**2.5 Xenografts: An intermediary model between cell lines and** *in vivo* **models**

We previously saw that some *in vitro* models tend to provide essential information on the inter-cellular interactions, by taking more or less into account the 3-dimensional structure of the tumour, but none of them benefit from the nervous and hormonal regulations found in the living organism.

There are particular models which can do perfectly the junction between *in vitro* and *in vivo* models, the xenografts. They are obtained by injecting cancer cells, usually derived from established cell lines, into a living organism. They are called xenografts because the injected cells are of human origin but are introduced into an animal organism, usually an immunodeficient rodent. The injection can be orthotopic (in breast gland) or heterotopic (localised in another part of the body, usually subcutaneously).

The xenograft model has the advantage of using cells from human tumour cell lines for which a significant amount of data was collected *in vitro*, and to study their behavior *in vivo*. There are several models available for research on breast cancer, principally using immunodeficient mice. The model nude is by far the most commonly used (Kim *et al.* 2004a). It is characterised by an absence of a functional thymus and active T cells (Kindred 1971). The second common model is the SCID mouse (severe combined immunodeficiency). These mice have a deficit in VDJ recombinases that allow the binding of specific and non-specific parts of immunoglobulin and T cell receptor (Bosma & Carroll 1991). See Figure 6 illustrating the two common models of mice used for breast cancer xenografts: nude and SCID.

Fig. 6. **Nude (A) and SCID (B) mice models** xenografted respectively with MCF-7 and MDA-MB-231 breast cancer cell lines. (A) was taken from Nizamutdinova *et al*. 2008, by permission of Oxford University Press, and (B) was taken from Wang et al. 2010, with permission from ASBMB journals.

The injected breast cancer cells mostly come from established cell lines like MCF-7, MDA-MB-231, T-47-D or ZR-75-1. The first experiments of cell transplantations were made in the 80s, and opened onto success in the establishment of malignant tumours in nude mice (Ozzello & Sordat 1980; Kim *et al.* 2004a). Since then, a lot of models have been developed for investigation of new treatments, therapeutic targets and establishment of new cancer detection method by medical imaging.

This technique is widely used to test the effect of new antitumourous compounds or therapeutic methods, for example to test new virotherapies. Thus, a benign virus

We previously saw that some *in vitro* models tend to provide essential information on the inter-cellular interactions, by taking more or less into account the 3-dimensional structure of the tumour, but none of them benefit from the nervous and hormonal regulations found in

There are particular models which can do perfectly the junction between *in vitro* and *in vivo* models, the xenografts. They are obtained by injecting cancer cells, usually derived from established cell lines, into a living organism. They are called xenografts because the injected cells are of human origin but are introduced into an animal organism, usually an immunodeficient rodent. The injection can be orthotopic (in breast gland) or heterotopic

The xenograft model has the advantage of using cells from human tumour cell lines for which a significant amount of data was collected *in vitro*, and to study their behavior *in vivo*. There are several models available for research on breast cancer, principally using immunodeficient mice. The model nude is by far the most commonly used (Kim *et al.* 2004a). It is characterised by an absence of a functional thymus and active T cells (Kindred 1971). The second common model is the SCID mouse (severe combined immunodeficiency). These mice have a deficit in VDJ recombinases that allow the binding of specific and non-specific parts of immunoglobulin and T cell receptor (Bosma & Carroll 1991). See Figure 6 illustrating the two common models

A B

 Fig. 6. **Nude (A) and SCID (B) mice models** xenografted respectively with MCF-7 and MDA-MB-231 breast cancer cell lines. (A) was taken from Nizamutdinova *et al*. 2008, by permission of Oxford University Press, and (B) was taken from Wang et al. 2010, with

The injected breast cancer cells mostly come from established cell lines like MCF-7, MDA-MB-231, T-47-D or ZR-75-1. The first experiments of cell transplantations were made in the 80s, and opened onto success in the establishment of malignant tumours in nude mice (Ozzello & Sordat 1980; Kim *et al.* 2004a). Since then, a lot of models have been developed for investigation of new treatments, therapeutic targets and establishment of new cancer

This technique is widely used to test the effect of new antitumourous compounds or therapeutic methods, for example to test new virotherapies. Thus, a benign virus

**2.5 Xenografts: An intermediary model between cell lines and** *in vivo* **models** 

(localised in another part of the body, usually subcutaneously).

of mice used for breast cancer xenografts: nude and SCID.

permission from ASBMB journals.

detection method by medical imaging.

the living organism.

