**2. Isolation and identification of breast cancer stem cells**

In most tumor tissues, including breast cancer, CSCs are rare. As we know, breast cancer is a histologically and molecularly heterogeneous disease, with six different subtypes, including luminal A, luminal B, normal breast-like, basal-like, claudinlow and HER2 overexpressing, which are characterized by distinct histology, gene expression patterns, and genetic alterations 32-35. The molecular heterogeneity between breast cancers has been revealed to issue from different targets of transformation. Recent studies found that basal-like breast cancers with BRCA1 mutations were more likely to arise from luminal progenitors rather than the basal stem cells 36, 37. However, further studies that focus on breast CSCs and mammary stem/progenitor cells as well as their potential relationship are needed for determining the exact origin of luminal versus basal-like cancers, with the aim of developing targeted therapies for different subtypes of breast cancers. Moreover, CSCs was found to be the main culprit for the failure of chemo- and radiation therapy, as well as the seeds for the distant metastasis and relapse in breast cancers 20, 32, 38-40. Taken together, in order to better understand the properties and biology of breast CSCs and eventually cure breast carcinoma, it is absolutely necessary and important to identify and separate breast CSCs prospectively.

#### **2.1 Isolation of breast CSCs with cell-surface marker profiles**

Since Dick, *et al* isolated a specific subpopulation of leukemia cells (that expressed surface markers similar to normal hematopoietic stem cells) which was consistently enriched for clonogenic activity in NOD/SCID immunocompromised mice from acute myeloid leukemias in the 1990s 5, 30, scientists attempted to see if they could enrich CSCs in human solid tumors by sorting for different cellular markers. CD24, a ligand for P-selectin in both mouse and human cells, was identified as a significant marker for human breast carcinoma invasion and metastasis 41, 42, and another adhesion molecular CD44 was found to correlate with cellular differentiation and lymph node metastasis in human breast cancers 43, 44, whereas B3.8 was described as a breast / ovarian cancer-specific marker 45. Based on these

Breast Cancer Stem Cells 277

studies showed that SP cells takes advantage of their ability to pump out the fluorescent dye Hoechst 33342 (H33342) through the ABCG2 (also known as breast cancer resistance protein-1), which was regarded as a major mediator of dye efflux in various stem cells 54, 62. As the ability to efflux substrates is particularly important for the protection of CSCs, and CSCs survive after chemotherapy partially by effluxing cytotoxic drugs, ABCG2 seems to protect stem cells from toxins. This is evident in *ABCG2* knockout mice that are more sensitive to compounds such as vinblastine, ivermectin, topotecan, and mitoxantrone 63-65. Taken together, SP cells have the capacity to efflux toxic substances out of breast cancer stem like cells via an ABCG2-mediated cytoprotective mechanism and seem to contribute to chemotherapy-resistance. In addition, it is important to consider that identification of cancer stem like cells by selecting for SP cells is not limited to breast carcinomas. Similar observations have been made in other solid tumors (such as glioma, ovarian and pancreatic cancers) where the isolated SP cells proliferated infinitely and could regenerate heterologous

**2.3 Propagation of breast CSCs by isolating ''mammospheres'' from suspension** 

researchers for isolating and studying the breast tumor–initiating cells (BT-IC) 72-74.

As we mentioned in the first part of this chapter, the cancer stem cell hypothesis suggests that many cancers are maintained in a hierarchical organization of rare, slowly dividing CSCs (or T-IC), rapidly dividing amplifying cells (early precursor cells, EPC) and postmitotic differentiated tumor cells 22. Thus, the complex scheme which operates in most tumor tissues seems to be that the slowly dividing CSCs give birth to EPC, which then undertake a program of exponential growth for a limited period of time before the descendant cells differentiate and become post-mitotic (Figure 1). Although the above three

