Sinclair miniature swine: an animal model of human melanoma

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Veterinary Immunology and Immunopathology 43 (1994) 167-175

Sinclair miniature swine: an animal model of human melanoma M.L. Misfeldt*, D.R. G r i m m Molecular Microbiology and Immunology, School of Medicine, Universityof Missouri-Columbia, Columbia, MO 65212, USA

Abstract

Sinclair swine display cutaneous melanoma lesions and develop a generalized depigmentation subsequent to tumor regression. Sinclair swine represent a valuable animal model to study the factors influencing the development of melanoma and also the factors which lead to the development of vitiligo. Therefore, information obtained in studies of Sinclair swine should facilitate our understanding of the mechanisms by which melanoma and vitiligo develop and provide us with possible therapeutic treatments for these human diseases.

1. Introduction Skin cancer accounts for approximately one-third o f all cancers diagnosed in the U n i t e d States. Well over 700 000 new cases o f skin cancer are reported every year. O f these 700 000 cases, basal cell carcinomas and squamous cell carcinomas, both o f which are treatable and rarely metastasize, make up the majority o f the cases. Approximately 5% o f skin cancer cases are malignant melanomas, which are m o r e lethal and account for an estimated 32 000 new cases and more than 6800 deaths per year (Boring et al., 1993 ). Between 1973 and 1989 the incidence rate for m e l a n o m a increased significantly and the mortality rate during the same period also increased (Grin-Jorgensen et al., 1992). *Corresponding author. Fax ( 314) 882-4287. 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

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2. Animal models

One of the limitations in the study of malignant melanoma has been the lack of suitable animal models which display spontaneous melanomatous lesions similar to human melanoma. Animal models of cutaneous melanoma have been described, including rodent, fish, canine, horse, and swine (Garma-Avina et al., 1981 ). However, most of these animal models of melanoma have certain limitations. For instance, in the horse model, the development of the melanoma lesions requires a lengthy period of time, several years. Most domestic swine models also have limitations. Cutaneous melanoma occurs infrequently in domestic swine and is limited almost exclusively to the Duroc-Jersey breed of swine (GarmaAvina et al., 1981 ). This infrequent nature makes it less desirable as a model for human melanoma. However, melanoma is heritable in two breeds of miniature swine, the Sinclair miniature swine and the Munich Troll miniature swine (Strafuss et al., 1968; Biittner et al., 1991 ). The Sinclair miniature swine have a significant incidence of melanoma with a histopathology similar to human melanoma (Strafuss et al., 1968; Millikan et al., 1974). Examples of Sinclair miniature swine displaying cutaneous melanomas are shown in Fig. 1. 2.1. Sinclair swine The Sinclair melanoma bearing swine were first described in 1967 by Strafuss and coworkers, who reported the occurrence of pigmented lesions in a herd of miniature swine maintained at the University of Missouri Comparative Medicine Research Farm, i.e. Sinclair Farm (Strafuss et al., 1967, 1968 ). Strafuss and his coworkers described two types of pigmented lesions; exophytic lesions which rapidly enlarge and frequently ulcerate during the first 6-7 weeks of life and a deeply pigmented, fiat, lentigo-like spot (Strafuss et al., 1968; Millikan et al., 1974). Although most tumors demonstrated a benign regression course, a few animals expressed tumors that metastasized and induced internal melanotic lesions which contributed to the animal's death (Millikan et al., 1974). Light microscopic studies of the cutaneous tumors indicated that the exophytic lesions shared many of the histopathological features of human melanoma lesions. These same studies also revealed that the swine tumors were heavily melanized and the tumors contained melanin-laden macrophages (Millikan et al., 1973 ). A separate colony of Sinclair miniature swine maintained at Texas A&M University, originally obtained from stock at the University of Missouri-Columbia, has also been extensively studied. A thorough histologic evaluation of the porcine melanoma has been performed and compared to human melanoma. Of the 129 tumors examined from Sinclair swine, 70% of the tumors were classified as superficial spreading melanoma with dendritic cells (SSM-D), 20% were classified as nodular melanomas (NM), and 13% as superficial spreading melanomas (T.K. Das Gupta et al., 1989). Eighty-nine percent of the tumors were classified as deep tumors which corresponded to Clark's levels IV and V, a classification given to invasive human melanomas (T.K. Das Gupta et al., 1989). The swine melano-

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Fig. 1. Examples of Sinclair swine cutaneous melanoma lesions.

