**2.1.2.1 Hypoxia**

It has been suggested that hypoxia contributes to the generation aggressive cancer by selecting tumor cells and results into growth of cells that can survive compromised levels of oxygen and nutrients (Graeber *et al.*, 1996). Further the growth of tumor results in hypoxic microenvironment, which is followed by periods of reoxygenation. Hence to mimic the invivo environment and to assess the fate of cells undergoing periods of hypoxiareoxygenation Louie E etal., exposed breast cancer cells (MDA-MB-231 and BCM2) to cycles of hypoxia and nutrient deprivation. They discovered that after the first cycle of hypoxia a small fraction of cells survived and that repetitive exposure of the same cells to hypoxia and reoxygenation led to increased viability under hypoxia and to proliferate either as monolayer or tumor spheres. They also found increase in the number of cells expressing CD44+/CD24-/ESA+ cell surface markers, and hence the cancer stem cell content. Therefore repetitive cycling of hypoxia and re-oxygenation can increase the stem cell content of metastatic breast cancer cell lines indicating that microenvironment plays an important role in selectively increasing CSC (Louie *et al.*, 2010).

#### **2.1.2.2 Stromal cells**

#### **Carcinoma associated fibroblasts (CAF)**

For a long time scientist have primararily focused on epithelial component of breast cancer, however recently, the critical importance of tumor stroma has been realized. Literature

Fig. 1. Microenvironment of normal breast epithelium and breast cancer cells.

in selectively increasing CSC (Louie *et al.*, 2010).

**Carcinoma associated fibroblasts (CAF)** 

is hypoxia. **2.1.2.1 Hypoxia** 

**2.1.2.2 Stromal cells** 

**2.1.2 Influence of microenvironment on development of breast cancer stem cells**  A limited number of factors have been studied to understand the interaction of microenvironment generated by tumors and its effect on development and maintenance of cancer stem cells. One of the widely studied environment which the solid tumors reside in,

It has been suggested that hypoxia contributes to the generation aggressive cancer by selecting tumor cells and results into growth of cells that can survive compromised levels of oxygen and nutrients (Graeber *et al.*, 1996). Further the growth of tumor results in hypoxic microenvironment, which is followed by periods of reoxygenation. Hence to mimic the invivo environment and to assess the fate of cells undergoing periods of hypoxiareoxygenation Louie E etal., exposed breast cancer cells (MDA-MB-231 and BCM2) to cycles of hypoxia and nutrient deprivation. They discovered that after the first cycle of hypoxia a small fraction of cells survived and that repetitive exposure of the same cells to hypoxia and reoxygenation led to increased viability under hypoxia and to proliferate either as monolayer or tumor spheres. They also found increase in the number of cells expressing CD44+/CD24-/ESA+ cell surface markers, and hence the cancer stem cell content. Therefore repetitive cycling of hypoxia and re-oxygenation can increase the stem cell content of metastatic breast cancer cell lines indicating that microenvironment plays an important role

For a long time scientist have primararily focused on epithelial component of breast cancer, however recently, the critical importance of tumor stroma has been realized. Literature documents important interaction between mammary epithelia and the adjacent tumor stroma. One of the reports demonstrates that CAF increases the number of CD44+CD24- cells in mammospheres, whereas normal fibroblasts (NFs) down-regulated it in mammospheres. They also demonstrate increase in the ability to form epithelial tumors in immunocompromised mice in presence of CAF. This indicates that CAFs can increase the cancer stem cell population in breast cancer (Huang *et al.*, 2010). Furthermore since, CXCR4 expression on carcinoma cells is known to correlate with a poor prognosis for several types of carcinomas (Balkwill, 2004), the authors assessed CXCR4 gene expression in mamosphere co cultured with CAF. They found increase expression of CXCR4 and it was speculated that increase in cancer stem cell population could be because of CXCR4 signaling (Huang *et al.*, 2010).

The normal fibroblasts on the contarary have a inhibitory effect on the tumor growth. For e.g Coculture studies using different mesenchymal cells and MCF10A and preneoplastic MCF10AT1-EIII8 mammary epithelial cells showed that fibroblasts derived from normal reduction mammoplasty inhibit or retard the morphological conversion and growth of MCF10A and EIII8 cells, whereas tumor derived fibroblasts evoke ductal-alveolar morphogenesis of both cell types (Shekhar *et al.*, 2001). Further caveolin-1 deficient (Cav1-/-) mammary stromal fibroblasts were shown to mimic the effects of human breast cancer associated fibroblasts as they show similar profile of RB/ E2F-regulated genes that are upregulated and confer a poor prognosis with enhanced epithelial-mesenchymal transition (EMT) (Sotgia *et al.*, 2009).