**2.4 Novel strategies for enrichment of breast CSCs** 

Colonial growth in nonadherent culture was used to test for self-renewal capacity in cultures of neural cell in 1996, and in the experiment, suspension culture led to formation of "neurospheres", which consisted of 4% - 20% normal neural stem cells 69. Based on this approach, Galli *et al.* succeeded in the characterization and isolation from human glioblastoma multiform of "cancer neurospheres", which were highly enriched in long-term self-renewing, multi-lineage-differentiating, and tumor-initiating cells 70. According to these successful procedures, researchers tried to extend this technology to the identification and propagation of mammary epithelial stem cells and breast CSCs. In 2003, Dontu *et al.* demonstrated that nonadherent mammospheres are enriched in human mammary epithelial progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems 55. More encouragingly, two years later (2005), Ponti and colleagues reported the isolation and *in vitro* propagation of spherical clusters of self-replicating cells (''mammospheres'') with stem/progenitor cell properties in suspension cultures from three breast cancer lesions and from an established breast carcinoma cell line MCF-7 71. They found that the isolated cells which overexpressed neoangiogenic and cytoprotec-tive factors showed CD44+CD24- and Cx43-, and expressed the stem cell marker OCT-4, and could form tumors *in vivo* when as few as 103 cells were implanted. This was the first time showing that breast tumorigenic cells with stem/progenitor cell properties can be propagated *in vitro* as nonadherent mammospheres, and accordingly, this experimental system was then frequently used by

NSP cells in culture 59, 66-68.

**cultures** 

observations, in 2003, Al-Hajj *et al* tried to determine whether these surface markers could distinguish tumorigenic from nontumorigenic cells, and flow cytometry was used to isolate cells that were positive or negative for each marker. They demonstrated that a small population of tumorigenic cells, isolated from human breast tumors and characterized by the expression of the cell surface markers CD44+CD24−*/*lowLineage−, was capable of regenerating the phenotypic heterogeneity of the original tumor when injected subcutaneously into NOD/SCID mice 7. They showed that as few as 100 cells with CD44+CD24−/low phenotype could form tumors in immunodeficient mice, while thousands of cells with fungible phenotypes failed to do so. Since then, CD44 and CD24 are widely accepted as surface markers for breast CSCs, and lots of studies have focused on roles of CD44+CD24− tumor cells in breast cancers. For example, Abraham *et al.* conducted immunohistochemical studies of CD44+CD24− tumor cells in human breast tumors and showed that breast tumors containing a high proportion of CD44+CD24− cells were associated with distant metastases 46.

Nevertheless, besides CD24 and CD44, there are other surface marker candidates for the enrichment of breast CSCs. Ginestier *et al.* reported that they separated breast cancer stem/progenitor cells by sorting for Aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme responsible for the oxidation of intracellular aldehydes 47, 48, and they found that fewer ALDH1-positive than CD44+CD24− tumor cells are required to produce tumors in immunodeficient mice 49. Additionally, recent studies revealed that ALDH1-positive seemed to be a more significantly predictive marker than CD44+CD24− for the identification of breast CSCs, in terms of resistance to chemotherapy and more metastatic 39, 50. Moreover, it has been reported that the surface marker CD133 could isolate a group of breast CSCs that doesn't overlap with CD44+CD24<sup>−</sup> cells 51; and another recent study demonstrated that in a basal breast cancer cell line MDA-MB-231 (known as triple-negative), PROCR and ESA, instead of CD44+CD24−*/*low and ALDH, could be used to highly enrich breast cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically 52.

#### **2.2 Separation of breast CSCs by selecting for side-population (SP) cells**

Advances in the separation of breast CSCs was accelerated by the identification of side population (SP) cells, due to lack of dye retention and chemotherapy efflux 53. The method is based on cells incubated with Hoechst dye 33342 or rhodamine, after which the cells are analyzed by flow cytometry for dye exclusion and size, and SP cells would not retain dye. Isolation of SP cells facilitates purification of adult tissue stem cells comprising human and murine hematopoietic stem cells and a population of putative mammary epithelial stem cells 54-57. Moreover, because some evidence revealed that breast CSCs and mammary epithelial stem cells represent biologically related entities 58, scientists thought to apply this technique to isolate breast CSCs. In 2005, Patrawala *et al* successfully isolated SP cells from an ER-positive human breast cancer cell line MCF-7, and they demonstrated that these small subset (0.2%) SP cells preferentially express stemness-associated genes (such as Notch1 and β-catenin) and verapamil-sensitive ATP-binding cassette (ABC) transporter ABCG2 mRNA 59. More interestingly, MCF-7 SP cells were highly tumorigenic, whereas MCF-7 non-SP cells could not give rise to tumors in mice at al59. Researchers then took advantage of similar method to separate SP cells with stem cell properties from an ER-negative human breast cancer cell line Cal-51 and an triple-negative human breast cancer cell line MDA-MB-231, respectively, and they both found the SP cells expressed high levels of ABCG2 60, 61. Previous

