**2.1 Expression of MMPs in the conditioned medium**

The MDA-MB-231 cell line is an estrogen receptor alpha (ER)-negative human breast cancer cell line (Liu *et al*., 2003). It was derived from a metastatic adenocarcinoma of the mammary gland of a 51-year-old Caucasian woman, according to the data sheet of the American Type Culture Collection (ATCC). This adherent epithelial cell line that likely contains more than one cell populations is a highly aggressive, invasive and poorlydifferentiated human breast cancer cell line. Similar to other invasive cancer cell lines, the MDA-MB-231 cells display the invasiveness by mediating the proteolytic degradation of the extracellular matrix (ECM), including basement membrane and several mechanical barriers to the ECM, through the increased expression of matrix metalloproteinases (MMPs), including gelatinases, en route to their destinations (Fig. 3).

Fig. 3. Illustration depicts the ECM in relationship to the epithelium, endothelium and connective tissues. To reach their destination, the invasive cancer cells must penetrate the mechanical barriers of the ECM and basement membrane through proteolytic degradation. The figure was modified from the Wikimedia.

Type IV collagen, which is the main component of the basement membrane, is the first component that must be degraded to allow the invasion process (Boutaud *et al*., 2000). The ability to degrade and penetrate the basement membrane is related with an increased potential of the cells for invasion and metastasis (Castro-Sanchez *et al*., 2011). Tumour cells are able to produce MMPs that degrade the matrix barriers surrounding the tumour, including basement membrane, permitting invasion into connective tissues, entry and exit from blood and lymphatic vessels, and metastasis to distant organs. MMPs are family of zinc-dependent endopeptidases that collectively are capable of degrading all components of the ECM, including the basement membrane. Binding of breast cancer cells to type IV collagen in the basement membrane induces Discoidin domain receptor 1 (DDR1) activation and then it triggers signal transduction pathways and cellular processes that promotes secretion of MMPs which contributes to basement membrane degradation and cancer cell

The MDA-MB-231 cell line is an estrogen receptor alpha (ER)-negative human breast cancer cell line (Liu *et al*., 2003). It was derived from a metastatic adenocarcinoma of the mammary gland of a 51-year-old Caucasian woman, according to the data sheet of the American Type Culture Collection (ATCC). This adherent epithelial cell line that likely contains more than one cell populations is a highly aggressive, invasive and poorlydifferentiated human breast cancer cell line. Similar to other invasive cancer cell lines, the MDA-MB-231 cells display the invasiveness by mediating the proteolytic degradation of the extracellular matrix (ECM), including basement membrane and several mechanical barriers to the ECM, through the increased expression of matrix metalloproteinases (MMPs),

Epithelial cells

Endothelium lining the capillary

Connective tissues

Basement membrane

Fibroblast

**2. Soluble growth factors in the conditioned medium of the MDA-MB-231** 

Fig. 3. Illustration depicts the ECM in relationship to the epithelium, endothelium and connective tissues. To reach their destination, the invasive cancer cells must penetrate the mechanical barriers of the ECM and basement membrane through proteolytic degradation.

Type IV collagen, which is the main component of the basement membrane, is the first component that must be degraded to allow the invasion process (Boutaud *et al*., 2000). The ability to degrade and penetrate the basement membrane is related with an increased potential of the cells for invasion and metastasis (Castro-Sanchez *et al*., 2011). Tumour cells are able to produce MMPs that degrade the matrix barriers surrounding the tumour, including basement membrane, permitting invasion into connective tissues, entry and exit from blood and lymphatic vessels, and metastasis to distant organs. MMPs are family of zinc-dependent endopeptidases that collectively are capable of degrading all components of the ECM, including the basement membrane. Binding of breast cancer cells to type IV collagen in the basement membrane induces Discoidin domain receptor 1 (DDR1) activation and then it triggers signal transduction pathways and cellular processes that promotes secretion of MMPs which contributes to basement membrane degradation and cancer cell

**2.1 Expression of MMPs in the conditioned medium** 

including gelatinases, en route to their destinations (Fig. 3).

The figure was modified from the Wikimedia.

**cells** 

invasion. A previous study demonstrated that gelatinase B or MMP-9, which degrades the type IV collagen in the basement membrane, plays a crucial role in the invasion process of the MDA-MB-231 cells (Liu *et al*., 2003). This phenomenon can be observed by determining the metastatic potential of the MDA-MB-231 cells in an experimental model that is closely correlated with the expression of the MMP-9 and the activities of the gelatinases in the conditioned medium of the MDA-MB-231 cells. According to the study, the invasion of the MDA-MB-231 cells was blocked by MMP-9-neutralising antibodies that reduced the gelatinolytic activities in the conditioned medium, as detected using Enzyme-linked immunosorbent assay (ELISA). This phenomenon also led to the significant inhibition of the invasive capacities of the MDA-MB-231 cells. This inhibition was induced by specific drugs e.g., peroxisome proliferator-activated receptor gamma ligands and all-trans-retinoic acid that were administered on a reconstituted basement membrane in a Matrigel® chamber *in vitro*. Therefore, MMP-9 was shown to play a crucial role in the invasion process of the MDA-MB-231 cells and it was shown to be absolutely required for the transmigration of this cell line.

