**2.3 CCL2 and CCL5 in the conditioned medium**

Chemokines or chemotactic cytokines are small proteins that are classified into four conserved groups, CXC, CC, C and CX3C, based on the position of the first two cysteines that are adjacent to the amino acid (Balkwill, 2004; Lu & Kang, 2009). Among more than 50

The Mesenchymal-Like Phenotype of the MDA-MB-231 Cell Line 393

The overexpression of chemokine decoy receptor proteins, such as D6 and Duffy antigen receptor for chemokines (DARC), have been demonstrated to inhibit the proliferation and invasion of the human breast cancer *in vitro*, tumorigenesis and lung metastasis *in vivo* (Wu *et al*., 2008). This inhibition is associated with decrease in chemokines, such as CCL2 and CCL5, vessel density and tumour-associated macrophage infiltration. The inhibition of CCL5 expression by short interfering RNA (siRNA) or by the use of neutralising antibodies against CCL5 impaired the tumour-supporting roles that were mediated by the CCL5-CCR5 loop; this significantly inhibited the metastatic potential of the MDA-MB-231 cells (Karnoub *et al*., 2007; Soria & Ben-Baruch, 2008). Moreover, CCL2-neutralizing antibodies inhibited bone resorption *in vitro* and bone metastasis *in vivo* as well as the tumour conditioned media-induced osteoclast formation *in vitro* and bone metastasis *in vivo*, indicating a role of the CCL2 and CCL5 in metastasis (Lu & Kang, 2009). The MDA-MB-231 cells are obviously having its own progenitor factor, which is in the cell population that produce the soluble growth factors in order to maintain the invasive and progressive phenotypes in the cells. Further identification and functional characterisation of CCL2 and CCL5, as well as MMP-9 and IL-6, would provide an effective treatment for systemic metastasis. Perhaps, the effects of certain inhibitors or drugs on the inhibition of proliferation and the reduction of invasion of breast cancer cell growth can be easily determined using these growth factors as they can be detected *via* serum and conditioned culture medium. Thus, all four molecules mentioned in this chapter could be considered as potential therapeutic targets for the development of a

Most of the cancer cell lines have recently been demonstrated by flow cytometry to contain a subpopulation of CD44+/CD24- where the MDA-MB-231 cells are found to contain a high percentage of the CD44+/CD24- subpopulation (85±5%) in the cells (Sheridan *et al*., 2006). Other cell lines that contain a high level of this subpopulation are MDA-MB-436 (72±5%), Hs578T (86±5%) and SUM1315 (97±3%) (Table 1). The subpopulation is shown to possess the capacity for self-renewal and the generation of heterogeneous progeny in the cells. Moreover, the subpopulation of the breast cancer cells has been reported to have stem/progenitor cell properties that contribute a unique ability to allow these cells to invade. Similar to the ability of the MSCs that was described above, the inherent properties of this subpopulation may impart their transformed counterparts with the ability to evade traditional antitumour therapies and to establish breast cancer metastasis (Reya *et al*., 2001; Behbod *et al*., 2005; Dean *et al*., 2005). Several studies suggested that this subpopulation of cells, as a subset of human breast cancer cells, possessed an enhanced ability to form tumour in immunocompromised mice (Al-Hajj *et al*., 2003; Ponti *et al*., 2005). However, the potential of this subpopulation to establish

The expression levels of pro-invasive genes, such as interleukin-1-alpha (IL-1), IL-6, interleukin-8 (IL-8) and urokinase plasminogen activator (UPA), are higher in the cell lines

invasive is consistent with the studies that demonstrate the metastatic process in breast cancer cells requires the following: (1) ECM degradation-associated proteins, including the

subpopulation (Sheridan *et al*., 2006). The results

subpopulation are more

detection assay for human breast cancer.

**3. The subpopulation in the MDA-MB-231 cells** 

breast cancer metastasis in the cell line remains unclear.

indicate that the cell lines with a significant number of CD44+/CD24-

that contained a significant CD44+/CD24-

identified human chemokines, chemokine (C-C motif) ligand 2 (CCL2 or MCP-1) and chemokine (C-C motif) ligand 5 (CCL5 or RANTES) are of particularly important. CCL2 is a potent chemoattractant for monocytes, memory T lymphocytes and natural killer cells whereas CCL5 is a potent inducer of leukocyte motility (Lu & Kang, 2009; Melgarejo *et al*., 2009; Yaal-Hahoshen *et al*., 2006). Both chemokines stimulate migration of leukocytes in response to inflammatory signals. The roles of CCL2 and CCL5 in breast malignancy have been extensively addressed in breast cancer studies (Goldberg-Bittman *et al*., 2004; Soria *et al*., 2008; Soria & Ben-Baruch, 2008; Wu *et al*., 2008; Fujimoto *et al*., 2009). Overexpression of the CCL2 and CCL5 are stimulated during breast cancer development and progression. They are also frequently associated with advanced tumour stage and metastatic relapse in breast cancer. Both chemokines act directly on the tumour cells to promote their pro-malignancy phenotype by increasing their migratory and invasion-related properties (Soria & Ben-Baruch, 2008). The chemokines are expressed by the cells of the tumour microenvironment osteoblasts and MSCs. In breast cells, the chemokines are highly expressed by breast tumour cells at primary tumour sites and minimally expressed by normal breast epithelial duct cells (Soria *et al*., 2008; Soria & Ben-Baruch, 2008). The chemokines are soluble growth factors that can be easily detected in serum and conditioned culture medium. Consistently, our recent study demonstrated that high levels of CCL2 and CCL5 were detected in the conditioned medium of the MDA-MB-231 cells, as determined by ELISA (Fig. 6). The results indicated that the CCL2 and CCL5 were present in the conditioned media of all of the tested cell lines. As expected, elevated levels of CCL2 and CCL5 were observed in the MSCs and MCF-10A cells. CCL2 was additionally more stably expressed in the non-tumorigenic cells, such as MCF-10A, than in the MDA-MB-231 cells. However, an opposite event was observed for CCL5 in the MCF-10A and MDA-MB-231 cells. Both CCL2 and CCL5 displayed relatively higher expression levels in the MDA-MB-231 cells than that in the weakly metastatic cells, such as MCF-7 and BT-474. However, the CCL2 level in the MDA-MB-231 cells was only slightly higher than in the MCF-7 and BT-474 cells.

