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

Abstract

PurposeTo test the hypothesis that antitumoral immunity can be induced after cryoablation (cryo) of hepatocellular carcinoma (HCC) through coadministration of the immunostimulant CpG and an immune checkpoint (programmed cell death 1 [PD-1]) inhibitor. Materials and MethodsSixty-three immunocompetent C57BL/6J mice were generated with 2 orthotopic HCC tumor foci: 1 for treatment and 1 to observe for antitumoral immunity. Tumors were treated with incomplete cryo alone or intratumoral CpG and/or a PD-1 inhibitor. The primary endpoint was death or when the following criteria for sacrifice were met: tumor > 1 cm (determined using ultrasound) or moribund state. Antitumoral immunity was assessed using flow cytometry and histology (tumor and liver) as well as enzyme-linked immunosorbent assay (serum). Analysis of variance was used for statistical comparisons. ResultsAt 1 week, the nonablated satellite tumor growth was reduced by 1.9-fold (P = .047) in the cryo + CpG group and by 2.8-fold (P = .007) in the cryo + CpG + PD-1 group compared with that in the cryo group. Compared with cryo alone, the time to tumor progression to endpoints was also prolonged for cryo + CpG + PD-1 and cryo + CpG mice, with log-rank hazard ratios of 0.42 (P = .031) and 0.27 (P < .001), respectively. Flow cytometry and histology showed increased cytotoxic T-cell infiltration (P = .002) and serum levels of the proinflammatory cytokine interferon-γ (P = .015) in tumors and serum of cryo + CpG mice compared with those in tumors and serum of mice treated with cryo alone. High serum levels of the anti-inflammatory cytokine tumor growth factor-β and the proangiogenesis chemokine C-X-C motif chemokine ligand 1 were correlated with a shorter time to endpoints and faster tumor growth. ConclusionsCryo combined with the immunostimulant CpG promoted cytotoxic T-cell infiltration into tumors, slowed tumor growth, and prolonged the time to progression to endpoints in an aggressive murine HCC model.