Note: In this chapter, conditioned medium is denoted as culture supernatant that is withdrawn from feeder layer. To accomplish this, a culture of feeder layer e.g., BMSCs is maintained with fresh growth medium. After certain duration, the growth medium is withdrawn from the feeder layer as conditioned medium. The conditioned medium is believed to contain growth factors released by the feeder layer.

#### **2.2 Activation of STAT3 and soluble IL-6 in the conditioned medium**

In addition to MMP-9 in the conditioned medium, the MDA-MB-231 cells are also demonstrated to contain elevated level of signal transducer and activator of transcription 3 (STAT3) in the cells (Sasser *et al*., 2007b). STAT3 is typically maintained in the cytoplasm as an inactive monomer. Once it is phosphorylated, the STAT3 forms homodimers and enters into nucleus where it activates the transcription of multiple genes associated with cell proliferation and survival (Heinrich *et al*., 1998; Zinzalla *et al*., 2010). The activation of STAT3 has been correlated with enhanced breast cancer cell growth, survival and immune evasion (Selander *et al*., 2004; Ling *et al*., 2005; Yu *et al*., 2007). According to a previous study, exposure of MSCs-conditioned medium to MCF-7 and T-47D activated the levels of pTyr705 STAT3 in the cells (Sasser *et al*., 2007a). Correlatively, the enhancement of the cancer cell growth rates was observed in ER-positive human breast cancer cell lines, including MCF-7 and T-47D, in the presence of the MSCs-conditioned medium. The growth rates of BT474 and ZR-75-1 cells were also observed to increase after the cancer cells were co-cultured with the MSCs-conditioned medium. All cancer cell growth rates were enhanced by approximately 2-3 fold, after the exposure to the conditioned medium (Fig. 4). The growth rate of an ER-negative breast cancer cell line, MDA-MB-468, was also elevated in the presence of the MSCs-conditioned medium, albeit to a lesser extent than the other ER positive cell lines that were tested (Sasser *et al*., 2007a; Sasser *et al*., 2007b). However, this induction was not observed when the MDA-MB-231 cell line was exposed to the MSCsconditioned medium.

Few effects were observed when the MDA-MB-231 cells were co-cultured with the MSCsconditioned medium because the cell line likely contained a subpopulation in the cell population that secreted a standard level of soluble growth factors in the conditioned medium (Sasser *et al*., 2007b). In this non-adhesive co-culture study, paracrine interleukin-6

The Mesenchymal-Like Phenotype of the MDA-MB-231 Cell Line 391

MCF-10A cells, as indicated in the previous study, may not correct as both MDA-MB-231 cells and MCF-10A cells secret IL-6 in the conditioned medium. Indeed, our study demonstrated that the conditioned media of the MDA-MB-231 cells and MCF-10A cells contained a high level of IL-6, although the level was not as high as in the MSCs-conditioned

medium (Fig. 5).

**MSC 1**

**0**

negative or express lower level of ER.

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

**100**

**200**

**300**

**Concentration of IL-6 (pg/ml)**

**400**

**500**

**600**

**MSC 2**

**MCF-10A**

Fig. 5. The activity of IL-6 in the conditioned media of MSCs, non-tumorigenic cells and human breast cancer cells. The culture supernatants were withdrawn from one-week-old feeder layer of above cultures. The level of IL-6 in the conditioned medium was assayed using ELISA. The values were expressed as the mean±SD from three replicates, and the determination was carried out from three replicates each of three independent experiments. Therefore, the finding indicates that the soluble growth factors within the MDA-MB-231 conditioned medium to stimulate an increase in IL-6 production from the MCF-10A cells might be due to combine of both conditioned media. Anyhow, this result indicates that the MDA-MB-231 cells may contain similar progenitor factor as MSCs in the cell population. This factor likely expresses the high level of IL-6 where it contributes to the induction of STAT3 phosphorylation and appears to be associated with cell proliferation of the MDA-MB-231 cells. Although the secretion of IL-6 allows for the activation and maintenance of the level of STAT3 as well as the growth in the MDA-MB-231 cells have been demonstrated, its role in invasiveness of the MDA-MB-231 remains unclear. Nevertheless, targeting the IL-6 in the conditioned medium can be an idea to diagnose patients with tumour that are ER

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

**MCF-7**

**MDA-MB-231**

**BT474**

Fig. 4. Breast cancer cell growth in the presence or absence of MSCs-conditioned medium was assessed for MDA-MB-231, MCF-7, BT474, T47D and ZR-75-1 cells. The MDA-MB-231 cell growth was unaltered by MSCs-conditioned medium, whereas the growth of the four remaining ER-positive cell lines in the presence of MSCs-conditioned medium was significantly elevated when compared to the cell lines growing alone for eight days (Sasser *et al*., 2007b).

