Tumor-derived acetylcholine promotes immune evasion in hepatocellular carcinoma by protecting regulatory T cells from ferroptosis.

Hepatocellular carcinoma (HCC) is characterized by immune suppression and limited durability of systemic therapy, yet how tumor metabolic remodeling supports regulatory T-cell (Treg) persistence remains unclear. Here, we identify a cholinergic immune-evasion pathway that links acetylcholine (ACh) accumulation to Treg ferroptosis resistance in HCC. Sorafenib-resistant patient-derived xenografts showed enhanced Treg-associated signatures together with enrichment of ferroptosis- and cholinergic signaling-related pathways. Clinical HCC tissues exhibited cholinergic remodeling, characterized by reduced BCHE, increased CHKA, elevated intratumoral ACh, increased Treg infiltration, and poor patient survival. Functionally, exogenous ACh and HCC cell-conditioned media protected MT-2 Treg-like cells from erastin-induced ferroptosis by reducing Fe2+ accumulation, reactive oxygen species (ROS) generation, lipid peroxidation and mitochondrial injury while restoring antioxidant capacity. ChAT knockdown in HCC cells weakened this protection, whereas ACh supplementation restored it. Mechanistically, ACh acted through α7 nicotinic acetylcholine receptor (α7nAChR) to activate signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation. STAT3 directly bound the ubiquitin-specific peptidase 11 (USP11) promoter and induced USP11 transcription. USP11 interacted with ferroptosis suppressor protein 1 (FSP1) and stabilized FSP1 by suppressing ubiquitin-proteasome-dependent degradation, thereby limiting ferroptosis and reinforcing an immunosuppressive marker profile. Inhibition of STAT3, USP11 or FSP1 abolished the ferroptosis-protective effect of ACh. In vivo, targeting the Ach-Treg ferroptosis pathway restrained HCC growth, reduced intratumoral Treg accumulation, and enhanced anti-PD-1 efficacy. Collectively, these findings identify a tumor-derived cholinergic mechanism that promotes Treg ferroptosis resistance and immune escape in HCC, and highlight this process as a potential therapeutic vulnerability for combination immunotherapy.