Additive anti-Tumor Response to the Rabbit VX2 Hepatoma by Combined Radio Frequency Ablation and Toll like Receptor 9 Stimulation

Introduction: Radiofrequency ablation (RFA), a palliative therapeutic option for solid hepatic tumors, stimulates localized and systemic anti-tumor cytotoxic T cells. We studied how far addition of CpG B oligonucleotides, toll like receptor (TLR) 9 agonists, may increase the anti-tumoral T cell response of RFA in the highly aggressive VX2 hepatoma.

Methods: 21 days after hepatic tumor implantation, rabbits were randomized to receive RFA, CpG B, their combination or no therapy. The antitumor efficacy of RFA alone or in combination with CpG B was tested by rechallenging a separate group with i.v. injected VX-2 tumor cells after 120 d. Animals were assessed for survival, tumor size and spread, and tumor- and immune-related histological markers after 120 d. Peripheral blood mononuclear cells pre and post tumor implantation and treatment were tested for tumor-specific T cell activation using 3 H-thymidine-incorporation and for hepatoma-specific cytotoxicity using luciferase activity for adenylate kinase. ELISAS for Il2, 8, 10, 12, TNF alpha und IFN gamma serum levels were performed and justified by Western blotting from tumor tissue lysates.

Results: Mean survival of untreated animals was 35.9 d, as compared to 97.3 d, 77.6 d and 113.9 d for RFA, CpG and combination therapy, resp. Compared to untreated controls, T cell stimulation/cytotoxicity increased 25.5-/16.4-, 31.7-/17.3- and 50.4-/37.8-fold post RFA, CpG and combination treatment after 2 w, resp. The combination also inhibited tumor spread to lungs and peritoneum significantly better than the other therapies. Only animals treated with the combination of RFA and CpG showed a suppression of VEGF and down regulation of Cytokine leading to immunotolerance (Il 10). Animals receiving a local single treatment or no treatment showed significantly upregulated cytokines leading to angiogenesis and tumor spread.

Conclusion: The combination of RFA and TLR9 stimulation by CpG B oligonucleotides elicited an additive T cell stimulation and cytotoxic anti-tumor effect in the VX2 tumor model, resulting in a significantly improved survival. Only the combination of RFA and CpG B prevented subsequent tumor spread and should be tested in clinical trials for liver cancer. A single treatment (RFA or CpG) could enhance tumor spread and growth.