Multimodal thermal therapy to promote the expansion of tumor-reactive–like CX3CR1 ⁺ GPR56 ⁺ T cells in hepatocellular carcinoma patients.

e16286 Background: Local ablation represents the minimal treatment option for patients with hepatocellular carcinoma (HCC) who cannot be treated with surgical resection. This approach could facilitate the release of tumor antigens, thereby potentially activating systemic antitumor immunity. Nevertheless, clinically observation indicated that the systemic response was rather transient and weak, insufficient to induce protective immunity. Multimodal thermal therapy (MTT), a novel local ablation technology developed that involves liquid nitrogen freezing followed by radiofrequency heating, has been proven to stimulate robust and sustained anti-tumor immunity. The primary aim of this study was to evaluate whether MTT could induce an effective anti-tumor immunity and prolong progression-free survival of HCC patients who progressed under prior therapies, including hepatic tumor resection, local-regional treatment, targeted therapy or immunotherapy. Methods: Four patients underwent MTT and the other three underwent conventional radiofrequency ablation (RFA). The primary endpoints included the antitumor immune response and the 6-month disease control rate (DCR). After treatment, patients were evaluated by triphasic contrast-enhanced MRI in 3, 30, 90, 180 and 360 days. Also, the immune landscapes of the patients' pre-treatment tumors and PBMCs, as well as PBMCs in 7 and 30 days posttreatment, were characterized using single-cell RNA sequencing and flow cytometry. Further verification was also performed using Hepa1-6 mouse models. Results: Compared to RFA, MTT significantly prolonged patients' progression-free survival (PFS). MTT, but not RFA, promoted the release of DAMPs such as DNA, HSP70, and remodeled the systemic immune environment, as evidenced by increased dendritic cell maturation, natural killer cell cytotoxicity, peripheral plasma cell levels and T-cell activation. In peripheral blood, the proportion of CX3CR1 + GPR56 + effector T cells was increased in patients receiving MTT, whereas it remained unchanged in patients receiving RFA. CX3CR1 + GPR56 + effector T cells expressing high levels of cytotoxic molecules, have increased ability to migrate, and share TCR sequences with tumor-reactive-like T cells and its proportion was positively correlated with the PFS of the patients. Furthermore, MTT facilitates the interaction between CX3CR1 + GPR56 + T cells and antigen-presenting cells, thereby promoting their expansion. Conclusions: The results presented that MTT sufficiently stimulated systemic anti- tumor immunity and robustly activated the expansion of peripheral CX3CR1 + GPR56 + effector T cells playing a patrolling and surveillance role contributing favorable prognosis, which warrants further clinical investigation at a large scale. Clinical trial information: NCT06588569 .