigenic than W0069-229 cells (Fig. 3B). The same results were obtained when the different cell lines were injected directly to the mammary fat pad of nude mice. After 106 cells of either W0069 or W0069-228 cells were injected, all mice formed tumors (6/6) in both cases after one month. On the other hand, when W0069-229 cells were injected, only half of the mice (3/6) formed tumors. In addition, tumors generated from W0069 and W0069-228 cells were much larger (>1cm) than tumors formed from the same number of W0069-229 cells (<0.5cm) when the tumors were measured one month after injection (Fig. 3C). Based on previous studies it is believed that tumors are composed of a heterogeneous population of cells, with differences in their phenotypic characteristics and proliferative potential, including cancer cells with indefinite proliferative potential and those with limited or no proliferative potential 11, 31. Thus, our data indicates that the presence of CD24+CD29+ cells may drive tumorigenicity of these cell lines, implying that this subpopulation shares the characteristics of cancer initiating 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