**2.1 Role of microenviroment in mammary gland development**

The breast tissue is composed of multiple cell types for proper functioning of tissue and the primary function of which is production of milk. During lactation milk is produced by the luminal epithelial cells and secreted in the hollow cavity. The luminal epithelial cells are surrounded by myoepithelial cells, which synthesize the basement membrane. Together the luminal epithelia and the myoepithelia form the milk duct. Different cell types whose function is to maintain the homeostasis surround milk duct. These cells include fibroblasts, leucocytes and endothelial cells.

The environment of epithelial cells plays a critical role in shaping their function. For eg. When the epithelial cells from breast tissue were placed on plastic, they were unable to produce milk and exhibited different phenotype as compared to the cells when plated in 3 dimentional reconstituted basement membrane (Matrigel) which led to proper function of epithelial cells (Howlett and Bissell, 1993; Petersen *et al.*, 1992). Hence proper cellular interaction and spatial localization of cells with the right constituents are required for

A number of theories have been proposed for the generation of these CSCs. (1)CSC can originate from genetic/ epigenetic alteration of normal stem cells or from the progenitor cells. (2) They can be derived from somatic tumor cells by de differentiation or reprogramming into a stem- like cell (Visvader and Lindeman, 2008). (3) And recently it has been suggested that CSC can be generated from non-CSC through production secretary

The existence of cancer stem cells was first demonstrated in solid tumors by Al Hajj et al.,

cellular markers (Al-Hajj *et al.*, 2003). They isolated the cells from primary breast cancer or metastatic pleural effusions and injected them directly in to mice or after cellular sorting

while CD44- , CD24- were unable to form tumors in immunocompromised mice. Further they performed repopulation assays where they found that the tumorigenic population (CD44+ / CD24- Lin-) was able to give rise to phenotypic heterogeneity of the initial tumor. This suggested that the breast cancer stem cells undergo self-renewal and differentiation as in the case of normal stem cells. After this report a large number of studies identified CSC from various other malignancies (Curley *et al.*, 2009; Fang *et al.*, 2005; Kondo *et al.*, 2004; Liu

The normal stem cells reside in a distinct environment called the "stem cell niche". This stem cell niche consists of complex composition of ECM, soluble factors, stromal cells, immune cells which are responsible for maintaining the self renewal ability of stem cells. Similarly the CSCs also depend on similar environment, which may be altered in many ways. Moreover in some of the tumors, the tumor niche has been shown to have a protective role from genotoxic insults (Garcia-Barros *et al.*, 2003). Although much research has been done on understanding the cancer stem cells, very few studies have been carried out on understanding the microenvironment of breast cancer stem cells and their targeting. We believe that understanding the breast cancer microenvironment will offer easily tractable

The breast tissue is composed of multiple cell types for proper functioning of tissue and the primary function of which is production of milk. During lactation milk is produced by the luminal epithelial cells and secreted in the hollow cavity. The luminal epithelial cells are surrounded by myoepithelial cells, which synthesize the basement membrane. Together the luminal epithelia and the myoepithelia form the milk duct. Different cell types whose function is to maintain the homeostasis surround milk duct. These cells include fibroblasts,

The environment of epithelial cells plays a critical role in shaping their function. For eg. When the epithelial cells from breast tissue were placed on plastic, they were unable to produce milk and exhibited different phenotype as compared to the cells when plated in 3 dimentional reconstituted basement membrane (Matrigel) which led to proper function of epithelial cells (Howlett and Bissell, 1993; Petersen *et al.*, 1992). Hence proper cellular interaction and spatial localization of cells with the right constituents are required for

 Lin as

were able to form tumors

where CSCs were identified from human breast cancer tissue using CD44+ / CD24-

with the above mentioned markers. They found that CD44+, CD24-

**2.1 Role of microenviroment in mammary gland development** 

*et al.*, 2007; Prince *et al.*, 2007; Singh *et al.*, 2004).

**1.1 Origin of CSC** 

molecules (Iliopoulos *et al.*, 2011).

**2. Breast cancer stem cells** 

solutions to cancer therapy.

leucocytes and endothelial cells.

correct functioning of epithelial tissue. This was explained by the fact that invivo normal mammary gland are in contact with myoepithelial cells and not the basement membrane. Further luminal epithelial cells display apical–basal polarity as demonstrated by MUC 1, ESA and occludin expression on the apical membrane and ß4 integrin on the basolateral membrane. However such a polarity is observed when luminal cells are grown in matrigel but not in collagen(Gudjonsson *et al.*, 2002). The polarity is restored when the myoepithelial cells are co- cultured with luminal epithelial cells even in collagen, which is mediated by laminin 1 secreted by myoepithelial cells. These studies demonstrate the role of 3D environment and is important for optimal function of epithelial cells.

#### **2.1.1 Microenvironment of breast cancer cells**

A large number of reports demonstrate that breast tumor progression is facilitated by stromal cells and that their presence is critical for survival of cancer cells. However it is also important to note that the normal mammary gland microenvironment has inhibitory effect on breast cancer progression (DeCosse *et al.*, 1973). This indicates that cancer cells can maintain their properties only in an abnormal microenvironment. One of the recent reports underlies the role of mesenchymal stem cells in amplifying the metastatic potential of weakly metastatic cells. Karnoub A et al mixed a weakly metastatic cell line MDA MB 231 with bone marrow derived human MSC and found that the metastatic potential of the cell line is dramatically increased (Karnoub *et al.*, 2007). To further understand the mechanism of this increase in metastatic potential they used a cytokine array to identify soluble factors. They found CCL5 release, which was induced by physical interaction between breast cancer cells and the MSC, and that it renders the breast cancer cells more metastatic.

Another seminal report by Kaplan et al demonstrate that bone marrow- derived hematopoietic progenitors may localize to future sites of metastasis and "prepare" the sites for the arrival and growth of disseminated cancer cells (Kaplan *et al.*, 2005). This has been proposed a new concept in metastasis, which is called the "premetastatic niche". The precise mechanism and the factors responsible for such localization of bone marrow derived hematopoietic progenitors is unclear however it appears to be derived from the serum (Kaplan *et al.*, 2005).

One of the extensive study in understanding the breast cancer microenvironment, Allinen et al. performed genome wide gene expression analysis of stromal cells (Endothelial cells, infiltrating leukocytes, fibroblasts, and myofibroblasts) and breast epithelial cells (luminal epithelial and myoepithelial cells) from normal, insitu carcinoma and invasive carcinoma. The authors found that alterations in gene expression takes place in all cell types however clonally selected genetic alterations are confined to tumor epithelial cells. Further there were consistent and significant alterations in myoepithelial cells from DCIS as compared to normal myoepithelial cells and many of these changes were in secreted proteins and cell surface receptors (Allinen *et al.*, 2004). This further underlines the importance of soluble factors in breast cancer progression.

Although a large amount of literature is present on microenvironment of breast cancer cells, there are few studies on cancer stem cell microenviroenment. This is ascribed to the age of this new field however research in this direction will significantly impact the therapy of breast cancer.

The Microenvironment of Breast Cancer Stem Cells 241

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

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

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"

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

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

phenotype, leading to drug-resistance and treatment failure (Sotgia *et al.*, 2009).

population could be because of CXCR4 signaling (Huang *et al.*, 2010).

(EMT) (Sotgia *et al.*, 2009).

(Trimboli *et al.*, 2009). **Mesenchymal stem cells** 

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