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mas were also frequently observed to invade blood vessels and nerves (T.K. Das Gupta et al., 1989). In Sinclair swine, the growth phase of the swine melanoma is accelerated and the superficial growth phase probably is completed in utero (T.K. Das Gupta et al., 1989). Sinclair swine melanoma cells have characteristics of human melanoma(s) at the light and Em level(s), but differences in melanosome structure (T.K. Das Gupta et al., 1989). The histologic prognostic markers of human melanoma, including levels of invasion, thickness, ulceration, and regional node involvement, are similar in Sinclair swine melanoma (T.K. Das Gupta et al., 1989). Thus, Sinclair melanoma swine represent a valuable laboratory model to study melanoma.

3. Inheritance

Genetic studies of human cutaneous melanoma have failed to identify the mode of inheritance of malignant melanoma and researchers now believe that more than one mode of inheritance may be involved in human melanoma expression. Human melanoma cells may exhibit a variety of non-random cytogenetic alterations. The non-random involvement of chromosomes 1, 6, and 7 in advanced melanoma cells is now well established (P. Dasgupta et al., 1989 ). These abnormalities involving chromosomes 6 and 7 have been observed to be a characteristic feature in cutaneous malignant melanoma cells (P. Dasgupta et al., 1989 ). Since Sinclair swine melanoma is an inherited neoplasm and selective breeding increases the incidence of melanoma, this animal model provides a unique opportunity to determine the mode of inheritance of swine melanoma (Millikan et al., 1974). Tissot and coworkers reported that the expression of cutaneous malignant melanoma in Sinclair swine involves at least two loci (Tissot et al., 1987, 1989). One locus was shown to be associated with the swine leucocyte antigen (SLA) complex. The presence of one particular SLA haplotype, arbitrarily assigned haplotype B and identified by mixed leukocyte assay, was associated with a high incidence of the swine cutaneous melanoma (Beattie et al., 1988; Tissot et al., 1989 ). The involvement of the B haplotype in melanoma expression was substantiated in a study of eight litters in which the parents had at least one B haplotype. It was observed that a significantly greater number of tumors developed between birth and weaning in homozygous B/B animals when compared to B/X animals further demonstrating the association of the B haplotype with tumor development (Tissot et al., 1989). In addition to the SLA locus, a second independently segregating autosomal locus has been implicated in swine melanoma development. This second locus must be homozygous for the mutant allele in order for tumor initiation and development but as yet this locus remains unidentified (Beattie et al., 1988).

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4. Immune response to melanoma

Immune mechanisms are thought to play a critical role in the resistance to human melanoma. The most convincing evidence that immune mechanisms are involved in the resistance to human melanoma is the marked and specific increase in resistance to melanoma after active immunization to melanoma cells or vaccines (Bystryn, 1978; Bystryn and Oratz, 1991 ). In addition, in swine that have been immunosuppressed by thymectomy or by treatment with anti-lymphocyte serum, one observes an aggressive growth of melanoma (Bystryn and Oratz, 1991). 4.1. I m m u n e response to melanoma in humans

Melanoma represents a solid tissue tumor which is capable of stimulating immune responses in vivo. Man and animals have been observed to develop both cellular immune responses and antibody responses to melanoma antigens. However, a large proportion of the immune responses are directed towards normal tissue antigens expressed on melanoma cells (Bystryn and Oratz, 1991 ). Studies of patients with melanoma have indicated that circulating T cells are not involved in tumor cell lysis (Itoh et al., 1992). Furthermore, studies have shown that tumor-infiltrating lymphocytes (TILs) freshly isolated from melanoma lesions were also unable to lyse autologous tumor cells (Itoh et al., 1992). However, TILs isolated from subcutaneous metastases and cultured with interleukin 2 (IL-2) were fully capable of lysing autologous tumor cells (Itoh et al., 1992). Therefore, even though one can detect cellular immune responses to melanoma antigens, the exact role of T cells in controlling tumor growth has not been completely elucidated. In addition to cellular immune responses, patients with melanoma also develop antibodies to melanoma antigens. Sera from melanoma patients has been shown to contain antibodies against unique antigens, antigens expressed only on autologous melanoma cells. In addition, antibodies to shared antigens, antigens expressed not only by autologous melanoma cells but also allogeneic tumors, normal melanocytes, and other selected tissues, particularly tissues derived from the neuroectoderm have also been detected (Itoh et al., 1992). Thus, antibodies directed to antigens expressed on melanoma cells are induced in patients with melanoma. However, the role of antibodies in controlling tumor growth has not been clearly defined. 4.2. I m m u n e response to melanoma in swine