observations, in 2003, Al-Hajj *et al* tried to determine whether these surface markers could distinguish tumorigenic from nontumorigenic cells, and flow cytometry was used to isolate cells that were positive or negative for each marker. They demonstrated that a small population of tumorigenic cells, isolated from human breast tumors and characterized by the expression of the cell surface markers CD44+CD24−*/*lowLineage−, was capable of regenerating the phenotypic heterogeneity of the original tumor when injected subcutaneously into NOD/SCID mice 7. They showed that as few as 100 cells with CD44+CD24−/low phenotype could form tumors in immunodeficient mice, while thousands of cells with fungible phenotypes failed to do so. Since then, CD44 and CD24 are widely accepted as surface markers for breast CSCs, and lots of studies have focused on roles of CD44+CD24− tumor cells in breast cancers. For example, Abraham *et al.* conducted immunohistochemical studies of CD44+CD24− tumor cells in human breast tumors and showed that breast tumors containing a high proportion of CD44+CD24− cells were

Nevertheless, besides CD24 and CD44, there are other surface marker candidates for the enrichment of breast CSCs. Ginestier *et al.* reported that they separated breast cancer stem/progenitor cells by sorting for Aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme responsible for the oxidation of intracellular aldehydes 47, 48, and they found that fewer ALDH1-positive than CD44+CD24− tumor cells are required to produce tumors in immunodeficient mice 49. Additionally, recent studies revealed that ALDH1-positive seemed to be a more significantly predictive marker than CD44+CD24− for the identification of breast CSCs, in terms of resistance to chemotherapy and more metastatic 39, 50. Moreover, it has been reported that the surface marker CD133 could isolate a group of breast CSCs that doesn't overlap with CD44+CD24<sup>−</sup> cells 51; and another recent study demonstrated that in a basal breast cancer cell line MDA-MB-231 (known as triple-negative), PROCR and ESA, instead of CD44+CD24−*/*low and ALDH, could be used to highly enrich breast cancer stem/progenitor cell populations which exhibited the ability to self renew and divide

**2.2 Separation of breast CSCs by selecting for side-population (SP) cells** 

Advances in the separation of breast CSCs was accelerated by the identification of side population (SP) cells, due to lack of dye retention and chemotherapy efflux 53. The method is based on cells incubated with Hoechst dye 33342 or rhodamine, after which the cells are analyzed by flow cytometry for dye exclusion and size, and SP cells would not retain dye. Isolation of SP cells facilitates purification of adult tissue stem cells comprising human and murine hematopoietic stem cells and a population of putative mammary epithelial stem cells 54-57. Moreover, because some evidence revealed that breast CSCs and mammary epithelial stem cells represent biologically related entities 58, scientists thought to apply this technique to isolate breast CSCs. In 2005, Patrawala *et al* successfully isolated SP cells from an ER-positive human breast cancer cell line MCF-7, and they demonstrated that these small subset (0.2%) SP cells preferentially express stemness-associated genes (such as Notch1 and β-catenin) and verapamil-sensitive ATP-binding cassette (ABC) transporter ABCG2 mRNA 59. More interestingly, MCF-7 SP cells were highly tumorigenic, whereas MCF-7 non-SP cells could not give rise to tumors in mice at al59. Researchers then took advantage of similar method to separate SP cells with stem cell properties from an ER-negative human breast cancer cell line Cal-51 and an triple-negative human breast cancer cell line MDA-MB-231, respectively, and they both found the SP cells expressed high levels of ABCG2 60, 61. Previous

associated with distant metastases 46.

asymmetrically 52.

studies showed that SP cells takes advantage of their ability to pump out the fluorescent dye Hoechst 33342 (H33342) through the ABCG2 (also known as breast cancer resistance protein-1), which was regarded as a major mediator of dye efflux in various stem cells 54, 62. As the ability to efflux substrates is particularly important for the protection of CSCs, and CSCs survive after chemotherapy partially by effluxing cytotoxic drugs, ABCG2 seems to protect stem cells from toxins. This is evident in *ABCG2* knockout mice that are more sensitive to compounds such as vinblastine, ivermectin, topotecan, and mitoxantrone 63-65. Taken together, SP cells have the capacity to efflux toxic substances out of breast cancer stem like cells via an ABCG2-mediated cytoprotective mechanism and seem to contribute to chemotherapy-resistance. In addition, it is important to consider that identification of cancer stem like cells by selecting for SP cells is not limited to breast carcinomas. Similar observations have been made in other solid tumors (such as glioma, ovarian and pancreatic cancers) where the isolated SP cells proliferated infinitely and could regenerate heterologous NSP cells in culture 59, 66-68.

