Immune response induced by irradiated B16F0 vaccine diminish melanoma lung metastasis in mouse model

2848 Background and Purpose: Melanoma has had a rising incidence in the United States over the past years. The aim of this study is to develop a novel protocol of stimulating immune response of melanoma carrying mice by irradiating and then surgical remove tumor to reduce melanoma metastatic disease.Methods:B16F0, mouse melanoma, in cell culture were irradiated with 25 Gy and incubated for 0, 6, and 24 hours, and then the cells growth on histological slides were fixed for CRT (calreculin), a potential immunogenicity of tumor cells, immunofluorescence staining. According to CRT expression following radiation from experiment 1, 1x 106 irradiated B16F0 cells were injected subcutaneously into the back neck of C57 mouse at 6 hours after 25 Gy. Control group were only received medium injection. 12 days after irradiated B16F0 vaccine injection, 0.8 x10 untreated B16F0 cells were implanted subcutaneously into the right hind limb on control and vaccine injection C57BL6/J mice. Tumor growth delay study were performed, and meanwhile B16F0 tumor tissues from control and vaccine injection mice were sampled for tumor-infiltrating lymphocytes (TIL) assay with whole mount histology technique for CD8+ T cell (cytotoxic T lymphocyte) detection. 0.5 x 106 B16F0 cells in 50 μl of medium were injected into the small previous made skin pouch on the right lateral abdomen of C57 mice. B16 F0 tumors (about 5-6 mm in diameter) of the irradiation + surgical remove group (6 mice) at 6 days after implantation were irradiated with 25 Gy. 300 ng of interleukin 2 was injected subcutaneously near tumor for the irradiation + surgical remove group at day 3 and 4 after irradiation. The tumor surgical remove group (5 mice) received shamed irradiation. The tumors were removed by surgery at day 5 after irradiation or shamed irradiation. 0.25 x 106 untreated B16F0 were injected each mouse through tail vein. The mice were sacrificed at day 12 after tail vein injection and the lungs were sampled for surface B16F0 nodes count. Results: CRT strongly expressed on the cell membrane surface of B16F0 after 25 Gy, 6 hours point had maximum expression. Irradiated B16F0 vaccine caused 5-7 days tumor growth delay comparing with the control group. The CD8+ T cell IHF staining showed that tumor infiltrating lymphocytes significantly appeared in B16F0 tumor tissues after irradiated B16F0 vaccine injection but control B16F0 tumor tissues had very few of CD8 + T cells positive staining. The counting of B16F0 nodes on lung surface resulted respectively the irradiation + surgical remove group: 4.2 ± 2.7, surgical remove group: 53.2 ± 9.5, and control group: 93.2 ± 17.1. Conclusion:Irradiated B16F0 vaccine can induce immune response that diminishes melanoma lung metastasis in mouse model.