**1. Introduction**

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

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Over 150 years ago, Cohnheim and Durante formalized the concept that cancers might arise from a small subset of cells with stem cell properties 1-3, and in 1961, Till and McCulloch demonstrated for the first time that the existence of hematopoietic stem cells (HSC) in the bone marrow, which was postulated that stem-like cells might be the origin of cancer 4. However, only recently did an increased interest in cancer stem cells (CSC) occur, thus spurring great advances in cancer stem cell biology. The CSC model was first developed in 1994 when malignant initiating cells were discerned in human acute myeloid leukemia (AML) 5. Afterwards, similar CSC model was extended to some solid tumors that originated in the breast, brain, lung, prostate, colon, head and neck, and pancreas 6-12. Most importantly, the development of CSC hypothesis has fundamental implications in terms of understanding the biology of muti-step tumorigenesis, the prevention of cancer, and the creation of novel effective strategies for cancer therapy.

#### **1.1 The definition of cancer stem cells**

It is well documented that tumors contain cancer cells with heterogeneous phenotypes reflecting aspects of their apparent state of differentiation. In a tumor, the mutable expression of normal differentiation markers by cancer cells implies that some of the heterogeneity arises as a result of this altered manifestation. Also, cancer is known to be the product of the accumulation of multiple genetic mutations and epigenetic alterations in a single target cell, the occurrences of which can sometimes take place over many decades. Furthermore, chemotherapy and radiation therapy for cancers have limited effectiveness in long-term scenarios, and the possible recurrence of tumors after years of disease-free survival exists in great majority of cancers. All these observations provide persuasive evidence that tumors are not mere monoclonal expansions of cells but might contain a subset of long-lived tumor-initiating cells with the ability to self-renew indefinitely and to regenerate the phenotypic diversity of original tumor 13. This subpopulation is now widely termed as cancer stem cells (CSCs), also named tumor-initiating cells (T-IC). The exist of CSCs within a tumor was also supported by *in vitro* ''clonogenic assays'' that showed subpopulations of tumor cells (with increased proliferative capacity) using cells isolated from tumor specimens, as well as by *in vivo* self-renewal assays that indicated only a small specific subset of cancer cell population had tumorigenic potential when injected into immunodeficient mice 13, 14.

Breast Cancer Stem Cells 275

subpopulation of cells was further enriched by sorting for those that expressed epithelial surface antigen (ESA). More interestingly, 200 of the enriched ESA+CD44+CD24−*/*low cells were able to form a tumor following injection into a NOD/SCID mouse, while 20,000 of the CD44+CD24+ cells failed to do so 7. In summery, these results opened a new chapter in the

Soon after, Michael F. Clarke's group published similar data about CSCs in brain tumors 8, 31. They carried out studies to enrich tumorigenic cells in glioblastoma multiforme and medulloblastoma by sorting for those that express positive / high levels of CD133, a neural cell surface stem cell antigen. CD133high cells formed numerous colonies in suspension culture, and injection of as few as 1000 of these cells into an immunocompromised mouse successfully form a tumor. Conversely, CD133low cells showed very limited proliferative potential *in vitro*, and as many as 10,000 of these cells failed to seed tumors in host mice 8. Furthermore, tumors developing from orthotopic, intracerebral injection of the minority of CD133+/high cells (about 5% - 30% of total tumor cells) reproduced the phenotypic diversity

As mentioned earlier, comparable results have been obtained in other solid tumors, like

In most tumor tissues, including breast cancer, CSCs are rare. As we know, breast cancer is a histologically and molecularly heterogeneous disease, with six different subtypes, including luminal A, luminal B, normal breast-like, basal-like, claudinlow and HER2 overexpressing, which are characterized by distinct histology, gene expression patterns, and genetic alterations 32-35. The molecular heterogeneity between breast cancers has been revealed to issue from different targets of transformation. Recent studies found that basal-like breast cancers with BRCA1 mutations were more likely to arise from luminal progenitors rather than the basal stem cells 36, 37. However, further studies that focus on breast CSCs and mammary stem/progenitor cells as well as their potential relationship are needed for determining the exact origin of luminal versus basal-like cancers, with the aim of developing targeted therapies for different subtypes of breast cancers. Moreover, CSCs was found to be the main culprit for the failure of chemo- and radiation therapy, as well as the seeds for the distant metastasis and relapse in breast cancers 20, 32, 38-40. Taken together, in order to better understand the properties and biology of breast CSCs and eventually cure breast carcinoma, it is absolutely necessary and important to identify and separate breast