Sinclair swine display cutaneous melanoma lesions at birth or develop lesions shortly thereafter. Most, if not all, Sinclair swine undergo regression of the tumors and subsequently remain free of tumors. The tumor regression observed in Sinclair swine appears to be associated with an increase in host leukocyte mediated reactivity. Examination of swine melanoma lesions during the latter stages

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of melanoma growth has revealed that large numbers of pigment-laden macrophages have invaded the lesions with an apparent decline in the number of tumor cells (T.K. Das Gupta et al., 1989). Furthermore, other studies have reported that melanoma bearing swine exhibited enhanced leucocyte reactivity to 3 M KC1 extracts of swine melanoma cells using an antigen stimulated active rosette assay (Aultman and Hook, 1979; Berkelhammer et al., 1982; Hook et al., 1983 ). This in vitro leukocyte reactivity appeared to correlate with tumor volume as leukocyte reactivity was greater in swine with a slower rate of tumor growth and lower tumor volume (Berkelhammer et al., 1982 ). Thus, Sinclair swine develop an immune response to the melanoma. Our laboratory has examined the peripheral blood cytotoxic activity of Sinclair swine displaying melanoma. We observed that Sinclair swine bearing melanoma display an age-dependent peripheral blood cellular cytotoxic response (Richerson and Misfeldt, 1989; Richerson et al., 1989). Peripheral blood lymphocytes (PBL) collected from 2-4 week old Sinclair swine displayed minimal cytotoxic activity against K562 cells and semi-allogeneic porcine melanoma cells. However, peripheral blood lymphocytes from pigs greater than 6 weeks old demonstrated significant cytotoxicity against K562 cells and semi-allogeneic porcine melanoma cells (Richerson et al., 1989). This enhanced PBL cytotoxic activity corresponded to the visual observation of tumor regression suggesting that a cellular immune response may be associated with the melanoma regression observed in Sinclair swine, although maturation of the porcine cellular immune system may also contribute to these findings. Since K562 cells and semi-allogeneic porcine melanoma cells are lysed by the Sinclair swine peripheral blood lymphocytes, it would suggest that the porcine effector cells may be non major histocompatibility complex (MHC) restricted cytotoxic cells. One cell that can function as a non-MHC restricted cytotoxic cell is a T cell which expresses the alternative form of the T cell receptor (TCR), the gamma/delta (7~) form (Brenner et al., 1988 ). T lymphocytes which express 7~ TCR have been reported to constitute a major T cell subpopulation in ruminants and swine (Hein and Mackay, 1991 ). 7~ T lymphocytes circulate continuously between blood, solid lymphoid tissues, lymph, and also can be found in the skin and other epithelial tissues (Hein and Mackay, 1991 ). Since 7~ T lymphocytes predominate in ruminants and swine, it has been suggested that 7~ T cells play a significant role in the immune system of ruminants and swine. Recently, our laboratory has described the isolation and characterization of peripheral blood lymphocyte cell lines from Sinclair swine peripheral blood (Grimm et al., 1993 ). The peripheral blood cell lines were examined for cell surface antigen expression by flow microfluorimetry and shown to lack expression of the lymphocyte surface markers CD2, CD4, CDS, and surface immunoglobulin (sIg) (Grimm et al., 1993). However, the cell lines were recognized by the monoclonal antibodies, MAC320 and 86D. MAC320 has been reported to recognize null lymphocytes in the pig (Binns et al., 1992) and 86D has been reported to recognize an external epitope on a subset of swine 7~ T lymphocytes (Reddehase et al., 1991; Binns et al., 1992 ). In addition, the cell lines expressed CD44, the endothelial lymphocyte adhesion marker (E-LAM) which

