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

Abstract

Abstract. Bacterial DNA contains unmethylated cytosinephosphate-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-γ secretion by rat splenocytes. Then, we compared tumorinfiltrating 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 . 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.

Short- and long-term changes in gene expression mediated by the activation of TLR9

“…1; p < 0.0001 for all 3 groups). These functional classes are consistent with the known biological effects of TLR9-dependent cell stimulation (Ballas et al , 1996; Broide et al , 1998; Klinman et al , 1996; Roman et al , 1997; Sparwasser et al , 1997; Sparwasser et al , 1998; Bertin et al , 2008; Haining et al , 2008; Stacey et al , 1996; Sun et al , 1998). …”

A Combined Chemoimmunotherapy Approach Using a Plasmid−Doxorubicin Complex

“…This early method of immunotherapy resulted in clinical efficacy in 10% of the patients . The immune stimulating component responsible was discovered to be “unmethylated CpG motifs” in bacterial DNA. , With the discovery of toll-like receptors (TLRs) that detect highly conserved pathogen-expressed molecules, tumor immunology for cancer treatment has recently seen the development of various synthetic agonists for several TLRs. − For cancer treatment, unmethylated CpG dinucleotides recognized by TLR-9 has shown promise as it elicits the most favored Th1 immune response. − Short oligonucleotides containing such CpG motifs are used as adjuvants or in combination with cytotoxic chemotherapy, and their antitumor effects are well documented in various experimental animal models. − …”

Comparative Proteomics Study Reveals That Bacterial CpG Motifs Induce Tumor Cell Autophagy in Vitro and in Vivo

“…6B). As bacterial plasmids, which present unmethylated CpG motifs, are widely used in preclinical and clinical cancer gene therapy, the same experiment was performed in tumors injected with a P, bearing active CpG sequences, or a Met-P in which the effect of CpG sequences has been abolished by in vitro methylation (25). Similarly the median number of autophagosomes per field increased significantly in P-compared with Met-P-injected tumors (Fig.…”

“…The plasmid (200 g in a 1-ml final volume) was methylated with SssI CpG methylase (New England Biolabs, Ozyme, St. Quentin en Yvelines, France) as described previously (25).…”