Stoking an anti-liver cancer immune response with cryoablation plus an intratumoral TLR9 agonist and dual checkpoint inhibitors.

The rising incidence of hepatocellular carcinoma (HCC) is partly driven by metabolic dysfunction-associated steatohepatitis (MASH). Recurrence after treatment is high, and advanced disease carries a poor prognosis. The immunosuppressive tumor microenvironment (TME) in HCC limits the efficacy of immunotherapy, necessitating innovative approaches. To evaluate the efficacy of a novel therapeutic strategy designed to stimulate an antitumor immune response by combining cryoablation (Cryo) to release tumor antigen with an intratumoral immunostimulant (CpG) to facilitate immune recognition and dual immune checkpoint inhibitors (CPI) to disinhibit T cells. RIL-175 cells were orthotopically injected into two liver lobes in mice on a MASH-inducing diet. One tumor in each mouse was treated with partial Cryo, systemic dual CPI (anti-PD-1 and anti-CTLA-4), intratumoral TLR9 agonist CpG, or combinations thereof, while the second tumor was untreated. Tumor growth was monitored prior to and after ablation. Immune cell profiles and cytokine levels in both treated and untreated tumors were analyzed postmortem. The combined treatment of Cryo with CpG and dual CPI achieved the strongest tumor control and survival benefit. Cryo alone increased growth of the untreated tumor but addition of CpG and CPI reversed this effect. Immunologically, CPI primarily drove CTL expansion and PD-1 modulation, whereas CpG suppressed Tregs. Cryo monotherapy leads to immunosuppression, characterized by elevated Tregs, MDSCs, and PD-1 expression. Combining Cryo with CpG and CPI counteracts the inherent limitations of each therapy and enhances systemic anti-tumor immune responses.