- Stability/instability

In practice, these strains, although cryopreserved, undergo dedifferentiation resulting from multiple subcultures, and leading to the lost of special characteristics. Moreover, differences in the culturing practices (medium composition, time between subcultures, subculturing technique, etc.), can explain some divergences observed for a same strain in different laboratories.

#### - Simplicity

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

Significant amounts of data on breast cancer have been collected over the past 40 years, thanks to the use of established cell lines. The first breast cancer cell lines (BCCL) have been established in the sixties-seventies and very few new cell lines have been developed since. Only a hundred of BCCL are currently available and three of them have been extensively studied and represent now nearly 80% of the 35 000 publications mentioning breast cancer

Most of the cell lines were created from cells derived from metastasis or from pleural effusion. Pleural effusions contain large amounts of well isolated tumour cells and few contaminating cells such as fibroblasts, thus making their recovery and growing easier than those of cells directly derived from primary tumours or metastasis. Moreover, metastatic cells are highly dedifferentiated cells, which allow their cultivation more successfully than

The three more used BCCL (MCF-7, MDA-MB-231 and T47D) are issued from pleural effusion of an invasive ductal carcinoma (Soule *et al.* 1973; Cailleau *et al.* 1974; Keydar *et al.* 1979), and they mainly differ by their oestrogen receptor (ER) and progesterone receptor (PgR) status: MCF-7 and T47D are ER+ PgR+ while MDA-MB-231 is ER- PgR-. Among these three cell lines, MCF-7 was the most often used during the last ten years: it has been cited in 53% of all the scientific papers mentioning BCCL, while MDA-MB-231 and T47D were respectively cited in about 18% and 7% of these articles (calculation made on the basis of a

The complete control of environmental conditions and standardised culture conditions

 Maintaining cells in culture is much less costly than working on animal models. Besides the fact that some animal models are expensive by themselves, the care of animals and the staff necessary to a good work in an animal house are the main drain of resources. Conversely, the medium and the staff time required to growth cells are cheaper, thus

Cryopreservation enables long-term conservation of the same strain and can

These advantages have allowed to gather essential data for the study of breast cancer in the last 40 years, making these cell lines reference models in the field with the establishment of a

In practice, these strains, although cryopreserved, undergo dedifferentiation resulting from multiple subcultures, and leading to the lost of special characteristics. Moreover, differences in the culturing practices (medium composition, time between subcultures, subculturing technique, etc.), can explain some divergences observed for a same strain in different

ensures the reproducibility of results between experiments and laboratories.

**2. Models for investigation on breast cancer** 

**2.1.1 The different cell lines and their main properties** 

**2.1 Established breast cancer cell lines** 

cell lines (Lacroix & Leclercq 2004).

Medline-based survey in March 2011).

The use of these lines has many technical advantages.

allowing the widespread use of BCCL.

complete genetic and proteinic profile.


laboratories.

**2.1.2 The main drawbacks of these models** 

theoretically permit the use of these cell lines indefinitely.

the primary tumour cells.

The relevance of cellular models is controversial since their over-simplicity implies difficulties in extrapolating results from the cell line to the tumour in humans and thus raises the question of their representativeness.

Indeed, cell lines are homogeneous, theoretically consisting only of a single cell type (pure and clonal) due to the way they are established:


These particularities reduce the similarity with the primary tumour.


The hundred of available cell lines do not cover all of the tumour features found in patients. Furthermore, the proportions of some characteristics are sometimes reversed, such as the ER and PgR status which is very different in cell lines, compared to that found in the patient population (Lacroix & Leclercq 2004). These dissimilarities can be explained by the fact that most lines are derived from pleural effusion and metastases containing cells which are already different from the original tumour and thus, more or less representative of this tumour. Indeed, the ER/PgR status sometimes differs between the metastasis and the original tumour from the same patient. Based on these observations, several teams have worked on the development of cell lines derived from a primary tumour (Amadori *et al.* 1993; Gazdar *et al.* 1998; Shen *et al.* 2009), which are much more representative of the *in vivo* cancerous tissues that lines derived from metastases, but which suffer from the same problems related to their relative homogeneity and instability in a long term use. Moreover, the establishment of cell lines from primary tumours remains a difficult achievement, failures mainly being the result of contamination by the stroma surrounding the tumour.


Besides these previous drawbacks, many criticisms have been made against BCCL because some of them have been proven not being from breast cancer origin. Indeed, some lines were contaminated by other cell types during their first years of use, then spread to other laboratories, and used on a large scale without further verifications of their true origin. Several cell lines were denounced as false, whereas it was not the case (Fogh *et al.* 1977; Nelson-Rees & Flandermeyer 1977). These contaminations have been subjects of controversial for a long time. However, studies have shown with certitude that two cell lines were not from their supposed origin.

