The role of interferon gamma and NK cells in the eradication of pulmonary osteosarcoma metastases by IL-12

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Osteosarcoma is a primary bone tumor that occurs predominantly in adolescents and young adults. Despite aggressive chemotherapy and surgical intervention, the 2- year disease-free survival of 60% has not changed significantly in the past 20 years. Salvage chemotherapeutic regimes has had little efficacy. Consequently, new approaches are needed. We are interested in stimulating the body’s own immune system to eradicate pulmonary metastases, and have previously demonstrated in mice that gene therapy with IL-12 resulted in a dramatic decrease in the size and number of pulmonary metastases. The purpose of this study is to identify the molecular and immunological events responsible for the eradication of pulmonary metastases in mice treated intranasally with an adenoviral vector containing the IL-12 gene. Balb/c mice were injected intra-osseously or intravenously (i.v.) with K7M3 cells, which resulted in the development of spontaneous, or experimental, pulmonary metastases, respectively. To assess the role of NK cell activation by IL-12 in this model, mice were treated with 50μl of asialoGM-1 antibody (this antibody inactivates NK cell function) or saline intraperitoneally twice weekly for 4 weeks. Mice then received AdmIL-12, Ad Bgal (control viral vector), or saline intranasally two times per week for 4 - 6 weeks. Analysis of the number of pulmonary metastases at the end of therapy demonstrated a two-fold increase in the size and number of pulmonary metastases in saline treated mice compared to IL-12 treated mice whose NK cell activity was blocked. Interestingly, the number of metastases in the IL-12 group (anti-asialo GM treated) was similar to control groups not receiving the antibody. These results suggest that NK cells are essential and partially responsible for the IL-12 induced eradications of pulmonary OS metastases. In another series of experiments, the role of IL-12 induction of interferon-gamma (IFNγ) was assessed. Some of IFNγ activities include inducing anti-angiogenic activity, activating macrophage iNOS activity, activation of NK cells, and IFN-γ dependent induction of Th1 cell differentiation and activation of T cells. IFNγ activation by IL-12 therapy was blocked using 100μg of anti-IFNγ intraperitoneally twice weekly. The mice received intranasal gene therapy with mIL-12, Ad Bgal, or saline as described above. Blocking IL-12’s induction of IFNγ resulted in a greater than two fold increase in the size and number of metastases compared to the group receiving intranasal mIL-12 and a control antibody intraperitoneally. In addition, in the IL-12 treated mice a five-fold increase in CD4+ T cells were recruited to the lungs day 3 after i.v. injection compared to control mice. In mice treated with anti-IFNγ, no significant difference in CD4+ T cell recruitment was noted between groups.