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has been reported to function as a lymphocyte homing receptor for the skin and other epithelial surfaces (Picker et al., 1991; Grimm et al., 1993). We also observed that the y~ T lymphocyte cell lines possessed cytotoxic potential as indicated by the detection of azurophilic lysosomal granules (Grimm et al., 1993 ). Finally, we observed that these y~ T lymphocytes adhered to cultured semi-allogeneic melanoma cells which resulted in the lysis of the melanoma cells (Grimm et al., 1993). Therefore, these results suggest that ),~ T lymphocytes may be involved in the tumor regression observed in Sinclair swine. Sinclair swine not only develop a cellular immune response to melanoma, but recently it has been observed that Sinclair swine develop antimelanoma antibodies. Sera collected from thirteen swine born with melanoma were tested for antimelanoma antibodies by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 125I-labelled swine melanoma macromolecules. Antibodies to melanoma macromolecules were present in the sera of all 13 swine. In addition, these swine antibodies were also capable of immunoprecipitating human melanoma macromolecules (Cui et al., 1994). Therefore, these results indicate that Sinclair swine can develop antibodies to melanoma antigens. Whether these antibodies play a role in the melanoma regression has not yet been determined. 5. Significance as an animal model

Sinclair swine represent a useful animal model in which to study the genetic aspects of melanoma development as well as the influence of immune factors on the growth of melanoma. In addition to its value in the study of melanoma, Sinclair swine represent a valuable animal model in the study of vitiligo.

5.1. Human vitiligo Vitiligo represents a human disease that displays selective destruction of melanocytes, the cells that are responsible for making pigment (Bystryn, 1989 ). The selective destruction of melanocytes, a dendritic cell of neuroectodermal origin, which upon transformation develops into melanoma, is the immediate cause of vitiligo. Since the selective destruction of pigmented cells, i.e. malignant melanocytes, is the therapeutic goal for the treatment of melanoma, an understanding of the immune mechanisms in vitiligo may provide clues for the treatment of melanoma. The actual link between vitiligo and melanoma is not known. However, patients with melanoma who develop vitiligo seem to have a better prognosis (Bystryn, 1989). These observations suggest that an immune response to pigment cell antigens may be responsible for the destruction of melanocytes in the normal skin of patients with melanoma and in the control of melanoma cell growth. This hypothesis has some merit since antibodies to melanocyte cell surface antigens has been observed in a large number of vitiligo patients (Naughton et al., 1983 ). These antibodies which were demonstrated by indirect immunofluorescence or immunoprecipitation have been shown to be directed to surface pro-

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teins, 'vitiligo antigens', of molecular weights ranging from between 50 and 250 kDa (Cui et al., 1992 ). These antigens appear to be selectively expressed on pigmented cells and can be detected on melanocytes and on allogeneic pigmented melanoma ceils but not on nonpigmented melanomas or other unrelated normal and malignant human cells (Bystryn and Naughton, 1985). Even though antibodies to vitiligo antigens can be detected, the question still remains whether these vitiligo antibodies are the cause or the result of the disease.

5.2. Swine vitiligo Animals also have the capacity to develop vitiligo. Sinclair swine develop vitiligo subsequent to tumor regression. Sinclair swine PBL were observed to mediate the lysis of semi-allogeneic uveal melanocytes (Richerson et al., 1989 ). This in vitro melanocyte cytotoxicity data suggests that cell-mediated melanocyte destruction may be associated with the generalized depigmentation which occurs in Sinclair swine subsequent to tumor regression. Sinclair swine also develop antibodies to vitiligo antigens. Antibodies directed to vitiligo antigens with approximate molecular weights of 45 and 68-75 kDa have been detected in the serum of Sinclair swine (Cui et al., 1994). Therefore, both a cellular and humoral immune response to melanocytes and their antigens can be demonstrated in Sinclair swine. Whether lymphocytes or antibodies function alone or in combination remains unanswered.

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