B16 as a Mouse Model for Human Melanoma

unit 20.1

Malignant melanoma is the sixth most common cancer in the U.S., with an estimated 44,200 new cases reported in 1999 (Centers for Disease Control and Prevention, 1999). A subset of patients with metastatic melanoma can be successfully treated by the administration of recombinant interleukin-2 (rIL-2), sometimes given together with autologous melanoma-reactive lymphocytes that have been expanded ex vivo (Rosenberg, 1997; Rosenberg, 1999). Recently, a number of different laboratories have used these anti-tumor lymphocytes to clone melanoma-associated antigens, which have generally been nonmutated melanocyte differentiation antigens (MDA), a group that includes MART-1/Melan-A, gp100, tyrosinase, and tyrosinase related proteins (TRP)-1 and -2 (Rosenberg, 1997; Rosenberg, 1999), and others listed in Table 20.1.1. With the possible exception of MART-1/Melan-A, each member of this group of proteins is an enzyme directly involved in the synthesis of eumelanin pigment (Winder et al., 1994; Kameyama et al., 1995).

Since MDA are expressed by most melanoma cells, they are an attractive target for melanoma vaccines. However, from an immunological perspective, MDA are "self" proteins, to which central and/or peripheral tolerance may exist, potentially hampering the induction of powerful, therapeutic anti-melanoma immune responses. Yet clinical observations suggest that some degree of autoreactivity can be induced and may even contribute to prolonged survival of patients. In a prospective study of patients receiving IL-2, vitiligo was seen in ~20% of melanoma patients that had objective responses. None of the nonresponding patients developed vitiligo, nor did any of the more than 100 patients receiving IL-2 for the treatment of kidney cancer (Rosenberg and White, 1996).

These observations prompted the authors of this unit to investigate the anti-tumor effect of inducing autoimmune responses to the mouse homologs of human MDA. Several models of melanoma exist in common mouse strains, including BALB/c and C57BL/6 (Donawho et al., 1996; Overwijk et al., 1998; Overwijk et al., 1999). As a model system, the authors used the spontaneous C57BL/6-derived B16 melanoma, a well established and widely used tumor model in which treatment is notoriously difficult. Results by the authors and others indicate that autoimmunity to MDA can indeed be induced, and can result in protection against and/or treatment of B16 melanoma (Naftzger et al., 1996; Bloom et al., 1997; Overwijk et al., 1998; Overwijk et al., 1999).

This unit will detail protocols for in vivo models of subcutaneous growth and pulmonary metastases of B16 melanoma (see Basic Protocols 1 and 2). Therapeutic approaches include the use of B16.GM-CSF (see Basic Protocol 3) and rVVmTRP-1 (see Basic Protocol 4) to induce autoimmune vitiligo and tumor protection. The induction and use of gp100-specific therapeutic cytotoxic T lymphocytes (CTL) are discussed. Methods are also included for CTL induction (see Support Protocol 3), isolation and testing (see Support Protocol 4), CTL maintenance (see Support Protocol 5), and adoptive transfer (see Basic Protocol 5). Support Protocols 7 and 8 detail the testing of mouse sera for presence of MDA-specific antibodies by immunoblotting and ELISA, respectively. Additional sections, including growing B16 melanoma (see Support Protocol 1), enumerating pulmonary metastases (see Support Protocol 2), and use of recombinant viruses for vaccination, are discussed together with safety concerns (see Support Protocol 6).

Contributed by Willem W. Overwijk and Nicholas P. Restifo

Current Protocols in Immunology (2000) 20.1.1-20.1.29 Copyright © 2000 by John Wiley & Sons, Inc.

Tumor Immunology

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