Plasmidic CpG sequences induce tumor microenvironment modifications in a rat liver metastasis model

Bacterial DNA contains unmethylated cytosine-phosphate-guanine (CpG) motifs which are recognized by mammalian immune cells as a danger signal indicating an infection. These immunostimulatory properties led to the use of oligodeoxynucleotides bearing CpG motifs (CpG-ODN) for cancer treatment in preclinical and clinical studies. Although naked DNA administration presently represents 18% of the gene therapy clinical trials worldwide, most of the work regarding the effects of unmethylated CpG sequences was performed using CpG-ODN. In the present study, we analyzed early induced tumor microenvironment modifications in a rat liver metastasis model after intratumoral injection of a plasmid used in suicide gene therapy. We first showed that plasmidic CpG motifs were active, i.e. able to induce IFN-gamma secretion by rat splenocytes. Then, we compared tumor-infiltrating immune cells 24 h after injection of native or SssI-treated plasmid, in which immunostimulatory CpG motifs have been inactivated by methylation. The presence of active plasmidic CpG sequences within the tumor was associated with a decrease in the number of tumor-infiltrating conventional dendritic cells and an upregulation of the CCR7 chemokine receptor responsible for lymph node homing. We also observed an increase in plasmacytoid dendritic cells and natural killer cell infiltration within the tumors as well as an increased mRNA expression of three cytokines/chemokines (IL-1beta, IL-10 and IL-18). These data suggest that, although suicide plasmid injection without prodrug treatment is not sufficient to observe a therapeutic effect, the presence of plasmidic CpG motifs within the tumor induces the recruitment and activation of the immune cells involved in antitumor response. These early cellular and molecular events should facilitate the induction of the immune response against tumor antigens released after in situ drug production.