(IL-6) was found to be the principal mediator of the STAT3 phosphorylation in the cells. MSCs-induced STAT3 phosphorylation was lost when the IL-6 was depleted from the MSCs-conditioned medium. A similar phenomenon was observed when the IL-6 receptor in the cancer cells was blocked. This secretion of IL-6 from the MDA-MB-231 cells allowed for the activation and maintenance of the level of STAT3 as well as the growth in the MDA-MB-231 cells. Therefore, the conditioned medium of MDA-MB-231 cells has similar effect as the conditioned medium withdrawal from the MSCs, as evidenced by previous study, where the conditioned medium from the MDA-MB-231 cells with constitutively active STAT3 is sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels, such as MCF-10A cells, a non-tumorigenic cell line (Lieblein *et al*., 2008). This signalling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and was persistent for at least 24 hours, indicating that a correlation between elevated levels of IL-6 production and p-STAT3 in the cells, as confirmed by ELISA analysis. Neutralisation of the IL-6 ligand or gp130 was sufficient to block the increased levels of p-STAT3 (Y705) in the treated cells. These results demonstrate that the STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signalling through soluble growth factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The finding of growth factors within the MDA-MB-231 conditioned media was also sufficient to stimulate an increase in IL-6 production from

\*

\*

\* \*\*\*

\*

**0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0**

**Relative Tumour Cell Growth**

*et al*., 2007b).

(bead:tumour cell ratio)

MDA-MB-231 alone

MDA-MB-231 + MSCs-CM

MCF-7 alone

MCF-7 + MSCs-CM

BT474 alone

Fig. 4. Breast cancer cell growth in the presence or absence of MSCs-conditioned medium was assessed for MDA-MB-231, MCF-7, BT474, T47D and ZR-75-1 cells. The MDA-MB-231 cell growth was unaltered by MSCs-conditioned medium, whereas the growth of the four remaining ER-positive cell lines in the presence of MSCs-conditioned medium was significantly elevated when compared to the cell lines growing alone for eight days (Sasser

(IL-6) was found to be the principal mediator of the STAT3 phosphorylation in the cells. MSCs-induced STAT3 phosphorylation was lost when the IL-6 was depleted from the MSCs-conditioned medium. A similar phenomenon was observed when the IL-6 receptor in the cancer cells was blocked. This secretion of IL-6 from the MDA-MB-231 cells allowed for the activation and maintenance of the level of STAT3 as well as the growth in the MDA-MB-231 cells. Therefore, the conditioned medium of MDA-MB-231 cells has similar effect as the conditioned medium withdrawal from the MSCs, as evidenced by previous study, where the conditioned medium from the MDA-MB-231 cells with constitutively active STAT3 is sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels, such as MCF-10A cells, a non-tumorigenic cell line (Lieblein *et al*., 2008). This signalling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and was persistent for at least 24 hours, indicating that a correlation between elevated levels of IL-6 production and p-STAT3 in the cells, as confirmed by ELISA analysis. Neutralisation of the IL-6 ligand or gp130 was sufficient to block the increased levels of p-STAT3 (Y705) in the treated cells. These results demonstrate that the STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signalling through soluble growth factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The finding of growth factors within the MDA-MB-231 conditioned media was also sufficient to stimulate an increase in IL-6 production from

BT474 + MSCs-CM

T47D alone

MSCs-CM

ZR-75-1 alone

ZR-75-1 + MSCs-CM

T47D + MCF-10A cells, as indicated in the previous study, may not correct as both MDA-MB-231 cells and MCF-10A cells secret IL-6 in the conditioned medium. Indeed, our study demonstrated that the conditioned media of the MDA-MB-231 cells and MCF-10A cells contained a high level of IL-6, although the level was not as high as in the MSCs-conditioned medium (Fig. 5).

Fig. 5. The activity of IL-6 in the conditioned media of MSCs, non-tumorigenic cells and human breast cancer cells. The culture supernatants were withdrawn from one-week-old feeder layer of above cultures. The level of IL-6 in the conditioned medium was assayed using ELISA. The values were expressed as the mean±SD from three replicates, and the determination was carried out from three replicates each of three independent experiments.

Therefore, the finding indicates that the soluble growth factors within the MDA-MB-231 conditioned medium to stimulate an increase in IL-6 production from the MCF-10A cells might be due to combine of both conditioned media. Anyhow, this result indicates that the MDA-MB-231 cells may contain similar progenitor factor as MSCs in the cell population. This factor likely expresses the high level of IL-6 where it contributes to the induction of STAT3 phosphorylation and appears to be associated with cell proliferation of the MDA-MB-231 cells. Although the secretion of IL-6 allows for the activation and maintenance of the level of STAT3 as well as the growth in the MDA-MB-231 cells have been demonstrated, its role in invasiveness of the MDA-MB-231 remains unclear. Nevertheless, targeting the IL-6 in the conditioned medium can be an idea to diagnose patients with tumour that are ER negative or express lower level of ER.
