**6.** *In vitro* **and** *in vivo* **breast cancer models and their relevance to human disease**

In 2007, Vargo-Gogola and Rosen summarized appropriately, within their title, the challenge of modelling breast cancer; "One size does not fit all"[31]. Considering the heterogeneity nature of this disease, these researchers rather addressed the reasonable question of the most powerful way to investigate breast cancer with respect to cell lines and animal models. We agree with their conclusion that an integrated and multi-systems approach is the strongest way to model this disease. Here, we briefly discuss the most commonly utilized *in vivo* and *in vitro* models and their relevance in human breast cancer.

#### **6.1. Mammary carcinoma cell lines**

Our current knowledge of breast cancer is mainly based on *in vivo* and *in vitro* studies com‐ pleted with breast cancer cell (BCC) lines. The first BCC line, namely BT-20, was established in 1958 [119] and since then the number of permanent lines sustained are relatively low despite continuous research in breast cancer [120]. In fact, the number of commercially available BCC *5.2.1. The role of estrogens and breast cancer*

354 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

of genes, including those coding for proliferation.

**disease**

is not representative of the term hormone replacement therapy [118].

*vitro* models and their relevance in human breast cancer.

**6.1. Mammary carcinoma cell lines**

The link involving estrogen and breast cancer can be traced back to as early as 1896 when a surgeon in England reported an improvement in condition in three young female breast cancer patients after the removal of their ovaries [111]. Since estrogen had not yet been discovered, the surgeon had unknowingly removed the source of estrogen that promotes the survival and division of cancer cells [111]. Since that time, a lot of evidence has shown that interactions between estrogens and their receptors influence the pathogenesis of breast cancer. Estrogen promotes proliferative effects on cultured human breast cancer cells [112]. Estradiol affects breast cancer risk by controlling the mitotic rate of breast epithelial cells and high levels of estradiol in post-menopausal women are also known to increase the risk of breast cancer [113]. Estradiol has also been shown to increase breast cancer risk via its metabolite, catechol estrogen 4-hydroxyestradiol, causing direct DNA damage through the formation of free radicals [114]. Estradiol has also been shown to modulate breast cancer cell apoptosis [115]. ER is a major determinant of the cellular response of estrogen and has been indicated in breast cancer promotion [116, 117]. The binding of estrogen to the ER modulates the transcription of a series

The close relationship between the etiology of breast cancer and exposure to estrogen warrants examination of key variables that may affect estrogen homeostasis, in particular those exhibiting anti-estrogen activity. Because of their impact on the primary and metastatic aspects of disease, anti-hormonal drugs are the mainstay breast cancer treatment. The goal in treating hormone receptor +ve breast cancers is to utilize drugs which suppress production of estrogen in the body. Estrogens in naturally occurring dietary compounds such as soy are used as an alternative to hormone therapy because of their anti-proliferative effects. This practice, in breast cancer treatment, is widely known as hormonal therapy, or anti-estrogen therapy, but

**6.** *In vitro* **and** *in vivo* **breast cancer models and their relevance to human**

In 2007, Vargo-Gogola and Rosen summarized appropriately, within their title, the challenge of modelling breast cancer; "One size does not fit all"[31]. Considering the heterogeneity nature of this disease, these researchers rather addressed the reasonable question of the most powerful way to investigate breast cancer with respect to cell lines and animal models. We agree with their conclusion that an integrated and multi-systems approach is the strongest way to model this disease. Here, we briefly discuss the most commonly utilized *in vivo* and *in*

Our current knowledge of breast cancer is mainly based on *in vivo* and *in vitro* studies com‐ pleted with breast cancer cell (BCC) lines. The first BCC line, namely BT-20, was established in 1958 [119] and since then the number of permanent lines sustained are relatively low despite continuous research in breast cancer [120]. In fact, the number of commercially available BCC lines (ranging from 70 – 80 based on ATCC and similar commercial suppliers) represent mostly cell lines that were established more than 25 years ago. It has been suggested that this ineffi‐ ciency in producing new and improved lines is likely due to technical difficulties in extracting viable tumor cells from their neighboring stroma [120]. In the past, it has been estimated that more than two-thirds of all scientific abstracts related to studies on mentioned BCC lines include the MCF-7, T-47D, and MDA-MB-231 lines [120]. Although no single cell line is entirely representative of human breast cancer, cell lines have been widely used. As a result, we have a greater understanding of breast cancer biology. Additionally, these cell lines have advanced the ability of pre-clinical models to predict pharmaceutical activity and therapeutic applica‐ tions for further study. Despite this, the use of cell lines for application in human disease is limited. The single most significant question, considering the devastating effects or outcomes of metastasis, is the issue of whether these cell lines are representative of metastatic conditions, or contain tumor initiating subpopulations. There are studies to indicate distinctions between tumor and non-tumor initiating populations at the cellular and molecular level [31, 121]. Research indicates that the two luminal subsets (A and B) evident in tumors are not apparent in the cell lines, and the basal-like cell lines are actually representative of two distinctive clusters (A and B) that are not apparent in analyses of primary tumors [16]. The differences in cell culture may be due to the absence of stromal or physiological interactions and/or signaling [122] [123]. However, there is evidence that cell lines may be derived from subpopulations of tumor cells that are selected because they grow well. For example, it has been noted that differences between the genome aberration patterns for the basal-like and luminal clusters in the cell line system don't match differences in these subtypes in primary tumors. Furthermore, the highly invasive Basal B cells carry the distinctive phenotype associated with the subpo‐ pulation of tumorigenic stem cells identified in breast cancer [50, 124]. Also, one must consider the issue of variants of the same cell lines and the phenotypic changes that can occur due to environmental exposures, rate of passage and age of cells.