#### **2.3 Propagation of breast CSCs by isolating ''mammospheres'' from suspension cultures**

Colonial growth in nonadherent culture was used to test for self-renewal capacity in cultures of neural cell in 1996, and in the experiment, suspension culture led to formation of "neurospheres", which consisted of 4% - 20% normal neural stem cells 69. Based on this approach, Galli *et al.* succeeded in the characterization and isolation from human glioblastoma multiform of "cancer neurospheres", which were highly enriched in long-term self-renewing,

multi-lineage-differentiating, and tumor-initiating cells 70. According to these successful procedures, researchers tried to extend this technology to the identification and propagation of mammary epithelial stem cells and breast CSCs. In 2003, Dontu *et al.* demonstrated that nonadherent mammospheres are enriched in human mammary epithelial progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems 55. More encouragingly, two years later (2005), Ponti and colleagues reported the isolation and *in vitro* propagation of spherical clusters of self-replicating cells (''mammospheres'') with stem/progenitor cell properties in suspension cultures from three breast cancer lesions and from an established breast carcinoma cell line MCF-7 71. They found that the isolated cells which overexpressed neoangiogenic and cytoprotec-tive factors showed CD44+CD24- and Cx43-, and expressed the stem cell marker OCT-4, and could form tumors *in vivo* when as few as 103 cells were implanted. This was the first time showing that breast tumorigenic cells with stem/progenitor cell properties can be propagated *in vitro* as nonadherent mammospheres, and accordingly, this experimental system was then frequently used by researchers for isolating and studying the breast tumor–initiating cells (BT-IC) 72-74.

#### **2.4 Novel strategies for enrichment of breast CSCs**

As we mentioned in the first part of this chapter, the cancer stem cell hypothesis suggests that many cancers are maintained in a hierarchical organization of rare, slowly dividing CSCs (or T-IC), rapidly dividing amplifying cells (early precursor cells, EPC) and postmitotic differentiated tumor cells 22. Thus, the complex scheme which operates in most tumor tissues seems to be that the slowly dividing CSCs give birth to EPC, which then undertake a program of exponential growth for a limited period of time before the descendant cells differentiate and become post-mitotic (Figure 1). Although the above three

Breast Cancer Stem Cells 279

The complex scheme which operates in most tumor tissues seems to be that the slowly dividing CSCs give birth to the rapidly dividing amplifying cells (early precursor cells, EPC), which then differentiate into post-mitotic tumor cells after a small number of cell divisions.

Fig. 2. Breast Cancer Cells under Pressure of Chemotherapy Are Enriched for BT-IC.

(A and B) 1°breast cancers from patients who received neoadjuvant chemotherapy are substantially enriched for self-renewing cells with the expected properties of BT-IC. Representative images show increased numbers of mammospheres after 15 days of culture (A) and a higher percentage of CD44+CD24- cells in freshly isolated tumors (B) from a patient who received chemotherapy. (C) Similarly, passaging the human breast cancer line SKBR3 in epirubicin-treated NOD/SCID mice enriches for cells with BT-IC properties.

classical methods are widely used for the isolation and identification of breast CSCs, these methods purify both T-IC and some EPC 7 59, 71. To study the breast CSCs more accurately, our group was trying to search for new strategies to enrich more purified breast CSCs. We found that breast carcinomas from chemo-treated patients were highly enriched for cells with the properties of BT-IC. We then sequentially passaged tumor cells in epirubicintreated NOD/SCID mice to get a highly malignant breast cancer cell line (SK-3rd) using the chemo-therapeutic resistance of BT-IC. Our SK-3rd cell line showed all the tentatively defined properties of BT-IC, including enhanced mammosphere formation, multipotent differentiation, chemo-therapy resistance, as well as BT- IC

phenotype(OCT4+CD44+CD24−lin−)76 (Figure 2). We assess that about 16% of SK-3rd cells were T-IC, while the rest cells (also CD44+CD24−) were mostly EPC, and mammospheric SK-3rd cells were ~100-fold more tumorigenic *in vivo* than the parent cell line, metastasize, and can be serial xenotransplanted26. Additionally, SK-3rd cells was capable of providing unlimited numbers of cells for BT-IC studies. This method of *in vivo* chemotherapy may provide researchers a novel approach of selecting CSCs from other breast cancer lines or possibly for other cancers.

