**1. Introduction**

Ernst Haeckel first described the term "stem" as a concept for the evolution or organisms. For representation purpose he described the ancestor organism as a "stem" from which all the other organisms evolved. Arthur Pappenheim later adopted this concept in the context of cells, and he elegantly placed the "stem cell" in the centre in cartoon from which all the blood cells arise describing hematopoiesis (Ramalho-Santos and Willenbring, 2007).

The concept was carried forward and the term "cancer stem cell" was first coined in 1980 (Carney *et al.*, 1982) where the authors described the stem cell origin of lung cancer cells. The difficulty in isolation and the absence of specific markers of cancer stem cell stalled the research in this area. However a decade later Bonnet and Dick successfully isolated CSC in AML which then incited the development in the field of cancer stem cells (Bonnet and Dick, 1997). Their discovery was later supported by many groups, which also resulted in isolation of CSC from a variety of malignancies including solid tumors.

Now a large body of evidence suggests that cancer comprises of different population of cells with various tumorogenic potentials. The tumor cells follow a hierarchy, where the subset capable of self-renewal, generate the tumor heterogeneity and are called cancer stem cells (CSC). Very low number of these cancer stem cells generates tumors in immunocompromised mice whereas large number of non-CSCs fails to generate tumors.

CSCs have been characterized based on their ability to form colonies in soft agar and their ability to form spheres in serum free media. The generation of tumors in immunocompromised mice however remains the gold standard. Another characteristic of CSC is their ability to resist the action of common chemotherapeutic drugs which is attributed to higher expression of ABC transporters and their slow cycling nature. Further it has also been documented that these CSCs have activated signaling pathways as in the case of normal stem cells. Hence CSCs are distinct from other non-CSC in many respects.

Cancer stem cells have been isolated based on membrane markers. One of the characteristics is their ability to efflux the Hoechst dye. However this ability to efflux the dye is also attributed to membrane ABC transporter ABCG2. ABCG5 has been used as a cancer stem cell marker as it pumps out the drug doxorubicin. ALDH1 has the ability to convert retinol to retinoic acid, which has diverse role in cell physiology, and this activity is used as a marker for CSC. CD 44, CD 133, EpCAM and CD 90 are also abundantly expressed in CSCs and are used to isolate or enrich CSC (Visvader and Lindeman, 2008). A number of groups have isolated CSC based on these markers however a robust marker for CSC still remains to be identified.

The Microenvironment of Breast Cancer Stem Cells 239

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

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

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

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

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

environment and is important for optimal function of epithelial cells.

**2.1.1 Microenvironment of breast cancer cells** 

breast cancer cells more metastatic.

factors in breast cancer progression.

(Kaplan *et al.*, 2005).

breast cancer.
