[CANCER RESEARCH 52, 3931-3937. July 15. 1992] Genetic Modification of a Murine Fibrosarcoma to Produce Interleukin 7 Stimulates Host Cell Infiltration and Tumor Immunity1 William H. McBride,2 J. Dean Thacker, Sian Coinora, James S. Economou, David Kelley, Donna Hogge, Steven M. Dubinett, and Graeme J. Dougherty Departments of Radiation Oncology /(*'. H. M., S. C.J, Surgery- ¡J.S. £./.Pulmonary and Critical Care Medicine fS. M. D., D. A'./, and the Jonsson Comprehensive Cancer Center (H'. H. M., J. S. E., S. M. D.], University of California at Los Angeles School of Medicine, Los Angeles, California 90024; Terry Fox Laboratory [J. D. T., D. H., G. J. D.], British Columbia Cancer Research Centre, Vancouver, British Columbia, V5ZIL3, Canada; and Departments of Medical Genetics [J. D. T., D. H.l and Pathology ¡G. J. D./, University of British Columbia, Vancouver, British Columbia, l'6TIH'5, Canada ABSTRACT Retroviral-mediated gene transfer was used to introduce and express the gene for murine interleukin 7 (II,-7) in a fibrosarcoma tumor (FSA). The tumorigenicity of these genetically modified FSA cells was greatly decreased in immunologically intact syngeneic mice but was unaltered in T-cell-deprived mice. IL-7-infected tumors that did grow in intact ani mals from large size inocula did so slowly and had a high incidence of spontaneous regression. Furthermore, mice that had rejected tumors became specifically immune to challenge with uninfected parental tumor cells. IL-7-infected FSA growing in intact mice were heavily infiltrated with host T-cells that were presumably responsible for slow growth and tumor regression, and tumor cells were in the minority. Fluorescence- activated cell sorter analysis showed that there was a 530% increase in T-cells in IL-7-infected FSA compared with control tumors. CD8+ T- cells were particularly elevated, but CD4+ lymphocytes were also in creased in number, as were eosinophils and basophils. The CD4+:CD8+ ratio in IL-7-infected FSA was 1:1.7 in comparison to 1:0.6 in control tumors. Lymphocytes isolated from IL-7-producing tu mors had greatly enhanced cytotoxicity towards uninfected, parental FSA cells. Killing of non-cross-reacting fibrosarcoma line was also in creased but to a much lesser extent. Injection of recombinant human IL-7 directly into established FSA tumors slowed their growth and, in a significant number of instances, caused complete regression. Mice that had rejected tumor became spe cifically immune. The dose that was needed for this effect was, however, somewhat large: 20 *igtwice daily for 10 days. This result contrasts with the efficacy of IL-7 gene infection in stimulating responses to the same tumor. These considerations make IL-7 a good candidate for tumor- directed cytokine gene therapy. INTRODUCTION The insertion of genes encoding various cytokines into tumor cells has proven a useful approach with which to investigate the molecular mechanisms that regulate in situ immune responses. Murine tumor cells expressing genes for IL-2-1 (1-4), IL-4 (5, 6), IFN-7 (4, 7), and TNF-o (8, 9) have been found to have decreased tumorigenicity and slower growth in vivo. IL-2 (3, 10) and IFN-7 (7) were shown to mediate their effects through mechanisms that were largely T-cell-dependent. In contrast, tumor cells expressing IL-4 have been reported to induce re sponses that were mainly T-cell-independent with contributions Received 12/16/91; accepted 5/5/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accor dance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the National Cancer Institute, the British Columbia Health Care Research Foundation, and the National Cancer Institute of Canada. G. J. D. is a National Cancer Institute of Canada Career Scientist. 2 To whom requests for reprints should be addressed, at Department of Radia tion Oncology, UCLA School of Medicine. University of California, Los Angeles, Los Angeles. CA 90024-1714. 3 The abbreviations used are: IL. interleukin; TNF-«,tumor necrosis factor a; LAK, lymphokine-activated killer; TD?(). number of cells that had to be injected for tumors to form in 50% of s.c. sites; IFN-7, Interferon 7; FCS. fetal calf serum. from eosinophils and macrophages (5,6). TNF-a also appeared to function by recruiting inflammatory cells, in particular those bearing the complement receptor CR3 (8). Significantly, in these studies mice that had eliminated the primary tumor fre quently developed lasting tumor-specific immunity. These re ports raise the possibility that tumor cells, genetically engi neered to produce cytokines, may prove useful as immu- notherapeutic agents. Such cells could deliver cytokines in a prolonged fashion at high local concentrations without the side effects that are frequently associated with the systemic delivery of large bolus doses of these active biological agents. Moreover, because the cytokines are delivered near tumor-reactive lym phocytes, antitumor responses may be specifically enhanced. IL-7 was originally described as a bone marrow stromal cell- derived cytokine that stimulated the growth of pre-B-cells (11). It was subsequently found also to function as a growth factor for thymocytes (12, 13) and mature CD4+ and CD8+ T-cells (14, 15) and to stimulate resting T-cells to proliferate both directly and through an IL-2-dependent pathway (14-19). This latter activity can be attributed to IL-7-induced expression of IL-2 receptors. IL-7, therefore, acts to enhance T-cell competence and lymphokine responsiveness in a manner similar to, but independent of, IL-1 and IL-6 (15-18). IL-7 has also been shown to help in the generation of cytotoxic T-cells (12, 20-22) and LAK cells (21, 23, 24) and to stimulate the secretion of IL-10, IL-6, and TNF-a by monocytes (25). Taken together, these data suggest that IL-7 may play a role in the induction as well as the expression of antitumor responses and to be a good candidate for cytokine-mediated gene therapy. To test this pos sibility, we have examined the immunological effects that result from the introduction of the IL-7 gene into a murine fibrosar coma of moderate immunogenicity. MATERIALS AND METHODS Mice. Experiments were performed on female C3Hf/Sed//Kam mice aged 10-14 weeks. The animals were bred and maintained in our defined-flora, specific pathogen-free colony at University of California- Los Angeles. T-cell-deprived mice were prepared by thymectomy and concurrent splenectomy at 4 weeks of age followed by lethal whole body irradiation to 10.0 Gy and reconstitution with IO6 bone marrow cells that had previously been treated with anti-Thyl.2 (monoclonal 30-H- 12; American Type Culture Collection, Rockville, MD) (26, 27). These mice are immunocompromised and can support the growth of many human tumor cell lines. Tumor and Tumor Cell Lines. FSA is a fibrosarcoma that was in duced in C3Hf/Sed//Kam mice by treatment with methylcholanthrene (28). It is moderately immunogenic, grows progressively, and is well- characterized with respect to the immunological responses it generates, including its intratumoral host cell populations (26, 27, 29-31). We have failed to find evidence for endogenously expressed retroviruses in cultured FSA cell lines or antibody responses to viral antigens in mice bearing FSA tumors (32). For cytokine gene transfer experiments, an in vitro cell line was established from tumor that had been passed in vivo 3931 on June 29, 2015. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from