Since Dick, *et al* isolated a specific subpopulation of leukemia cells (that expressed surface markers similar to normal hematopoietic stem cells) which was consistently enriched for clonogenic activity in NOD/SCID immunocompromised mice from acute myeloid leukemias in the 1990s 5, 30, scientists attempted to see if they could enrich CSCs in human solid tumors by sorting for different cellular markers. CD24, a ligand for P-selectin in both mouse and human cells, was identified as a significant marker for human breast carcinoma invasion and metastasis 41, 42, and another adhesion molecular CD44 was found to correlate with cellular differentiation and lymph node metastasis in human breast cancers 43, 44, whereas B3.8 was described as a breast / ovarian cancer-specific marker 45. Based on these

understanding of the biology of CSCs in human solid tumors.

and differentiation pattern of the parent tumors 31.

CSCs prospectively.

lung, prostate, colon, head and neck, as well as pancreatic 6, 9-12.

**2. Isolation and identification of breast cancer stem cells** 

**2.1 Isolation of breast CSCs with cell-surface marker profiles** 

The definition of CSCs is defined by two main properties: 1) self-renewal that drives tumorigenesis: the ability to form new CSCs with potential for proliferation, expansion, and differentiation; 2) multipotent differentiation, which contributes to the cellular heterogeneity of a tumor: the ability to give rise to a heterogeneous progeny of tumor cells, which diversify in a hierarchical manner.

When distinguished from the majority of differentiated cancer cells, CSCs are resistant to many current cancer treatments, including chemo- and radiation therapy 15-20. This suggests that lots of cancer treatments, while targeting the majority of tumor cells, may fail in the end due to not eliminating CSCs, which survive by developing new tumors. However, this would open avenues for developing novel effective drugs targeting CSCs. Although CSCs share several properties (i.e. the ability to self-renew and to differentiate, increased membrane transporter activity, the capacity for migration and metastasis, the same intrinsic signaling pathways (Notch, Wnt, Hedgehog etc) for regulation of self-renewal etc) with the normal stem cells 21, they are found to have some particular characteristics. For instance, the proliferation and self-renewal of CSCs are uncontrolled and unlimited (sometimes referred to as "immortality"), and the CSCs always differentiate into abnormal cancer cells, thus they cannot give rise to mature somatic cells 22. This reveals that therapies targeted at extrinsic signals generated in the microenvironment (such as CXCR1, endothelial cell-initiated signaling, IL-6 and CXCL7) 23-25 or microRNAs (see Part 3 of this chapter) 26-29, which are found to specifically regulate self-renewal and/or differentiation of CSCs, might achieve clinical success with little adverse effects in cancer treatment.

#### **1.2 Leukemia stem cells: The first cancer stem cells identified**

In the early 1990s, Dick and his colleagues started a series of groundbreaking investigations to understand whether the functional hierarchy observed in normal hematopoiesis was conserved in leukemia 5, 30. They used magnetic separation techniques and purified cells from AML patients into several groups according to different surface markers. These groups of cells were then implanted into immunocompromised mice and assessed for the ability to produce leukemic colony forming units. Interestingly, only the CD34+ CD38 subpopulation of leukemic cells had the ability to generate substantially more leukemic colonies *in vivo*. As well, they found that CD34+ CD38 leukemic stem cells retained differentiative capacity, giving rise to CD38+ and Lin+ populations. These observations provided the first compelling evidence that in a human cancer, there was a small population of self-renewing, tumorigenic stem cells.

#### **1.3 Solid tumor stem cells**

Subsequent experiments extended the leukemic stem cell model to human solid tumors. In the year 2003, Al-Hajj *et al* reported the identification of CSCs in human breast cancer, the first solid tumor that the existence of a functional hierarchy stem cell system had been demonstrated 7. In their experiments, human breast cancer specimens obtained from primary or metastatic sites in nine different patients all engrafted in the NOD/SCID (nonobese diabetic/severe combined immune deficiency) mice. They observed that in most human breast cancers, only a minority subset of the tumor clones (defined as CD44+, CD24−*/*low and representing 11%–35% of total cancer cells) is endowed with the capacity to maintain tumor growth when xenografted in NOD/SCID mice. Importantly, tumors grown from the CD44+, CD24−*/*low cells were shown to contain mixed populations of epithelial tumor cells, recreating the phenotypic heterogeneity of the parent tumors. The small