Fig. 1. A Model of the Cellular Hierarchies that May Exist in Human Cancers.

Besides our strategy, there might be other new approaches for generating breast CSCs. The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression, invasion and metastasis. Associations between the breast CSCs and EMT hypothesis of cancer were established recently as similarities in these two ideas were noted (will be discussed in Part 4 of this chapter). Several very recent studies have found that the EMT could generate mammary epithelial stem cells and breast CSCs 77- 79. This may provide potential novel methods to generate and enrich relatively unlimited numbers of breast CSCs, whose biology may then be studied with far greater facility.

classical methods are widely used for the isolation and identification of breast CSCs, these methods purify both T-IC and some EPC 7 59, 71. To study the breast CSCs more accurately, our group was trying to search for new strategies to enrich more purified breast CSCs. We found that breast carcinomas from chemo-treated patients were highly enriched for cells with the properties of BT-IC. We then sequentially passaged tumor cells in epirubicintreated NOD/SCID mice to get a highly malignant breast cancer cell line (SK-3rd) using the chemo-therapeutic resistance of BT-IC. Our SK-3rd cell line showed all the tentatively defined properties of BT-IC, including enhanced mammosphere formation, multipotent

phenotype(OCT4+CD44+CD24−lin−)76 (Figure 2). We assess that about 16% of SK-3rd cells were T-IC, while the rest cells (also CD44+CD24−) were mostly EPC, and mammospheric SK-3rd cells were ~100-fold more tumorigenic *in vivo* than the parent cell line, metastasize, and can be serial xenotransplanted26. Additionally, SK-3rd cells was capable of providing unlimited numbers of cells for BT-IC studies. This method of *in vivo* chemotherapy may provide researchers a novel approach of selecting CSCs from other breast cancer lines or

Fig. 1. A Model of the Cellular Hierarchies that May Exist in Human Cancers.

Besides our strategy, there might be other new approaches for generating breast CSCs. The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression, invasion and metastasis. Associations between the breast CSCs and EMT hypothesis of cancer were established recently as similarities in these two ideas were noted (will be discussed in Part 4 of this chapter). Several very recent studies have found that the EMT could generate mammary epithelial stem cells and breast CSCs 77- 79. This may provide potential novel methods to generate and enrich relatively unlimited

numbers of breast CSCs, whose biology may then be studied with far greater facility.

differentiation, chemo-therapy resistance, as well as BT- IC

possibly for other cancers.

The complex scheme which operates in most tumor tissues seems to be that the slowly dividing CSCs give birth to the rapidly dividing amplifying cells (early precursor cells, EPC), which then differentiate into post-mitotic tumor cells after a small number of cell divisions.

Fig. 2. Breast Cancer Cells under Pressure of Chemotherapy Are Enriched for BT-IC.

(A and B) 1°breast cancers from patients who received neoadjuvant chemotherapy are substantially enriched for self-renewing cells with the expected properties of BT-IC. Representative images show increased numbers of mammospheres after 15 days of culture (A) and a higher percentage of CD44+CD24- cells in freshly isolated tumors (B) from a patient who received chemotherapy. (C) Similarly, passaging the human breast cancer line SKBR3 in epirubicin-treated NOD/SCID mice enriches for cells with BT-IC properties.

