Radiofrequency hyperthermia enhances the effect of OK-432 for Hepatocellular carcinoma by activating of TLR4-cGAS-STING pathway

•1. OK-432 could activate the cGAS-STING pathway of DC cells;•2. Radiofrequency hyperthermia could enhance the activation through enhancing the expression of TLR4 on DC cells;•3. The combination of OK-432 and Radiofrequency hyperthermia exhibited a strong anti-tumor effect and brought survival benefits. Radiofrequency ablation (RFA) has been used as an alternative to surgical management of early-stage hepatocellular carcinoma (HCC). However, when large and irregular HCCs are subjected to RFA, a safety margin is usually difficult to obtain, thus causing a sublethal radiofrequency hyperthermia (RFH) at the ablated tumor margin. This study investigated the feasibility of using RFH to enhance the effect of OK-432 on HCC, with the aim to generate a tumor-free margin during RFA of HCC. Our results showed OK-432 could activate the cGAS-STING pathway, and RFH could further enhance the activation. Meanwhile, RFH could induce a high expression of TLR4, and TLR4 might be an upstream molecular of the cGAS-STING pathway. The combined therapy of RFH with OK-432 resulted in a better tumor response, and a prolonged survival compared to the other three treatments. In conclusion, RFH in combination with OK-432 might reduce the residual and recurrent tumor after RFA of large and irregular HCCs, and serve as a new option for other solid malignancies treated by RFA.