1.4.1 Mouse models
Ideally tumour immunology should be studied in the host species and not a mouse model because the results from mouse models do not always correlate to the host species (Bierer 2009, Frese and Tuveson 2007). Nevertheless, it is often impractical, undesirable or unethical to conduct experiments in the host species in which case mouse models can provide valuable information and preliminary data prior to any clinical trials (Frese and Tuveson 2007).
1-24 While some would argue that promising in vitro trials should progress to the host species rather than an intermediary mouse model (Bailey 2011) all clinically approved agents for the treatment of human cancer have demonstrated positive activity in mice (Becher and Holland 2006). However, many agents that have
therapeutic benefit in the mouse model do not translate into effective treatment in the human host (Becher and Holland 2006).
The use of transgenic animals is intended to minimise the difference between the mouse and the host species and it has been noted in some genetically engineered mouse models that tumour growth is so similar to that in the host that clinical pathologists have difficulty telling them apart using a microscope (Becher and Holland 2006). Xenografts obtained directly from patient tumours have replicated the histology and biology of the primary tumour more faithfully than cell lines, some of which may have passed through more than 100 passages (Becher and Holland 2006, Borrell 2010).
When using mouse models for the study of tumour regression or rejection it is
paramount to consider the implications that the underlying rejection mechanism may be graft rejection rather than tumour specific rejection. In the case of DFTD this is less of an issue because in the wild the tumour is transmitted as an allograft which under normal circumstances should be rejected by a graft rejection mechanism (Azimzadeh et al 1996, Pearse and Swift 2006).
The shorter lifespan of mice compared to human disease development is significant (Kim et al 2003); however, this may be less of a problem in the DFTD model as the disease is particularly virulent leading to mortality of the natural host within months of infection (Lachish et al 2007).
There are usually differences in the progression of tumours between host species and mouse and this is often seen in variant cellular targets, size of tumour and metastatic disease progression. For example, the metastatic route in human breast cancer is usually lymphatic while in the mouse model the route is usually the blood vessels (Kim et al 2003).
The study of metastatic disease can be difficult in mice because most implantations are done subcutaneously rather than orthotopic (Becher and Holland 2006). This
1-25 may be less of a problem in the study of DFTD as the implantation of cells in the natural host is through biting which implants the cells close to the skin surface in the dermis or submucosal connective tissue in the mouth (Loh et al 2006a).
Mice consume higher amounts of oxygen per cell compared to larger animals and this may be significant in the tumour microenvironment were different expression of hypoxia-induced genes may occur altering proliferation and differentiation (Kim et al 2003). The development of blood supply by the process of neovascularisation is determined by the host not the tumour itself (Becher and Holland 2006). The interaction between stroma cells and cancer is artificial since the stroma is murine (Becher and Holland 2006). Species or class specific differences in the binding of proteins and metabolism can be another variable in the mouse model experiments (Becher and Holland 2006).
Mouse models for CTVT
Since the Russian veterinarian Norwinsky’s first experiments in 1876, CTVT has been studied experimentally by transferring viable cells into animals (Das and Das 2000). Animal studies of CTVT have provided an understanding of how CTVT is transmitted as an allograft, accepted by the new host and ultimately regresses leaving the dog immune to re-infection (Harmelin et al 2001). Since dogs are not an endangered species most of these studies have been conducted in the host species (Das and Das 2000).
Murine xenograft models for CTVT have also been used to reduce the need for maintaining allogeneic transfer in dogs (Harmelin et al 2001). Compared to dogs the murine model is relatively low cost to house and maintain. There would also be fewer problems with maintaining animal ethics approval and greater availability of
antibodies to study immune responses (Bierer 2009, Harmelin et al 2001).
CTVT has been engrafted into mice that have had their immune system suppressed with irradiation or into immunocompromised athymic nude mice and NOD/SCID mice (Harmelin et al 2001). The NOD/SCID model could be considered the model of choice as it allows CTVT to be established and progress with the typical
characteristics of CTVT in the natural host (Harmelin et al 2001). An inoculation of 1 x 106 cells will produce tumours within 47 days in the NOD/SCID model (Harmelin et al 2001).
1-26 The mouse models primarily can be used to test hypotheses and predict responses to treatments. Complex biological problems can be examined and predictive models focus on testing treatment responses including efficacy and toxicity (Coghlan 2013). The CTVT murine model provides a precedent and justification for using murine xenograft models to study the similarly infectious cancer DFTD. It could be expected that mouse models that have been successfully exploited for the study of CTVT would prove suitable for the study of DFTD since they are both transmissible neoplasms (Bierer 2009, Harmelin et al 2001, Loh et al 2006a).
1.4.2 Xenograft tolerant mouse strains
There is a variety of mice strains which have specific immunological impairments that clarify the immunological functions imperative to tumour engraftment, rejection, or regression (Frese and Tuveson 2007, Harmelin et al 2001).
