**2.1.2.4 Embryonic microenvironment**

242 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

are recruited to tumors and that IFN-beta inhibits tumor growth. (Ling X 2010). Such a reduction in tumor could also be attributable to decrease CSC content. Karnoub A et al., have shown increase in the metastatic potential of the breast cancer cells when they were mixed with bone marrow derived human MSC. Using a cytokine array they identified CCL5 is induced by physical interaction between breast cancer cells and the MSC, and that it renders the breast cancer cells more metastatic. These results indicate the importance of

IT has been documented that CSCs arise from mutant versions of normal stem cells. Alternatively, CSCs can also represent a stage in the path of transformation. CSCs are precursors of differentiated cancer cells (NSCCs), however CSCs can also be derived from NSCCs or can arise independently. The proportion of CSCs remains constant over multiple generations, but the basis of this phenomenon is unknown. Hence Iliopoulos D et al., assessed these issues using an inducible model of oncogenesis that MCF-10A cells which harbor a ligand-binding domain of estrogen receptor (ER-Src), a derivative of the Src kinase oncoprotein (v-Src) that is fused to the ligand-binding domain of the estrogen receptor. Treatment of these cells with tamoxifen (TAM) rapidly induces Src, results in transformation within 24-26 h. This property of the model helps in understanding the transition between normal and transformed cells. The authors then discovered that induction of CSC from non-CSC through activation of v-src. They also document that CSC formation depends on transformation however it is not required for transformation. Moreover because of the fact that breast CSCs have an enhanced inflammatory feedback loop compared with NSCCs, they treated the cells with IL6 which resulted in generation of CSC fron non-CSC (Iliopoulos *et al.*, 2011). This indicates the critical role of microenvironment as the CSC itself secrete IL6 which can maintain the stemness of a cancer cell population. Further the fact that macrophages and dendritic cells are potent IL-6 producers, which can be activated by molecular "danger" signals by cancer cells it is important to control the IL6 signaling to regenerate the CSC .

One of the elegant studies by Mani et al demonstrates the role of TGF beta in cancer stem cell through induction of EMT. The authors treated the immortalized HMEC cells with TGF beta which resulted in fibroblast like, mesenchymal like phenotype with concomitant downregulation of ephtielial markers like E-cadherin and upregulation of mesenchymal markers like vimentin, fibronectin and N-cadherin. Similar results were obtained through ectopic expression of TWIST or SNAI1. They further assessed the CD44 and CD24 population of these cells and found that CD44+ and CD24 low cells were increased which TGF beta treatment/ TWIST, SNAI1 expression. The rise in CD44+ and CD24 low population was accompanied by approximately 30-40 fold enrichment in mamosphere forming capability (Mani *et al.*, 2008). This was a clear demonstration of TGF beta induction

Yin X et al., showed that the activating transcription factor 3 (ATF3) is induced by TGF beta in breast cancer and is important for increasing the migration potential of the breast cancer cells. Further ATF3 can be induced by a number of stromal factors like TGF beta, IFN alpha, TNF alpha and hypoxia. And the fact that ectopic expression of ATF3 increases the cancer stem cell content of breast cancer cells (CD 24low/ CD 44high), it was hypothesized that tumor microenvironment has a significant effect in the development of cancer (Yin *et al.*, 2010).

mesenchymal stem cells in rendering the cells more metastatic (Karnoub *et al.*, 2007).

**2.1.2.3 Stromal factors** 

**IL-6** 

**TGF beta** 

of cancer stem cell population.

Four decades back it was documented that embryonic microenvironment can reprogram the cancer cells to a benign phenotype; however, the mechanisms underlying this phenomenon remains unclear (Hendrix *et al.*, 2007). The human embryonic stem cells (hESC) and cancer cells have various common features however hESC do not form tumors owing to the ability to differentiate in response to signals from the microenvironment. Normally the stem cell microenvironment or the stem cell niche controls the fate of the stem cells and that it provides the necessary constituents for maintaining homeostasis of tissue (Fuchs *et al.*, 2004). In cancer cells such control is lost and that restoring the niche may result in maintaining the homeostasis of growth and normal differentiation.

Hence to understand the mechanism Lynne-Marie Postovit et al (2006) developed an in vitro 3D model to investigate the capacity of hESC-derived factors to epigenetically influence metastatic cancer cells. They showed exposure of melanoma cells to a hESC microenvironment results in the reexpression of melanocyte-specific markers which are indicative of differentiation and a reduction in invasive potential.

Further (Lynne-Marie Postovit, 2006) they discovered that hESC microenvironments suppress the tumorigenic phenotype of human metastatic melanoma and breast carcinoma cells and that this effect is is brought about only by hESCs and not other stem cell types. Further they found that hESC microenvironment neutralize the aberrant expression of Nodal in metastatic melanoma and breast carcinoma cells and reprogram them to a less aggressive phenotype (Postovit *et al.*, 2006a; b). They also identified lefty which is sectreted by hESC (an inhibitor of Nodal signaling) as an important mediator of these phenomena. Hence the microenvironment of hESCs provides a previously unexplored therapeutic entity for the regulation of aberrantly expressed embryonic factor(s) in aggressive tumor cells (Postovit *et al.*, 2008).