The MCF-7-ADRr cell line was developed in 1986 by Batist. It is derived from the lineage of human mammary adenocarcinoma MCF-7 and was rendered resistant to adriamycin treatment after exposition to increased concentrations of this drug. The obtained resistant cell line was also resistant to other agents such as actinomycin D, vinblastine and vincristine. However in 1998, the lineage between MCF-7 and MCF-7-ADRr became controversial, as shown by DNA fingerprinting studies and genetic comparison, so that the true origin of the cell line was undetermined and the cell line was renamed NCI/ADR-RES. Liscovitch and Ravid, in 2007, have collected data showing that NCI/ADR-RES were carcinoma ovarian

*In Vitro* Breast Cancer Models as Useful Tools in Therapeutics? 25

 Under chemical pressure, normal cells in culture can also be immortalised. This is the case for some cell lines as 184A1 and 184B5 which were obtained by exposition to benzo[a]pyrène, a chemical carcinogen, leading to clonal events which are the origin of

The use of these "non cancerous" cell lines is important to give a comparison point to results obtained with cancerous cell lines. However, there are drawbacks and controversy to their use, the major one concerning the way they were obtained. Indeed, if they are still nontumourigenic, they suffer of genetic modifications which lead them to become immortal.

The metabolic equipment of a cell can explain its sensitivity/resistance to drugs. Indeed, any xenobiotic molecule (therapeutic drugs included) undergoes the same metabolic fate in the cells. Briefly, enzymes of Phase I (essentially cytochromes P450 (CYP) dependent enzymes) ensure a bioactivation of the molecules while enzymes of Phase II conjugate the metabolites issued from Phase I to endogenous molecules (glucuronic acid, glutathione, sulfates…) in order to make them more water-soluble and to facilitate their elimination. Finally, transporters of Phase III are responsible for exporting these last products out of the cells. Each human organ is equipped with these enzymes, but their expression pattern differs quantitatively and qualitatively. The liver is the most efficient organ in metabolising processes, even if we know that some enzymes are more specifically expressed in non

When considering the usefulness of breast cell lines as *in vitro* tools to predict sensitivity or resistance to a molecule, it is easy to perform, in first line, simple cytotoxicity tests. However, in order to explain the reasons of these cells behavior, or to predict the metabolism of a new compound, the knowledge of the metabolic equipment of the cells is necessary. As it is impossible, and not very interesting, to decline the results of the literature concerning breast cell lines and assays with the numerous chemical molecules which have been, precisely or not precisely, tested, we chose two examples of therapeutic drugs, used in breast cancer, that need to be bioactivated by CYP before exerting their deleterious effects in

The oxazaphosphorines generally used in pharmacology (*i.e.* cyclophosphamide (CPA), ifosfamide (IFO), and trofosfamide) represent an important group of chemotherapeutic agents. However, their use is limited by severe toxic side effects. New oxazaphosphorines derivatives have been developed in order to improve selectivity and to reduce toxicity but they won't be studied here, due to their bioactivation process which is different from that of

Both CPA and IFO, the most widely used as alkylating agents, are prodrugs whose metabolism involves different cytochromes P450 (CYPs) catalysing 4-hydroxylations leading to acrolein and nitrogen mustards capable of reacting with DNA molecules leading to cell apoptosis and/or necrosis. Another pathway consists in an N-dealkylation whose last product is the toxic chloroacetaldehyde (Figure 1) (Rooseboom *et al.* 2004; Zhang *et al.* 2005). All these metabolites are highly reactive metabolites responsible for urotoxicity, neurotoxicity and nephrotoxicity. As all the mechanisms underlying these toxicities are not

these immortal cell lines (Stampfer 1989).

They are looking like normal cells, but they are not.

the cells: oxazaphosphorines and ellipticine. - Metabolism of oxazaphosphorines

previous molecules (Zhang *et al.* 2005).


hepatic tissues.

**2.1.4 Breast cell lines and metabolism of therapeutic drugs** 

cells (Liscovitch & Ravid 2007), and experiments of Affymetrix SNP array analysis at the Sanger Institute (Cancer Genome Project) and of karyotyping, helped to put in evidence an indisputable resemblance of NCI/ADR-RES with the OVCAR-8 human ovarian carcinoma cell line. The most likely scenario is that the stock of MCF-7 cells from the National Cancer Institute used in 1986 for the development of the lineage, was contaminated with OVCAR-8 cells before the first generation of MCF-7-ADR-r. OVCAR-8 cells are naturally resistant to adriamycin, and the *in vitro* selection probably eliminated the MCF-7 cells and allowed the survival of OVCAR-8 cells (Liscovitch & Ravid 2007). It can be noted that MCF-7-ADRr are no longer distributed by the international cell bank ATCC.

The second misidentification concerns the MDA-MB-435 cell line established by Cailleau and colleagues in 1978. This cell line has been controversial in 2000, further to the results of DNA microarray analysis which suggested that these cells might be of melanocyte origin (Ross & Perou 2001). Some other results, obtained by microsatellite comparison analysis, karyotyping and comparative genomic hybridisation experiments (Rae *et al.* 2007), confirmed that MDA-MB-435 cells are in fact M14 melanoma cells.

However, these two cell lines, MCF-7-ADRr and MDA-MB-235, are still used as breast cancer cell lines for some studies and are used for publications in international journals, while it has been proven that they are not from breast cancer origin (Lacroix 2008). The verification of the origin of a cell line is essential, and a way of ensuring that the cell lines are really from a well-defined origin is to make a short tandem repeat (STR) profiling. This method is used to confirm the identity of a cell line by comparison to a known profile and a periodic re-authentication of cell lines is advisable. Moreover, banks of cell lines such as ATCC guarantee the exact origin of their cells. Several authors suggested to prove the authenticity of the cell lines used for each publications (Burdall *et al.* 2003; Lacroix 2008).
