Although oligodeoxynucleotides containing CpG motifs (CpG-ODN) are potent immune stimulators, the use of natural CpG-ODN--phosphodiester-backbone CpG--has been limited due to its instability by nuclease in vivo. The aim of this study is to investigate the anticancer efficiency of CpG-ODN capsulated using liposome, which enhances the stability of CpG-ODN. We formulated lipoplex, encapsulated natural CpG-ODN from Mycobacterium bovis with liposome, and tested its immune stimulatory activity in vitro and in vivo. The lipoplex induced a systemic innate immune response in vivo and stimulated dendritic cells, but not macrophages, to stimulate proinflammatory cytokines such as tumor necrosis factor alpha and interleukin-6 in vitro. As expected, the lipoplex effectively mediated the prolonged cancer-therapeutic activity against B16 melanoma, which was dependent on natural killer and CD8(+) T cells. The therapeutic activity was observed after only intratumoral administration of lipoplex among several treatment routes. Intratumoral treatment of lipoplex significantly increased the populations of natural killer and CD8(+) T cells and reduced regulatory CD4(+) T cell recruitment, which was correlated with expression profiles of chemokines (CCL1, CCL3, CXCL1, CXCL10, and CCL22). The antitumor therapeutic effect of lipoplex was dependent on the altered lymphocyte population that might be developed by the profile of intratumoral chemokine expression.