Perhaps the most highly debated topic regarding the use of breast cancer cell lines revolves around the MDA-MB-435 cell line, and by extension MDA-MB-435S (ATCC® HTB-129™) and derivatives. Spontaneously metastatic and originally designated as Basal B and being derived from a breast carcinoma, the MDA-MB-435 cell line has subsequently been questioned because of recent evidence indicating that it might originate from melanoma or may have been crosscontaminated with the M14 melanoma cell line [125-130]. While some researchers continue to use this cell line as a breast cancer mouse model [131] and conclude that this cell line is in fact breast cancer cells [132], others insist that the MDA-MB-425 cell line is a melanoma cell line and renders its use as improper in breast cancer studies [133]. Later in the chapter, we will describe some research that has been conducted using MDA-MB435 cell lines and/or its derivatives as breast cancer models in research involving natural, despite this controversy. This may not be all bad news for natural product researchers. On the bright side, natural product therapies indicated in the controversial cells line MDA-MB-435 and its derivatives then point toward activity in melanoma as opposed to breast cancer.

Nonetheless, there is clearly a need for new cell lines that are representative of all the known sub-types of breast cancer. Currently, researchers have the option of using a group (or panel) of subtype cell lines to increase power in their research, as proposed by Neve [2006] [129]. To date, and based on our knowledge, there has been no evidence of a male breast cancer derived cell line. With the incidence of male breast cancer rising [2], relevant models require further development.

Cell lines are easy to culture, inexpensive in comparison to animal models, and provide an unlimited source of homogenous material to work with. Comparisons between researcher and studies are usually fairly consistent and reproducible. A limitation of cell cultures is their inability to measure tumor-stromal cell interactions, however the growth of cell lines for transplantation allows for such *in vivo*.

#### **6.2. Animal models**

Animal models have allowed researchers to gain much insight into the disease progression, in particular metastasis. In addition to advancing the concept of metastasis [134, 135], our knowledge of the biological function of genes and signaling pathways has progressed as a result of the vast amount of information generated from animal models. Experimental systems based on mouse models that are currently used to study breast cancer can be categorized as tumor transplantation and genetically engineered mice (GEM) – often termed as transgenic. Here we will focus on commonly utilized models within each of these categories and discuss some of the advantages and limitations of each in relation to human breast cancer disease relevance.