The three strains discussed in the following paragraphs all lack functional T cells. Lack of T cells would limit the protection offered by macrophages (Bancroft et al 1986). As part of the surveillance of the innate immune system macrophages detect threats and present early signals to promote T cell proliferation and differentiation. In turn the T cells provide feedback signals that enhance the activity of the
macrophages (Bancroft et al 1986). C.B-17 scid/scid mice
C.B-17 scid/scid (scid) mice are homozygous for the severe combined
immunodeficiency (scid) mutation and this results in a lack of functionality of B and T cells; however, some young adults might generate a few functional B and T cells and by 10 to 14 months nearly all the older adults have developed a limited number of functional T cells (Bancroft et al 1986).
B and T cells are the only leukocytes that have impaired function in a scid mouse. NK cells, macrophages, APCs, monocytes, granulocytes and DCs are all normal in the scid mouse (Bosma and Carroll 1991). This would provide a model to study macrophage, NK cell and DC responses to DFTD cells independent of T and B cell interactions (Bancroft et al 1986).
Lymphoid tissues are underdeveloped. The thymus is typically less than 10% of the normal size and the lymph nodes are minuscule and contain few lymphocytes. The
1-27 spleen presents with an unusual histology and contains macrophages and large granular cells but only low numbers of lymphoid cells and plasmacytes. Red blood cell levels are normal and serum Ig concentrations are less than 20 ng/ml (Bancroft et al 1986, Bosma and Carroll 1991).
Xenogeneic tumours can be successfully engrafted into scid mice (Bancroft et al 1986, Bosma and Carroll 1991). The scid mutation inhibits the early development of B and T cells but does not affect the ability of the mice to support normal lymphocyte proliferation and this has allowed the reconstitution of the immune system with functioning lymphocytes from other mice and humans (Bosma and Carroll 1991, Pearson et al 2008).
NOD/SCID mice
The NOD/SCID mouse strain has been created by backcrossing mice with the scid mutation and mice that have a diabetes-susceptible non-obese diabetic (NOD) background (Prochazka et al 1992). NOD/SCID mice have a more compromised immune system than athymic nude mice and CB-17-scid mice. NOD/SCID mice lack T and B cells, lack effective levels of serum antibody, have no complement activity, impaired development and function of macrophages and other APCs but do
demonstrate limited NK cell function (Harmelin et al 2001).
The lack of functioning T cells from the scid background makes the mice diabetes resistant (Prochazka et al 1992). The impaired immunity means these mice must be maintained in a pathogen free environment and have a short life expectancy of about eight months (Harmelin et al 2001). A single injection with broad spectrum antibiotics will usually prevent bacterial infection in NOD/SCID mice (Bastide et al 2002,
Harmelin et al 2001).
The NOD/SCID strain has proved suitable for studying certain human cancers because there is no evident tumour immunity (Bastide et al 2002). CTVT tumours have demonstrated the ability to undergo numerous passages in these mice
providing a source of tumour cell lines maintained in-vivo (Harmelin et al 2001). The NOD/SCID xenograft model preserves the cytological, histological and molecular characteristics of CTVT. This facilitates the study of engraftment, disease
progression including metastasis, diagnosis and treatments for CTVT (Harmelin et al 2001).
1-28 Adoptive transfer of immune cells from competent mice to NOD/SCID mice can identify the contribution of individual components to effective immunity against challenges (Hicks et al 2006). It may be possible to apply this approach to DFTD engraftment in NOD/SCID mice to elucidate the ability and role of specific cell populations in rejection and or regression of DTFD.
Athymic nude mice
The nude mouse has an autosomal recessive mutation of the 11th chromosome that disrupts the FOXN1 gene resulting in failure to grow hair and lack of a functional thymus. The lack of a thymus means that nude mice are deficient in mature T cells including CD4 + and CD8+ cells which has negative implications for cell-mediated immune responses including the lack of CD4+ helper T cells to produce antibodies (Kim et al 2003).
The lack of effective anti-tumour immunity makes this strain suitable as a xenograft model without the need of additional immune system suppression (Kim et al 2003). Human tumours can be maintained through more than fifty passages and still demonstrate the same morphology and phenotype with no species hybridization (Spangthomsen and Visfeldt 1976).
Xenograft tumour growth is contained locally in a well defined capsule-like
connective tissue that is not attached to the skin or underlying tissues. If the athymic nude mice did engraft DFTD cells they may because of their hairless nature and translucent skin make visual monitoring of tumour growth and precise intratumoural injections easier. Another advantage of the athymic nude mice is that they retain some functional components of the immune system including macrophages, DCs, B cells and NK cells (Spangthomsen and Visfeldt 1976) and these may be stimulated with therapeutic agents to target the DFTD cells.