Breast Cancer Stem Cells 281

differentiated cells, it was found that let-7 regulated the key features of breast CSCs—self renewal in vitro, multipotent differentiation, and the ability to form tumors. Because the two targets of let-7 RAS and HMGA2 were responsible for the self renewal and multipotent differentiation, respectively, aberrant expression of let-7 in breast CSCs helps to maintain

Recently, Yu et al. found that similar to let-7, the expression of miR-30 was reduced in breast cancer stem-like cells (BT-ICs), and its target genes, Ubc9, an E2-conjugating enzyme essential for sumoylation, and integrin ß3(ITGB3), were upregulated at protein levels. Overexpression of miR-30 in BT-ICs inhibited their self-renewal ability by repressing Ubc9 and promoted apoptosis by inhibiting Ubc9 and ITGB3. Furthermore, ectopic expression of mir-30 or blocking the expression of Ubc9 in BT-ICs xenografts reduced their tumor-forming capacity and metastasis in NOD/SCID mice, while miR-30 inhibitor enhanced tumorigenesis and metastasis of SKBR3 breast cancer cells with low metastasis potential86. These results suggested that miR-30 could be one of the important miRNAs in regulating

MiR-15/ miR-16 are also tumor suppressors. It was first identified in B cell chronic lymphocytic leukaemia (B-CLL) that miR-15/ miR-16 was lower in their expression level while their target protein the anti-apoptosis Bcl-2 was overexpressed87. The downregulation or deletion of miR-15/miR-16 was also found in other cancer types, such as prostate cancer88, pituitary adenomas89, non-small cell lung cancer (NSCLC)90, and ovarian cancer91. Expression of these miRNAs inhibited cell proliferation, promoted apoptosis, and suppressed tumorigenicity both in vitro and in vivo by targeting multiple oncogenes, including Bcl-2, MCL1, CCND1, Wnt3A and Bmi-1. There has been growing evidence illustrated that the pivotal signaling pathways of the "stem cell genes": Notch, Hedgehog, Wnt, HMGA2, Bcl-2 and Bmi-1 were involved in the self-renewal of CSCs92. Since the oncogenic activation of Bmi-1, Bcl-2 and Wnt3A were frequently correlated with the downregulation of miR-15/miR-16, it was strongly suggested miR-15/miR-16 played a key

MiR-34 has been implicated in cell cycle control related to p5393. In p53 deficicent human gastric cancer cells, restoration of functional miR-34 inhibited the formation of tumorsphere in vitro and tumor initiation in vivo94. In parallel, miR-34 was reported to be involved in pancreatic CSCs self-renewal95. The mechanism of miR-34 mediated suppression of selfrenewal of CSCs was potentially related to the direct modulation of downstream targets Bcl-2 and Notch, suggesting that miR-34 might play an important role in gastric and pancreatic CSCs' self-renewal and/or cell fate determination. However, reduced expression of miR-34a in prostate cancer stem cells facilitated tumor development and metastasis by directly regulating CD44. Accordingly, CD44 knockdown inhibited prostate cancer growth and metastasis96. These results provided a solid experimental basis for developing miR-34a as a

MiR-128 is also a tumor suppressor involved in CSCs. Its expression was dramatically reduced in high grade gliomas, while application of miR-128 inhibited glioma proliferation and self-renewal by targeting Bmi-1 oncogene/stem cell renewal factor97. Same result was found in neural tumor medulloblastoma that miR-128a had growth suppressive activity in medulloblastoma and this activity was partially mediated by targeting Bmi-1 and thereby increasing the steady-state levels of superoxide and promoting cellular senescence. This data has implications for the modulation of redox states in CSCs, which are thought to be

their stemness26.

the stem-like features of breast cancer

role in the regulation of CSCs.

promising therapeutic agent against prostate CSCs.

resistant to therapy due to their low ROS states98.

Shown are numbers of 1°, 2°and 3°mammospheres on day 15 from 1000 cells. (D) Mammospheres generated from single-cell cultures of SK-3rd and SKBR3, imaged on indicated day of suspension culture. (E) The majority of freshly isolated SK-3rd cells are CD44+CD24-, while cells with this phenotype are rare in SKBR3. (F) SK-3rd and SKBR3 cells cultured as spheres are CD44+CD24-. When they differentiate in adherent cultures, they gradually assume the parental SBKR3 phenotype, but somewhat more rapidly for SKBR3 mammospheres. (G) When SK-3rd spheres are removed from growth factors, and plated on collagen for 8 hr (top), they do not express luminal (Muc1 and CK-18) or myoepithelial (CK-14 and a-SMA) differentiation markers, while after further differentiation (bottom), they develop into elongated cells with subpopulations staining for either differentiated subtype. (H) Freshly isolated SK-3rd cells are enriched for Hoechstlow SP cells compared with SKBR3 cells26. Adapted from Yu F, et al.*Cell*, 2007: 131:1109-23.