Coxsackievirus 21 (CVA21) was intravenously injected in SCID mice xenografted with MDA-MB-231 breast cancer cells. CVA21 virus targets the receptors ICAM-1 and DAF that are overexpressed in breast cancer cells. In this experiment a rapid lysis focused on cancer cells was observed in all mice, making this virus a good candidate for use in systemic therapy (Skelding *et al.* 2009). See Figure 7 illustrating the effect of the virus on xenografted mice, visualised by bioluminescent analysis.

Fig. 7. **Observation of the oncolytic activity of CVA21 virus in SCID mouse xenografted with MDA-MB-231-luc**. The breast cancer cells were xenografted into the mammary fat pad, mice were then treated with PBS or CVA21. Metastases were detected 3 weeks post-cell injection. The mice on the pictures are representative for bioluminescent observation at day 42 post treatment. From Skelding *et al*. 2009, with kind permission from Springer Science and Business Media B.V.

In the investigation of new treatments, the vitamin D3 receptors constitute good targets as they are present in over 80% of mammary tumours and they are negative growth regulator of both oestrogen-dependent and independent breast cancer cells *in vitro*. In a study published in 1998 it was shown that EB1089, a vitamin D3 analog, was able to highly reduce the growth of tumour in nude mice xenografted with MCF-7 cells (tumours were 4-fold smaller than those in untreated mice). This reduction was resulting from an enhancement of apoptosis and reducing proliferation of tumour epithelial cells, suggesting the great potential of vitamin D3 analogs such as EB1089 against human breast cancer (VanWeelden *et al.* 1998).

This model can also be used to explore new potential targets for anticancer therapies. A good example is the targeting of receptor ERβ. In an experiment, standard T47D ERα+ ERβand modified T47D ERα+ ERβ+ (T47D stably transfected with a plasmid allowing the expression of the receptor ERβ), were xenografted in SCID mice. 17β-estradiol was then injected into mice. The treatment triggered an acceleration of tumour growth in mice xenografted with the native T47D strain, and conversely a regression of tumours T47D ERβ+. These results emphasize the antagonistic role of ER receptors that appear to play an antitumourigenic role, and offered prospects for the development of ER-selective inhibitors. (Hartman *et al.* 2006).

The targets cited above are non exhaustive. Many other therapeutic targets are tested with xenografts models, as it is the case of the VEGF pathway implicated in tumour angiogenesis (Le *et al.* 2008), or of cell cycle regulating proteins such as CDK kinases (Fry *et al.* 2004).

The use of established cell lines for producing xenografts raises several questions about their relevance. The murine model presents considerable differences with the human body, concerning the biochemical and physiological regulation. Moreover, the stroma that will grow surround the tumour will be of murine origin and it will result in a chimeric tumour

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which biology may significantly differ from human one (Kim *et al.* 2004a). Furthermore, in humans, the immune system plays an important role in the fight against tumour, whereas in xenografts models the immune system is totally absent.

The xenograft model has some limitations but is the most accomplished of all models because it takes into account the complexity of the organism.

Besides the xenografts, there are also murine models which can develop tumours spontaneously or under the influence of inducing compounds (Russo & Russo 1996). Although the achievement of these models is easy, their use is largely debated because of their relevance to the clinical situation. Indeed, murine breast cancers are most often caused by viral infections and are not hormone dependent, whereas a considerable proportion of human cancers are oestrogen dependent. To date there is no evidence suggesting a viral induction of breast cancer in humans. The biology of spontaneous rodent tumours differs from the human ones. The size, the oncogenic targets or the degree of maturation and differentiation of cells differ between the two species, making them hardly comparable.