#### *6.2.1. Transgenic or (GEM) models*

Different GEM breast cancer models are useful for studying distinct signaling interactions and for testing therapies that target pathways involved. Genomic deletion of tumor suppressor genes or the transgenic insertion of oncogenes allow these mice to be a relevant tool to investigate the spontaneous initiation of breast tumors in each step of metastasis [136]. Specific to breast cancer, the expression of oncogenes in explicit breast regions are restricted via a mammary gland specific promoter [136]. MMTV (mouse mammary tumor virus) or WAP (Whey Acidic Protein) promoter control the expression of oncogenes (i.e: PyMT, ErbB2, Wnt1, or Ras) in transgenic mice and initiate mammary gland tumors which lead to metastasis in various organs [137]. The MMTV-PyMT transgenic mouse model represents hyperplasia, adenoma, and early or late carcinoma as seen in human cancer stages. In breast cancer, this model demonstrates short latency tumor development specific to the mammary gland with a high incidence pulmonary metastasis [138]. The deletion of mammary epithelial cell-specific tumor suppressor genes results in similar conditions observed in human cancer patients, including spontaneous tumors, bone metastasis, loss of estrogen receptor (ER) expression, and hormone-responsiveness [139, 140]. Crossing GEM mice with other transgenic mice has allowed researchers to investigate the role of genes and signaling pathways in tumorigenesis and metastasis. While tumor progression was delayed in MMTV-PyMT or MMTV-ErbB2/neu mice crossed into an Akt or PTP1B-deleted genetic background [141, 142], increased metastasis was observed in the MMTV-PyMT mouse in a CD44\_/\_ background; the later indicates the importance of receptors and other secreted factors in epithelial–stromal interactions or overall tumor environment [139]. In this regard, much insight has been gained through the use of GEM models to indicate the role of both innate and adaptive immune responses in the progression of breast cancer. For example, pulmonary metastasis is notably diminished via the selective loss of the interleukin (IL-) 4 cytokine in PyMT/IL-4\_/\_ mice through inhibition of IL-4 mediated EGFR signaling of mammary tumors [143]. The understanding of such interactions are important if we consider that tumor-associated immune cells release numerous growth factors, including cytokines, chemokines, and enzymes that promote tumor growth, angio‐ genesis, and metastasis, and thus are associated with poor prognosis [136]. Inducible GEM mouse models (those capable of expression or repression of certain genes) have advanced knowledge with respect to the association of multiple genetic mutations of oncogenes in relation to tumor establishment and maintenance [144]. There are several drawbacks to consider in the use of GEM models. There are inherent differences in mouse and human, for example: BRCA1 and p53 are on the same chromosome in mice, but not in humans [136, 144]. Technical discrepancies, depending on methods used to generate the model and the interpre‐ tation of results can present varying outcomes. Also, temporal and spatial tumor development and metastasis differ depending on the mouse strain used. For example the percentage of ER/ progesterone receptor (PR)+ve tumors in Wap-Cre/Trp53\_/\_ mice are high while MMTV-Cre/ Trp53\_/\_ mice report a low incidence of such [145]. The molecular profile of mammary tumors from GEM models represent the subtypes of human breast cancers, including those included in Table 1, however no single model to date is representative of all expression patterns and characteristics of human cancer.

#### *6.2.2. Tumor transplantation model*

of subtype cell lines to increase power in their research, as proposed by Neve [2006] [129]. To date, and based on our knowledge, there has been no evidence of a male breast cancer derived cell line. With the incidence of male breast cancer rising [2], relevant models require further

Cell lines are easy to culture, inexpensive in comparison to animal models, and provide an unlimited source of homogenous material to work with. Comparisons between researcher and studies are usually fairly consistent and reproducible. A limitation of cell cultures is their inability to measure tumor-stromal cell interactions, however the growth of cell lines for

Animal models have allowed researchers to gain much insight into the disease progression, in particular metastasis. In addition to advancing the concept of metastasis [134, 135], our knowledge of the biological function of genes and signaling pathways has progressed as a result of the vast amount of information generated from animal models. Experimental systems based on mouse models that are currently used to study breast cancer can be categorized as tumor transplantation and genetically engineered mice (GEM) – often termed as transgenic. Here we will focus on commonly utilized models within each of these categories and discuss some of the advantages and limitations of each in relation to human breast cancer disease

Different GEM breast cancer models are useful for studying distinct signaling interactions and for testing therapies that target pathways involved. Genomic deletion of tumor suppressor genes or the transgenic insertion of oncogenes allow these mice to be a relevant tool to investigate the spontaneous initiation of breast tumors in each step of metastasis [136]. Specific to breast cancer, the expression of oncogenes in explicit breast regions are restricted via a mammary gland specific promoter [136]. MMTV (mouse mammary tumor virus) or WAP (Whey Acidic Protein) promoter control the expression of oncogenes (i.e: PyMT, ErbB2, Wnt1, or Ras) in transgenic mice and initiate mammary gland tumors which lead to metastasis in various organs [137]. The MMTV-PyMT transgenic mouse model represents hyperplasia, adenoma, and early or late carcinoma as seen in human cancer stages. In breast cancer, this model demonstrates short latency tumor development specific to the mammary gland with a high incidence pulmonary metastasis [138]. The deletion of mammary epithelial cell-specific tumor suppressor genes results in similar conditions observed in human cancer patients, including spontaneous tumors, bone metastasis, loss of estrogen receptor (ER) expression, and hormone-responsiveness [139, 140]. Crossing GEM mice with other transgenic mice has allowed researchers to investigate the role of genes and signaling pathways in tumorigenesis and metastasis. While tumor progression was delayed in MMTV-PyMT or MMTV-ErbB2/neu mice crossed into an Akt or PTP1B-deleted genetic background [141, 142], increased metastasis was observed in the MMTV-PyMT mouse in a CD44\_/\_ background; the later indicates the importance of receptors and other secreted factors in epithelial–stromal interactions or overall

development.

**6.2. Animal models**

relevance.

transplantation allows for such *in vivo*.