The definition of CSCs is defined by two main properties: 1) self-renewal that drives tumorigenesis: the ability to form new CSCs with potential for proliferation, expansion, and differentiation; 2) multipotent differentiation, which contributes to the cellular heterogeneity of a tumor: the ability to give rise to a heterogeneous progeny of tumor cells, which

When distinguished from the majority of differentiated cancer cells, CSCs are resistant to many current cancer treatments, including chemo- and radiation therapy 15-20. This suggests that lots of cancer treatments, while targeting the majority of tumor cells, may fail in the end due to not eliminating CSCs, which survive by developing new tumors. However, this would open avenues for developing novel effective drugs targeting CSCs. Although CSCs share several properties (i.e. the ability to self-renew and to differentiate, increased membrane transporter activity, the capacity for migration and metastasis, the same intrinsic signaling pathways (Notch, Wnt, Hedgehog etc) for regulation of self-renewal etc) with the normal stem cells 21, they are found to have some particular characteristics. For instance, the proliferation and self-renewal of CSCs are uncontrolled and unlimited (sometimes referred to as "immortality"), and the CSCs always differentiate into abnormal cancer cells, thus they cannot give rise to mature somatic cells 22. This reveals that therapies targeted at extrinsic signals generated in the microenvironment (such as CXCR1, endothelial cell-initiated signaling, IL-6 and CXCL7) 23-25 or microRNAs (see Part 3 of this chapter) 26-29, which are found to specifically regulate self-renewal and/or differentiation of CSCs, might achieve

In the early 1990s, Dick and his colleagues started a series of groundbreaking investigations to understand whether the functional hierarchy observed in normal hematopoiesis was conserved in leukemia 5, 30. They used magnetic separation techniques and purified cells from AML patients into several groups according to different surface markers. These groups of cells were then implanted into immunocompromised mice and assessed for the ability to produce

leukemic cells had the ability to generate substantially more leukemic colonies *in vivo*. As well,

to CD38+ and Lin+ populations. These observations provided the first compelling evidence that in a human cancer, there was a small population of self-renewing, tumorigenic stem cells.

Subsequent experiments extended the leukemic stem cell model to human solid tumors. In the year 2003, Al-Hajj *et al* reported the identification of CSCs in human breast cancer, the first solid tumor that the existence of a functional hierarchy stem cell system had been demonstrated 7. In their experiments, human breast cancer specimens obtained from primary or metastatic sites in nine different patients all engrafted in the NOD/SCID (nonobese diabetic/severe combined immune deficiency) mice. They observed that in most human breast cancers, only a minority subset of the tumor clones (defined as CD44+, CD24−*/*low and representing 11%–35% of total cancer cells) is endowed with the capacity to maintain tumor growth when xenografted in NOD/SCID mice. Importantly, tumors grown from the CD44+, CD24−*/*low cells were shown to contain mixed populations of epithelial tumor cells, recreating the phenotypic heterogeneity of the parent tumors. The small

leukemic stem cells retained differentiative capacity, giving rise

subpopulation of

diversify in a hierarchical manner.

they found that CD34+ CD38-

**1.3 Solid tumor stem cells** 

clinical success with little adverse effects in cancer treatment.

**1.2 Leukemia stem cells: The first cancer stem cells identified** 

leukemic colony forming units. Interestingly, only the CD34+ CD38-

subpopulation of cells was further enriched by sorting for those that expressed epithelial surface antigen (ESA). More interestingly, 200 of the enriched ESA+CD44+CD24−*/*low cells were able to form a tumor following injection into a NOD/SCID mouse, while 20,000 of the CD44+CD24+ cells failed to do so 7. In summery, these results opened a new chapter in the understanding of the biology of CSCs in human solid tumors.

Soon after, Michael F. Clarke's group published similar data about CSCs in brain tumors 8, 31. They carried out studies to enrich tumorigenic cells in glioblastoma multiforme and medulloblastoma by sorting for those that express positive / high levels of CD133, a neural cell surface stem cell antigen. CD133high cells formed numerous colonies in suspension culture, and injection of as few as 1000 of these cells into an immunocompromised mouse successfully form a tumor. Conversely, CD133low cells showed very limited proliferative potential *in vitro*, and as many as 10,000 of these cells failed to seed tumors in host mice 8. Furthermore, tumors developing from orthotopic, intracerebral injection of the minority of CD133+/high cells (about 5% - 30% of total tumor cells) reproduced the phenotypic diversity and differentiation pattern of the parent tumors 31.

As mentioned earlier, comparable results have been obtained in other solid tumors, like lung, prostate, colon, head and neck, as well as pancreatic 6, 9-12.
