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

Khoo Boon Yin

*Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia* 

### **1. Introduction**

Mesenchymal stem cells (MSCs) are progenitor cells that can be isolated from all connective tissues such as bone, adipose, cartilage, blood and muscle (Wang *et al*., 2009). MSCs have recently been described to localise within breast carcinomas where the stem cells integrate into tumour-associated stromal tissues whereby the MSCs promote breast cancer cell invasion and metastasis (Karnoub *et al*., 2007). Previous studies have demonstrated that when combine with weakly metastatic human breast carcinoma cells, bone marrow-derived mesenchymal stem cells (BMSCs) increase the metastatic potency of the cancer cells greatly (Hombauer & Minguell, 2000). This phenomenon was significantly observed in MCF-7 cells where increase in cancer cell proliferation was observed when the cancer cells were co-cultured on the BMSCs feeder layer. Furthermore, light and epifluorescence microscopy studies revealed that the MCF-7 cluster grew in a dispersed fashion on the BMSCs feeder layer due to the decrease expression of adhesive molecules, such as E-cadherin and epithelial-specific antigen (ESA), in the cancer cells. The interaction between the MCF-7 cells and the BMSCs likely causes the loss of the adhesive molecules in the cancer cells. A phenomenon similar to this interaction was also observed in our recent study. Indeed, the study found that the growth of the MCF-7 cells was enhanced not only when the cancer cells were adhesively co-cultured with the BMSCs but also when they were co-cultured non-adhesively.

In the adhesive cell interaction, the growth or proliferation rate of the MCF-7 cells, which was measured by colony size, was observed to increase when the cancer cells were cocultured on the BMSCs feeder layer (Fig. 1A and Fig. 1B). The non-adhesive interaction of the MCF-7 cells with BMSCs was also found to increase the growth of the cancer cells. When the cancer cells were incubated with the conditioned medium (culture supernatant) of the BMSCs, the proliferation rate of the MCF-7 cells increased approximately 16.6% when compared to the proliferation rate of the cancer cells incubated with growth medium only (Fig. 1C). This phenomenon indicates that the increase in the proliferation rate of the cancer cells due to the presence of the BMSCs must not be related to a direct physical cell–cell interaction, as similar findings are observed in both the adhesive and non-adhesive coculture conditions.

Note: In this chapter, adhesive co-culture is defined as the growth of cancer cells on a nontumorigenic cell monolayer where direct physical cell–cell interaction occurs. Non-adhesive

The Mesenchymal-Like Phenotype of the MDA-MB-231 Cell Line 387

evidence of direct cell–cell contact (Fig. 2). Thus, the influence of the BMSCs on the changed of cell morphology and increased proliferation rate of the MCF-7 cells may be achieved *via* the culture medium without the need for any direct physical cell–cell interaction between the two cell lines. However, this phenomenon, in which the BMSCs increased the proliferation rate of the breast cancer cells, was not observed when the BMSCs were cocultured with highly invasive and metastatic human breast cancer cells, such as MDA-MB-231 cells. The MDA-MB-231 cell line likely contains its own source that similar to the MSCs as progenitor factor in the cell population that is able to secrete a standard level of the soluble growth factors into the conditioned medium of the MDA-MB-231 cells. Therefore, the activity of the MDA-MB-231 cells was not influenced by their exposure to the MSCsconditioned medium and few effects were observed when the cells were co-cultured with

Fig. 2. Unstained MCF-7 and MDA-MB-231 cells as visualised under an inverted light microscope. Panels A and B show clustered and single cell layer of MCF-7 cells that were incubated with cell growth medium and BMSCs-conditioned medium, respectively, whereas panels **C** and **D** show few effects on cell morphology were observed when the MDA-MB-231 cells were incubated with cell growth medium and BMSCs-conditioned medium, respectively. The pictures were taken after one week of cell incubations.

the MSCs (Sasser *et al*., 2007a).

**(A) (B)** 

**(C) (D)** 

co-culture is defined as the incubation of cancer cells with a conditioned medium that is withdrawn from the non-tumorigenic cells; here, the cells interact with one another *via* the culture medium.

Fig. 1. Colony formation of the MCF-7 cells cultured with or without BMSCs. The pictures show Oil-Red-O staining of **(A)** the MCF-7 colonies alone and **(B)** the MCF-7 colonies grown on the BMSCs feeder layer for one week. The pictures were visualised under an inverted light microscope using same magnification. **(C)** The proliferation rate of the MCF-7 cells incubated with cell growth medium (control) and conditioned medium of BMSCs for one week. One hundred cells were used as the input prior to incubation. The values were expressed as mean±SD from three replicates, and the determination was carried out from three replicates each of three independent experiments.

The BMSCs likely secreted or influenced the MCF-7 cells to secrete certain soluble growth factors into the conditioned medium whereby the growth factors stimulated the MCF-7 cells in cluster and grow into single cell layer, after which the cells dispersed without any

co-culture is defined as the incubation of cancer cells with a conditioned medium that is withdrawn from the non-tumorigenic cells; here, the cells interact with one another *via* the

 **Conditioned Medium of BMSC**

Fig. 1. Colony formation of the MCF-7 cells cultured with or without BMSCs. The pictures show Oil-Red-O staining of **(A)** the MCF-7 colonies alone and **(B)** the MCF-7 colonies grown on the BMSCs feeder layer for one week. The pictures were visualised under an inverted light microscope using same magnification. **(C)** The proliferation rate of the MCF-7 cells incubated with cell growth medium (control) and conditioned medium of BMSCs for one week. One hundred cells were used as the input prior to incubation. The values were expressed as mean±SD from three replicates, and the determination was carried out from

The BMSCs likely secreted or influenced the MCF-7 cells to secrete certain soluble growth factors into the conditioned medium whereby the growth factors stimulated the MCF-7 cells in cluster and grow into single cell layer, after which the cells dispersed without any

 **Cell Growth Medium**

culture medium.

**0**

three replicates each of three independent experiments.

**20**

**40**

**60**

**% Cell Proliferation**

**(C)** 

**80**

**100**

**120**

**(A) (B)** 

evidence of direct cell–cell contact (Fig. 2). Thus, the influence of the BMSCs on the changed of cell morphology and increased proliferation rate of the MCF-7 cells may be achieved *via* the culture medium without the need for any direct physical cell–cell interaction between the two cell lines. However, this phenomenon, in which the BMSCs increased the proliferation rate of the breast cancer cells, was not observed when the BMSCs were cocultured with highly invasive and metastatic human breast cancer cells, such as MDA-MB-231 cells. The MDA-MB-231 cell line likely contains its own source that similar to the MSCs as progenitor factor in the cell population that is able to secrete a standard level of the soluble growth factors into the conditioned medium of the MDA-MB-231 cells. Therefore, the activity of the MDA-MB-231 cells was not influenced by their exposure to the MSCsconditioned medium and few effects were observed when the cells were co-cultured with the MSCs (Sasser *et al*., 2007a).

Fig. 2. Unstained MCF-7 and MDA-MB-231 cells as visualised under an inverted light microscope. Panels A and B show clustered and single cell layer of MCF-7 cells that were incubated with cell growth medium and BMSCs-conditioned medium, respectively, whereas panels **C** and **D** show few effects on cell morphology were observed when the MDA-MB-231 cells were incubated with cell growth medium and BMSCs-conditioned medium, respectively. The pictures were taken after one week of cell incubations.

The Mesenchymal-Like Phenotype of the MDA-MB-231 Cell Line 389

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

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

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 MSCs-

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

believed to contain growth factors released by the feeder layer.

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

cell line.

conditioned medium.
