**4. Mesenchymal-like phenotype of the MDA-MB-231 cells**

Before the subpopulation and invasiveness of the MDA-MB-231 cells can be further elucidated, hierarchy of the breast cancer stem cells in the breast cancer cell compartment should be understood. A breast cancer stem cell, as described in the cancer stem cell compartment hierarchy, is capable of undergoing an asymmetric cell division to generate one cell that is identical to itself (orange colour) and one that it is more committed towards a certain differentiation pattern (a breast cancer cell, grey colour) (Cariati & Purushotham, 2008; Fig. 7).

Fig. 7. A breast cancer stem cells-breast cancer cells compartment hierarchy. A breast cancer stem cell is capable of going through an asymmetric cell division to generate one cell that is identical to itself (orange colour) and one that tends toward a certain differentiation pattern (breast cancer cells, grey colour) (Modified from Cariati & Rurushotham, 2008).

The formation of the identical cell ensures that the cancer stem cell compartment is maintained throughout its time in the subpopulation. These distinct cells undergo series of divisions and differentiation steps that result in the generation of a terminally differentiated

The Mesenchymal-Like Phenotype of the MDA-MB-231 Cell Line 397

cells compared with the MCF-7 cells; the expression levels of 2345 transcripts were significantly increased in the MCF-7 cells compared with the MDA-MB-231 cells (Forozon *et al*., 2000; Charafe-Jauffret *et al*., 2006). Moreover, 387 of the above transcripts have been defined by the gene expression profile to be mesenchymal-like cellular subtypes (Charafe-Jauffret *et al*., 2006). Recently, 31 mesenchymal-like and luminal-like subtype features of breast cancer cell lines were revealed in Charafe-Jauffret's study, which was based on the gene expression profiles. The study found that 680 transcripts were preferentially expressed in the group of mesenchymal-like cell lines, and 629 transcripts were expressed preferentially in the group of luminal-like breast cancer cell lines. In a recent expression study, 387 transcripts, which are also identified in the mesenchymal-like subtype gene list in Charafe-Jauffret's study, showed significantly higher expression levels in the MDA-MB-231 cells; and 328 transcripts, which were present on the luminal subtype gene list from Charafe-Jauffret's study, showed significantly higher expression levels in the MCF-7 cells (Li *et al*., 2009). These data revealed the differential expression profiles of mesenchymal-like and luminal-like subtypes of the breast cancer cell lines. These expression profiles can be utilised to effectively overcome the invasion and metastasis of

Fig. 8**. A:** The MDA-MB-231 cells, which were predicted to have a mesenchymal-like phenotype subpopulation, existed in the epithelial cell population. The cells were stained with Oil-red O and visualised using an inverted light microscope. **B:** The MDA-MB-231 cells were positive for the CD105 antibody staining as visualised using fluorescent microscope.

Our recent study proposes to isolate or withdraw the mesenchymal-like stem cells from the MDA-MB-231 population using CD105 and other known antibody-conjugated microbeads, thus allowing for a clearer understanding of the subpopulation of the cancer cells. Potential drugs will then be applied to the isolated CD105+ (mesenchymal-like stem

These images were captured using a digital camera.

**(A) (B)** 

**Mesenchymal-like cells** 

human breast cancer.

**5. Future prospects** 

population of breast cancer stem cells and breast cancer cells. The existence of these cancer stem cells explains why only a small minority of cancer cells are capable of extensive proliferate and transfer to the tumour. Chemotherapy can remove breast cancer cells, but it fails to eliminate the cancer stem cells that can revive the breast cancer cells. This allows the regrowth of the breast cancer after the treatment has ended (Sanchez-Garcia *et al*., 2008). This shortcoming explains the high recurrence of the disease. Therefore, the current strategy for the development of anti-breast cancer agents is to target both breast cancer stem cells and breast cancer cells. Moreover, the healthy breast cells transform into cancer cells *via* the formation of breast cancer stem cells is also a possibility, but, the precise mechanism of the transformation for this disease remains unclear. Therefore, study of the transformation remains warranted. By understanding the precise mechanism that transforms normal stem cells into cancer cells *via* the formation of the cancer stem cells, it would be possible to develop more effective tools for the ER-negative human breast cancer prevention, detection and treatment.

