LOCOREGIONAL DELIVERY OF UNMETHYLATED CPG-OLIGODEOXYNUCLEOTIDES TO CANCER THERAPY: PRECLINICAL STUDIES

Tumor cell growth, even in advanced stages of ovarian cancer, is nearly always restricted to the peritoneal cavity, therefore repeated intraperitoneal injections of CpG-ODN recruiting and activating innate effector cells throughout the abdominal cavity to the tumor site might control tumor cell growth and ascites formation. After a single CpG-ODN treatment, in IGROV-1 ovarian tumor ascites-bearing athymic mice, the number of tumor cells declined rapidly and markedly, and ascites volumes declined shortly after treatment (5 h), increasing thereafter at a slower rate than in controls. When administered every 7 days for 4 weeks, CpG-ODN had only a marginal effect on survival time, whereas administration 5 days/week for 3 or 4 weeks led to a significantly increased survival-time as compared to controls and completely controlled ascites growth without apparent toxicity, although a disorganization of lymphoid organs was observed. Depletion of NK or monocytes/macrophages only slightly influenced the CpG-ODN-induced reduction of ascites tumor cells, indicating that the antitumor activity might not be related to a specific cell/cytokine but rather to the repertoire of cells and cytokines accumulated in the peritoneal cavity. Thus, our data suggest a relevant role for repeated activation of cells and cytokines of innate immunity in the therapy of ovarian cancer patients with malignant ascites. However, daily i.p. administration of CpG-ODN induced a significant increase of survival-time but no cure of a single mouse, therefore we screened the effectiveness of CpG-ODN in combination with different agents, including bevacizumab, Poly(I):Poly(C) and cisplatin. Our data indicate that the combination of repeated i.p. CpG-ODN treatments plus bevacizumab and Poly(I):Poly(C) do not significantly increase median survival time, while the association of CpG-ODN and cisplatin revealed a significant increase in mice lifespan. Based on these results, we performed several experments in order to gain inside the molecular mechanisms by which CpG-ODN improves the therapeutic efficacy of cisplatin, a DNA-damaging drug. We demonstrate that an immunostimulatory Toll-like receptor-9 (TLR9) agonist CpG-oligodeoxynucleotide (CpG-ODN) oppositely modulates expression of DNA repair genes in tumor and immune cells. Analyses in silico and in a human ovarian tumor model by microarray revealed downregulation of these genes in tumors and upregulation in immune cells, with no detectable modulation in normal non-immune tissue. CpG-ODN induced activation of cells present in the tumor microenvironment was critical in inducing DNA-repair gene modulation in tumors. In conclusion, the combination of cisplatin with CpG-ODN was effective and well tolerated, and might represent a preclinical basis for the design of clinical studies, even considering the ability of CpG-ODN to down-modulate DNA repair genes in tumor cell.