Single-cell spatial landscape of aggrephagy activity stratifies hepatocellular carcinoma neutrophils and delivers a 5-gene diagnostic panel for patient stratification.

BACKGROUND: Hepatocellular carcinoma (LIHC) features a complex tumor microenvironment (TME) where tumor-associated neutrophils (TANs) show significant plasticity. The role of aggrephagy-selective autophagy of protein aggregates-in shaping neutrophil heterogeneity and LIHC progression remains poorly understood. METHODS: We integrated scRNA-seq (183,671 cells), spatial transcriptomics, and bulk datasets (TCGA, GSE39791). Neutrophils (n=12,547) were re-clustered into six subsets, and aggrephagy activity was quantified via UCell scores. Analysis included pseudotime trajectories, cell-cell communication, metabolic scoring, and machine-learning-based feature selection, followed by in vitro functional validation. RESULTS: Aggrephagy activity was significantly elevated in tumor tissues compared with adjacent normal tissues (P < 0.001) and showed strong cell-type specificity, with TANs among the most enriched populations. High-aggrephagy neutrophils exhibited an undifferentiated state, preferential tumor enrichment, and a positive correlation with transcriptomic risk scores. Trajectory analysis positioned these cells at an early differentiation branch and revealed dominant neutrophil-to-stroma signaling through the CCL3-CCR1, SPP1-CD44, and ANXA1-FPR1 axes. Metabolically, high-aggrephagy neutrophils displayed enhanced inflammatory and epithelial mesenchymal-transition programs alongside suppressed oxidative phosphorylation. Integrative network analysis identified a five-gene diagnostic panel (SQSTM1, WDFY3, DOCK4, CD177, LIMK2) with robust performance across bulk cohorts (AUC 0.83-0.91). Among these, LIMK2 marked a highly interactive neutrophil subset and functionally promoted tumor cell proliferation, survival, migration, and invasion in vitro. CONCLUSION: Aggrephagy is associated with a pro-tumorigenic, metabolically reprogrammed neutrophil state in LIHC. The LIMK2-centered gene panel provides a robust framework for subset identification and nominates candidate targets for future autophagy- and neutrophil-directed studies.