Fig. 6. The activities of **(A)** CCL2 and **(B)** CCL5 in the conditioned media of MSCs, human breast tumorigenic and non-tumorigenic cells. The levels of the soluble growth factors were assayed using ELISA. The values were expressed as mean±SD from three replicates, and the determination was carried out from three replicates each of three independent experiments.

identified human chemokines, chemokine (C-C motif) ligand 2 (CCL2 or MCP-1) and chemokine (C-C motif) ligand 5 (CCL5 or RANTES) are of particularly important. CCL2 is a potent chemoattractant for monocytes, memory T lymphocytes and natural killer cells whereas CCL5 is a potent inducer of leukocyte motility (Lu & Kang, 2009; Melgarejo *et al*., 2009; Yaal-Hahoshen *et al*., 2006). Both chemokines stimulate migration of leukocytes in response to inflammatory signals. The roles of CCL2 and CCL5 in breast malignancy have been extensively addressed in breast cancer studies (Goldberg-Bittman *et al*., 2004; Soria *et al*., 2008; Soria & Ben-Baruch, 2008; Wu *et al*., 2008; Fujimoto *et al*., 2009). Overexpression of the CCL2 and CCL5 are stimulated during breast cancer development and progression. They are also frequently associated with advanced tumour stage and metastatic relapse in breast cancer. Both chemokines act directly on the tumour cells to promote their pro-malignancy phenotype by increasing their migratory and invasion-related properties (Soria & Ben-Baruch, 2008). The chemokines are expressed by the cells of the tumour microenvironment osteoblasts and MSCs. In breast cells, the chemokines are highly expressed by breast tumour cells at primary tumour sites and minimally expressed by normal breast epithelial duct cells (Soria *et al*., 2008; Soria & Ben-Baruch, 2008). The chemokines are soluble growth factors that can be easily detected in serum and conditioned culture medium. Consistently, our recent study demonstrated that high levels of CCL2 and CCL5 were detected in the conditioned medium of the MDA-MB-231 cells, as determined by ELISA (Fig. 6). The results indicated that the CCL2 and CCL5 were present in the conditioned media of all of the tested cell lines. As expected, elevated levels of CCL2 and CCL5 were observed in the MSCs and MCF-10A cells. CCL2 was additionally more stably expressed in the non-tumorigenic cells, such as MCF-10A, than in the MDA-MB-231 cells. However, an opposite event was observed for CCL5 in the MCF-10A and MDA-MB-231 cells. Both CCL2 and CCL5 displayed relatively higher expression levels in the MDA-MB-231 cells than that in the weakly metastatic cells, such as MCF-7 and BT-474. However, the CCL2 level in the MDA-MB-231 cells was only slightly higher than in the MCF-7 and BT-474 cells.

**MSC**

**Concentration of CCL2 (pg/ml)**

**MCF-10A**

**MCF-7**

**MDA-MB-231**

**BT474**

**MSC** 

**Concentration of CCL5 (pg/ml)**

**(A) (B)** Fig. 6. The activities of **(A)** CCL2 and **(B)** CCL5 in the conditioned media of MSCs, human breast tumorigenic and non-tumorigenic cells. The levels of the soluble growth factors were assayed using ELISA. The values were expressed as mean±SD from three replicates, and the determination was carried out from three replicates each of three independent experiments.

**MCF-10A**

**MCF-7**

**MDA-MB-231**

**BT474**

The overexpression of chemokine decoy receptor proteins, such as D6 and Duffy antigen receptor for chemokines (DARC), have been demonstrated to inhibit the proliferation and invasion of the human breast cancer *in vitro*, tumorigenesis and lung metastasis *in vivo* (Wu *et al*., 2008). This inhibition is associated with decrease in chemokines, such as CCL2 and CCL5, vessel density and tumour-associated macrophage infiltration. The inhibition of CCL5 expression by short interfering RNA (siRNA) or by the use of neutralising antibodies against CCL5 impaired the tumour-supporting roles that were mediated by the CCL5-CCR5 loop; this significantly inhibited the metastatic potential of the MDA-MB-231 cells (Karnoub *et al*., 2007; Soria & Ben-Baruch, 2008). Moreover, CCL2-neutralizing antibodies inhibited bone resorption *in vitro* and bone metastasis *in vivo* as well as the tumour conditioned media-induced osteoclast formation *in vitro* and bone metastasis *in vivo*, indicating a role of the CCL2 and CCL5 in metastasis (Lu & Kang, 2009). The MDA-MB-231 cells are obviously having its own progenitor factor, which is in the cell population that produce the soluble growth factors in order to maintain the invasive and progressive phenotypes in the cells. Further identification and functional characterisation of CCL2 and CCL5, as well as MMP-9 and IL-6, would provide an effective treatment for systemic metastasis. Perhaps, the effects of certain inhibitors or drugs on the inhibition of proliferation and the reduction of invasion of breast cancer cell growth can be easily determined using these growth factors as they can

be detected *via* serum and conditioned culture medium. Thus, all four molecules mentioned in this chapter could be considered as potential therapeutic targets for the development of a detection assay for human breast cancer.