The MDA-MB-231 cell line is a good example of a breast cancer cell line that consists of the above mentioned populations of breast cancer stem cells and breast cancer cells. As described above, the MDA-MB-231 cells have been demonstrated to contain a subpopulation of CD44+/CD24- that provides stem/progenitor cell properties to enhance the invasiveness of the cancer cells. Surprisingly, in our present study, we found that the MDA-MB-231 cells were positive for CD105 staining, while the weakly metastatic breast cancer cell lines, such as MCF-7 and T47D, were negative for the CD105 staining; the staining was visualised by fluorescent microscopy (Fig. 8A). CD105, also known as Endoglin, is a type I integral trans-membrane glycoprotein and is an accessory receptor for transforming growth factor-alpha (TGF) superfamily ligands (Barbara *et al*., 1999). CD105 is found on activated monocytes, mesenchymal stromal cells and leukemic cells of lymphoid and myeloid lineages. The BMSCs, as well as other non-hematopoietic MSCs, are positive for the CD105 antibody staining, as visualised by fluorescent microscopy (Miao *et al*., 2006; Bernacki *et al*., 2008). Therefore, it is hypothesised that the MDA-MB-231 cells may have similar mesenchymal phenotypes as the progenitor factors that contribute to the metastatic potency of the cancer cells. Therefore, the cell line contains not only the stem/progenitor cell properties but also the mesenchymal-like stem/progenitor cell properties. This property likely contributes to the invasiveness of the cell line and causes the cell line to express high levels of MMP-9, IL-6, CCL2 and CCL5 in the conditioned medium. The Oil-Red-O-stained MDA-MB-231 cells were also observed to contain a mixture of epithelial cells and a mesenchymal-like subpopulation, as visualised by light microscopy (Fig. 8B).

These results focused our attention and research on the mesenchymal-like phenotype in the MDA-MB-231 cells. MDA-MB-231 and MCF-7 are cell lines that originate from pleural effusion metastatic cells in ductal invasive breast carcinomas (Burdall *et al*., 2003; Lacroix & Leclercq, 2004). These cells are among the most commonly used breast cancer cell lines in medical research laboratories. MDA-MB-231 is a mesenchymal-like cell line that is highly aggressive and invasive, whereas MCF-7 is classified as a luminal (epithelial)-like cell line with a relatively low invasive phenotype and potential (Lacroix & Leclercq, 2004; Charafe-Jauffret *et al*., 2006). A comparison of the two cell lines in terms of DNA copy number variation and gene expression profiles has been performed, and the expression levels of 2157 transcripts were shown to be significantly increased in the MDAMB-231

population of breast cancer stem cells and breast cancer cells. The existence of these cancer stem cells explains why only a small minority of cancer cells are capable of extensive proliferate and transfer to the tumour. Chemotherapy can remove breast cancer cells, but it fails to eliminate the cancer stem cells that can revive the breast cancer cells. This allows the regrowth of the breast cancer after the treatment has ended (Sanchez-Garcia *et al*., 2008). This shortcoming explains the high recurrence of the disease. Therefore, the current strategy for the development of anti-breast cancer agents is to target both breast cancer stem cells and breast cancer cells. Moreover, the healthy breast cells transform into cancer cells *via* the formation of breast cancer stem cells is also a possibility, but, the precise mechanism of the transformation for this disease remains unclear. Therefore, study of the transformation remains warranted. By understanding the precise mechanism that transforms normal stem cells into cancer cells *via* the formation of the cancer stem cells, it would be possible to develop more effective tools for the ER-negative human breast cancer prevention,

