Treatment of liver metastases from colon cancer by suicide gene therapy : role of the plasmidic CpG sequences in the triggering of antitumor immune response

PhD Pierrefite-Carle^a, F Anjuere PhD^b, M Samson PhD^a,d, P Baqué MD PhD^c, A Gavelli MD^a, N Brossette PhD^a, C Pons^a, PhD Coulomb^a, JM Guigonis PhD^d, N Nafati PhD^d, B Karimdjee MD^a, S Bertin^a, A Bourgeon MD^c, B Rossi PhD^a

^aFaculté de Médecine, Unité INSERM 364, IFR50, Nice, ^bFaculté de Médecine, IFR50, Nice, ^cService de Chirurgie Générale et Cancérologie Digestive, Hôpital l’Archet II, IFR50, Nice, ^dPlateau protéomique, Faculté de Médecine, IFR50, Nice, France

AIM: The cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). We have previously shown that, in rats bearing experimental metastases in right and left liver lobes, intratumor injection of a CD-expressing plasmid in one of the tumors followed by 5-FC treatment results in regression of both lesions. Moreover, this treatment also induced a potent distant bystander effect on extra-hepatic metastases, resulting in an increased survival compared to control animals. 5-FC administration in the absence of DNA injection or intratumor plasmid injection without 5-FC treatment had no therapeutic effect. However, plasmid DNA could be involved in the potent antitumor immune response observed via the presence of of unmethylated CpG sequences. Indeed, these motifs present in bacterial DNA are immunostimulatory in vertebrates and trigger pleiotropic effects in immune cells such as proliferation, activation and cytokine/chemokine secretion. In the present work, we hypothesized that these sequences present in the CD-expressing plasmid could set the ground for the immune response induction. METHODS: We compared native plasmid and SssI-treated plasmid, in which immunostimulatory motifs have been inactivated by methylation. Twelve or 24 hours after intratumor plasmid DNA injection, we analyzed tumor-infiltrating immune cells and tumor’s proteome by two-dimensional gel electrophoresis and mass spectrometry. RESULTS: The results suggest that native CD-plasmid injection is associated with tumor-infiltrating dendritic cells activation and with an increase in NK cells recruitement. Moreover, proteome analysis of tumors indicate that expression of specific proteins, such as "suffering" markers, is induced after native plasmid injection. CONCLUSIONS: These data indicate that although CD-expressing plasmid is not sufficient to observe a therapeutic effect, the presence of this bacterial DNA within the tumor induces the recruitment and activation of the immune cells involved in the antitumor response. In addition, presence of CpG motifs is associated with the triggering of danger signals potentially stimulating this antitumor response. These cellular and molecular events should facilitate the induction of the immune response when tumor’s antigens are released after in situ 5-FU production.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 995 (Immunotherapy).