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Radiofrequency ablation (RFA) is the most common approach to thermal ablation for cancer therapy. Unfortunately, its efficacy is limited by incomplete ablation, and further optimization of RFA is required. Here, we demonstrate that incubation at 65 °C triggers more EG7 tumor cell death by necrosis than treatment at 45 °C, and the 65 °C-treated cells are more effective at inducing antigen-specific CD8 + cytotoxic T lymphocyte (CTL) responses after injection in mice than the 45 °C-treated ones. Dendritic cells (DCs) that phagocytose 65 °C-treated EG7 cells become mature with upregulated MHCII and CD80 expression and are capable of efficiently inducing effector CTLs in mouse tumor models. RFA (65 °C) therapy of EG7 tumors induces large areas of tumor necrosis and stimulates CTL responses. This leads to complete regression of small (~100 mm 3 ) tumors but fails to suppress the growth of larger (~350 mm 3 ) tumors. The administration of the Toll-like receptor-9 (TLR9) agonist unmethylated cytosine-phosphorothioate-guanine oligonucleotide (CpG) to DCs phagocytosing 65 °C-treated EG7 cells enhances the expression of MHCII and CD40 on DCs as well as DC-induced stimulation of CTL responses. Importantly, the intratumoral administration of CpG following RFA also increases the frequencies of tumor-associated immunogenic CD11b - CD11c + CD103 + DC2 and CD11b + F4/80 + MHCII + M1 macrophages and increases CD4 + and CD8 + T-cell tumor infiltration, leading to enhanced CD4 + T cell-dependent CTL responses and potent inhibition of primary RFA-treated or distant untreated tumor growth as well as tumor lung metastasis in mice bearing larger tumors. Overall, our data indicate that CpG administration, which enhances RFA-induced CTL responses and ultimately potentiates the inhibition of primary tumor growth and lung metastasis, is a promising strategy for improving RFA treatment, which may assist in optimizing this important cancer therapy.

TLR9 agonist enhances radiofrequency ablation-induced CTL responses, leading to the potent inhibition of primary tumor growth and lung metastasis