Interestingly, genome-wide expression profiling of human breast cancer-associated fibroblasts and Cav-1 (-/-) mammary stromal fibroblasts indicates that they both show the upregulation of a number of ES-cell related genes and factors (Oct4, Nanog, Sox2 and Myctarget genes), indicating that they may behave like "cancer stem cells". Thus, the tumor stromal microenvironment may directly contribute to maintaining the "cancer stem cell" phenotype, leading to drug-resistance and treatment failure (Sotgia *et al.*, 2009).

Fibroblast synthesize growth and survival factors which are critical for the tumor. In breast cancer, stromal fibroblasts evolve with the tumor epithelial cells and assist the growth of tumor cells. Inspite of much known about role of stromal cells the mechanistic basis of such a requirement of fibroblast remains elusive. PTEN is a tumor suppressor and is a critical regulator of PI3K signaling whose activation is associated with activation of tumor stroma (Cully *et al.*, 2006). To understand the role of fibroblast in tumor formation Trimboli et al deleted PTEN from fibroblast in MMTV- ERBB2 mice model. They found that deletion of PTEN from fibroblast results in increase incidence and tumor load in the mice model. Extensive remodeling of ECM and increased recruitment of innate immune cells were some of the salient findings. Gene expression analysis revealed that PTEN deleted stromal fibroblasts consists of activation of Ets2 transcription factor. Further double transgenic mice having inactivation of Ets2 in mammary stroma reversed the increased malignancy caused by PTEN deficiency. These observations show the importance of the PTEN-Ets2 axis in stromal fibroblasts in the *MMTV-ErbB2* model in suppressing breast cancer growth and indicate the stromal pathway contributes to the complexity of human breast cancer stroma (Trimboli *et al.*, 2009).

#### **Mesenchymal stem cells**

Mesenchymal stem cells localize to the breast carcinoma and integrate into tumor associated stroma. A seminal report by Ling X et al., demonstrate that MSC overexpressing IFN-beta inhibit breast cancer growth and metastasis (Ling *et al.*, 2010). They demonstrate that MSC

The Microenvironment of Breast Cancer Stem Cells 243

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

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

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

CSC are rare cells and they are distinct from other bulk tumor cells. They generate the tumor and maintain the tumor hetrogenity. If the CSCs are elemiminated/differentiated to nonCSCs then cancer can be eradicated. The CSC niche maintains the CSC characteristics and increases the CSC potential, hence CSC niche offers a critical window treatment of cancer. Hence strategies that target the pathways critical for selfrenewal which are maintained through niche should be the focus of therapy. Notch, Wnt and Hedgehog pathways are known for maintaining self renewal of normal stem cells (Merchant and Matsui, 2010; Pannuti *et al.*, 2010; Takahashi-Yanaga and Kahn, 2010). These pathways offers targets in combination of other tumor specific markers for CSC targeting. For eg. Farnie, G et al., demonstrated that inhibiting notch signaling using gama secretase inhibitors in DCIS derived cells decreases their mamosphere forming efficiency (Farnie *et al.*, 2007). Further antibodies against the ECM Protein fibronectin receptor α4β1 integrin prevented the interaction of cancer cells with premetastatic niches and reduce the minimal residual disease (Kaplan *et al.*, 2005). Moreover antibodies to fibronectin and β1 integrin promoted epithelial phenotype of invasive breast cancer cells in organotypic three dimentional cultures (Sandal *et al.*, 2007). Hence when formulating such therapeutic modalities a combination of inhibitors/biomolecules which can efficiently inhibit the cancer stem cells self renewal

**2.1.2.4 Embryonic microenvironment** 

(Postovit *et al.*, 2008).

should be considered.

**3. Conclusion** 

homeostasis of growth and normal differentiation.

indicative of differentiation and a reduction in invasive potential.

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

## **2.1.2.3 Stromal factors**

### **IL-6**

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 .

#### **TGF beta**

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 of cancer stem cell population.

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).
