Stimulating Antitumoral Immunity by Percutaneous Cryoablation and Combination Immunoadjuvant Therapy in a Murine Model of Hepatocellular Carcinoma.

PURPOSE To test the hypothesis that antitumoral immunity can be induced after cryoablation of hepatocellular carcinoma (HCC) through co-administration of the immunostimulant CpG and an immune checkpoint (PD-1) inhibitor. METHODS 63 immunocompetent C57BL/6J mice were generated with two orthotopic HCC tumor foci: one for treatment and one to observe for antitumoral immunity. Tumors were treated with incomplete cryoablation (Cryo) alone or with intra-tumoral CpG and/or PD-1 inhibitor. The primary endpoint was death or when criteria for sacrifice were met: tumor >1 cm (by ultrasound) or moribund state. Antitumoral immunity was assessed by flow cytometry and histology (tumor and liver) and ELISA (serum). ANOVA was used for statistical comparisons. RESULTS At 1 week, non-ablated satellite tumor growth in the Cryo+CpG group was reduced by 1.9-fold (P=0.047) and in the Cryo+CpG+PD-1 group by 2.8-fold (P=0.007) compared to the Cryo group. Compared to Cryo alone, time to tumor progression to endpoints was also prolonged for the Cryo+CpG+PD-1 and Cryo+CpG mice with logrank hazard ratios of 0.42 (P=0.031) and 0.27 (P<0.001), respectively. Flow cytometry and histology showed increased cytotoxic T cell infiltration (P=0.002) and serum pro-inflammatory cytokine IFN-γ (P=0.015) in tumors and serum of Cryo+CpG mice compared to Cryo alone. High serum levels of the anti-inflammatory cytokine TGF-β and the pro-angiogenesis chemokine CXCL1 correlated with shorter time to endpoints and faster tumor growth. CONCLUSION Cryoablation combined with the immunostimulant CpG promoted cytotoxic T cell infiltration into tumors, slowed tumor growth, and prolonged time to progression to endpoints in an aggressive HCC model.