16 - Lactate-Modulating Nanoreactors Facilitate Self-Amplifying Pyroptosis and the cGAS-STING Cascade for TACE Therapy

Purpose: To convert intra-tumoral lactate from an immunosuppressive metabolite into a therapeutic fuel, we engineered LOCoF₂ nanoreactors that synchronously trigger pyroptotic cell death and potentiate cGAS-STING signaling, and to validate their efficacy as an in-situ immune primer for TAE/TACE in orthotopic hepatocellular carcinoma (HCC).

Materials and Methods: LOCoF₂ was prepared by electrostatic assembly of lactate oxidase (LOx) on DSPE-PEGylated cobalt fluoride (CoF₂) nanorods. Enzyme-mimetic activity was quantified with DPBF, NBT, TMB and OPD probes. In vitro: H22 and HuH7 cells were assessed for lactate consumption, ROS, mitochondrial membrane potential, pyroptosis morphology, LDH/ATP release, CRT/HMGB1 exposure, stemness markers, and cGAS-STING phosphorylation. In vivo: murine subcutaneous or bilateral H22 models, humanized HuH7 xenografts, and rat orthotopic N1-S1 HCC were used. LOCoF₂ was injected intratumorally or dispersed in Lipiodol for trans-arterial embolization (TAE/TACE) with or without oxaliplatin (Oxa) or α-PD-1. Immune profiling by flow cytometry, ELISA, RNA-seq, and MRI-guided tumor monitoring was performed.

Results: LOCoF₂ depleted lactate within 30 min and cascade-generated •OH/O₂•–, elevating intracellular ROS 3.8-fold and collapsing mitochondrial potential to 87 % monomers. Consequently, cleaved-caspase-1 and GSDMD-N increased 4.1- and 5.3-fold, provoking pyroptotic bubbles, IL-1β surge (240 pg mL⁻¹) and ICD signals (CRT 24 %, HMGB1 release 60 %). Released mtDNA plus Co²⁺ amplified cGAS-STING phosphorylation (p-TBK1, p-IRF3, p-STING each >2-fold), yielding 1 ng mL⁻¹ IFN-β. In mice, a single 5 mg kg⁻¹ LOCoF2 dose induced 33.5 % mature DCs, 40 % CD8⁺ T-cell infiltration, 20 % MDSC and 13 % Treg reduction, eradicating 40 % of primary tumors and 30 % of distant lesions. Combination with α-PD-1 achieved 80 % complete responses and memory immunity. In the rat orthotopic model, LOCoF₂-Lipiodol TACE reduced tumor volume 4.7-fold versus Lipiodol alone; adding Oxa (TACE) extended median survival from 14 to 32 days with augmented CD45⁺ immune infiltration and no systemic toxicity.

Conclusion: LOCoF₂ transforms lactate into H₂O₂, driving CoF₂-catalyzed ROS overproduction that ignites pyroptosis and releases mtDNA, which together with Co²⁺ sparks a self-amplifying cGAS-STING cascade. The resulting potent catalytic-immunotherapy synergizes with ICB and can be delivered locally via TAE/TACE, offering a clinically translatable strategy to convert metabolic vulnerability into robust, sustained anti-HCC immunity.