The MDA-MB-231 cell line is a good example of a breast cancer cell line that consists of the above mentioned populations of breast cancer stem cells and breast cancer cells. As described above, the MDA-MB-231 cells have been demonstrated to contain a subpopulation of CD44+/CD24- that provides stem/progenitor cell properties to enhance the invasiveness of the cancer cells. Surprisingly, in our present study, we found that the MDA-MB-231 cells were positive for CD105 staining, while the weakly metastatic breast cancer cell lines, such as MCF-7 and T47D, were negative for the CD105 staining; the staining was visualised by fluorescent microscopy (Fig. 8A). CD105, also known as Endoglin, is a type I integral trans-membrane glycoprotein and is an accessory receptor for transforming growth factor-alpha (TGF) superfamily ligands (Barbara *et al*., 1999). CD105 is found on activated monocytes, mesenchymal stromal cells and leukemic cells of lymphoid and myeloid lineages. The BMSCs, as well as other non-hematopoietic MSCs, are positive for the CD105 antibody staining, as visualised by fluorescent microscopy (Miao *et al*., 2006; Bernacki *et al*., 2008). Therefore, it is hypothesised that the MDA-MB-231 cells may have similar mesenchymal phenotypes as the progenitor factors that contribute to the metastatic potency of the cancer cells. Therefore, the cell line contains not only the stem/progenitor cell properties but also the mesenchymal-like stem/progenitor cell properties. This property likely contributes to the invasiveness of the cell line and causes the cell line to express high levels of MMP-9, IL-6, CCL2 and CCL5 in the conditioned medium. The Oil-Red-O-stained MDA-MB-231 cells were also observed to contain a mixture of epithelial cells and a mesenchymal-like subpopulation, as visualised by light

These results focused our attention and research on the mesenchymal-like phenotype in the MDA-MB-231 cells. MDA-MB-231 and MCF-7 are cell lines that originate from pleural effusion metastatic cells in ductal invasive breast carcinomas (Burdall *et al*., 2003; Lacroix & Leclercq, 2004). These cells are among the most commonly used breast cancer cell lines in medical research laboratories. MDA-MB-231 is a mesenchymal-like cell line that is highly aggressive and invasive, whereas MCF-7 is classified as a luminal (epithelial)-like cell line with a relatively low invasive phenotype and potential (Lacroix & Leclercq, 2004; Charafe-Jauffret *et al*., 2006). A comparison of the two cell lines in terms of DNA copy number variation and gene expression profiles has been performed, and the expression levels of 2157 transcripts were shown to be significantly increased in the MDAMB-231

detection and treatment.

microscopy (Fig. 8B).

cells compared with the MCF-7 cells; the expression levels of 2345 transcripts were significantly increased in the MCF-7 cells compared with the MDA-MB-231 cells (Forozon *et al*., 2000; Charafe-Jauffret *et al*., 2006). Moreover, 387 of the above transcripts have been defined by the gene expression profile to be mesenchymal-like cellular subtypes (Charafe-Jauffret *et al*., 2006). Recently, 31 mesenchymal-like and luminal-like subtype features of breast cancer cell lines were revealed in Charafe-Jauffret's study, which was based on the gene expression profiles. The study found that 680 transcripts were preferentially expressed in the group of mesenchymal-like cell lines, and 629 transcripts were expressed preferentially in the group of luminal-like breast cancer cell lines. In a recent expression study, 387 transcripts, which are also identified in the mesenchymal-like subtype gene list in Charafe-Jauffret's study, showed significantly higher expression levels in the MDA-MB-231 cells; and 328 transcripts, which were present on the luminal subtype gene list from Charafe-Jauffret's study, showed significantly higher expression levels in the MCF-7 cells (Li *et al*., 2009). These data revealed the differential expression profiles of mesenchymal-like and luminal-like subtypes of the breast cancer cell lines. These expression profiles can be utilised to effectively overcome the invasion and metastasis of human breast cancer.

**Mesenchymal-like cells** 

Fig. 8**. A:** The MDA-MB-231 cells, which were predicted to have a mesenchymal-like phenotype subpopulation, existed in the epithelial cell population. The cells were stained with Oil-red O and visualised using an inverted light microscope. **B:** The MDA-MB-231 cells were positive for the CD105 antibody staining as visualised using fluorescent microscope. These images were captured using a digital camera.