356 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

*6.2.1. Transgenic or (GEM) models*

Syngeneic transplantation refers to the transfer of cancer cells from one mouse into another with identical genetic background [146] and such models are used to establish organ specific metastasis [147]. The 4T1 breast cancer tumor model (a syngeneic mouse model) is considered excellent for testing experimental cell-based immunotherapy strategies and is comparable with stage IV human breast cancer [148]. The injection of 4T1 cells, originally derived from a spontaneous mouse mammary tumor are injected into the mammary fat pad of a syngeneic animal and the rapid proliferation of cells forms tumors and eventually metastasizes to the lungs, liver, bone and brain [146, 147, 149]. Furthermore, this model has been refined through the development of 4T1 cell lines to employ varying degrees of metastasis that are location specific and representative of distinct gene expression signatures [150]. Xenograft transplan‐ tation encompasses the use of human cancer cells into immuno-compromised mice via intravenous, intraperitoneal or subcutaneous injection, orthptopically or ectopically [136, 151]. First discovered in 1962, nude mice have been commonly used for xenotransplantation because they lack a thymus and are unable to mount most types of immune responses, including rejection of allografts and xenografts [152]. Severe Combined Immunodeficiency (SCID) mice present with impaired ability to make T or B lymphocytes and are used as model organisms for research into transplantation strategies as they cannot reject tumors and transplants [153]. In general, rodents with immunological defects are typically resistant to growth of mammary carcinomas, and despite improvements these approaches have yielded low percentage of successful breast tumor engraftment compared to other types of cancers [154]. Recent mouse models have contributed significantly to the understanding of breast cancer, however further research into suitable animal models will be required to advance development of new therapies for breast cancer. The growth and metastasis of human breast cancer cell lines *in vivo* allows the measurement of gene function relative to disease progression and has provided much insight into the use of investigational drugs intended interrupt or interfere with tumor growth [155].

Table 3 provides an overview of the defining characteristics of established breast cancer cell lines (mouse and human) commonly used in both *in vitro* and transplantation models.


**Table 3.** Breast Cancer Cell Lines for *in vitro* and transplantation models

It is unlikely that any one transplantation model will ever replicate the complexity of the whole cancer process; however studies to date demonstrate that xenographs are relevant in human breast cancer. Treatment with Herceptin was shown to improve the anti-tumor activity of paclitaxel and doxorubicin against HER2/neu-overexpressing human breast cancer xenografts leading to consecutive favorable clinical trials [169, 170]. Davis *et al.* [2004] reported the effective inhibition of tumor growth and metastasis in an orthotopic xenograft model by the use of combination therapy of paclitaxel and neutralizing antibodies targeting vascular endothelial growth factor receptor 2 (VEGFR2] [171]. The results of this research likely led to the development of bevacizumab, a humanized monoclonal antibody that targets vascular endothelial growth factor A (VEGF-A) [172]. Although, bevacizumab was removed as a breast cancer indication by the FDA [173], this is yet another example of how transplantation models lead to further development in the clinic. While these are merely a few cases for breast cancer and the use of xenograft studies, the information obtained from such has been translated into successful clinical trials for a variety of cancers [174-178]. Furthermore, useful information has been gained from transplantation models with respect to toxicity and in the identification of predictive biomarkers.

In spite of these successes, the xenograft model presents disadvantages in its ability to predict clinical response to therapy. Most animal tumors do not accurately model clinical metastatic disease. The use of immuno-compromised mice prevents an immune rejection response which is fundamentally different from the human system where the immune response promotes primary growth of tumor cells and their migration to secondary organs [136, 179]. Most xenograph models do not metastasize at the common sites of human breast cancer (such as lymph nodes, liver, bone and brain) as they prefer to colonize in the lungs [5]. However, improved models that have been created to represent human breast cancer metastasis from a primary orthotopic site to human bone, such as initially published in 2005 by Kuperwasser *et al* [167]. Consider that some xenograph models which utilize subcutaneous injection of tumor cells into the flank and mammary fat pad are not as representative of clinical disease as those that use orthotopic transplantation of cells into the mammary gland. Inherent discrepancies in the background of mice and humans should be considered, and this is of particular importance in predicting side effects for cancer therapy targets [180]. Additionally, the area of metastasis can change depending on the cells line utilized or methods of inoculation [167]; thus, there are considerable technical issues. One could overcome such restrictions by use of clinical isolates. However the utility of primary xerographs is inefficient; access is limited, results are restricted by sample size and thus, studies to date indicate only some degree of success [181]. Current research with aims to generate partial human immune systems or intact populations of human cells in mice systems may also overcome such limitations. We must consider that despite all advances to date, animals do not represent a complete model of human disease. For example, tumor relapse would be especially problematic to study as the usual life span in the majority of mice does not exceed two years. Aside from these underlying basic issues, there have been issues associated with the testing of natural products within these models. Further clarity on the fundamental mechanisms of tumor progression and metastasis and new drug targets will be unveiled as mouse models advance. Until then, we will continue to rely on current disease models and choose such based on disease state and therapeutic targets.
