The clinical efficacy of anti-PD-1 immunotherapy in hepatocellular carcinoma (HCC) remains limited due to the inherently immunosuppressive tumor microenvironment. This study aims to investigate the role of the spermatogenesis-associated serine-rich protein 2 (SPATS2) in mediating resistance to anti-PD-1 immunotherapy in HCC.

Five independent HCC anti-PD-1 therapy RNA-seq cohorts (n = 105) were included to identify key genes driving anti-PD-1 resistance. Multiplex immunofluorescence staining, single-cell RNA sequencing, spatial transcriptome analysis, flow cytometry, in vitro co-culture systems, RNA immunoprecipitation assays, mice spontaneous HCC models and patient-derived organoid models to elucidate the biological function of SPATS2 in tumor progression and immune escape.

Cross-cohort analysis identified SPATS2 as a critical driver of anti-PD-1 resistance in HCC. Elevated SPATS2 expression promotes immune evasion by impairing dendritic cell (DC) infiltration and phagocytic activity, leading to therapeutic resistance. Mechanistically, SPATS2 binds AU-rich elements (ARE) in the CD47 3’UTR to stabilize its mRNA and interacts with the eIF4F complex. These two interactions result in CD47 mRNA circularization, and facilitate recruitment of translation initiation factors to the translation start site, thereby enhancing CD47 translation. Increased CD47 suppresses DC uptake of tumor-derived mitochondrial DNA (mtDNA), thereby dampening DC cGAS-STING signaling and subsequently CD8⁺ T cell-mediated antitumor responses. Importantly, targeted silencing of SPATS2 via lipid nanoparticle-delivered siRNA, or combination with anti-CD47 therapy, significantly improved HCC anti-PD-1 therapeutic efficacy.

SPATS2 facilitates immune evasion and anti-PD-1 resistance in HCC by inhibiting DC infiltration and impairing phagocytic function. Targeting SPATS2 or combining with anti-CD47 monoclonal antibody strategy is expected to overcome immunotherapy tolerance and improve the immune response rate of HCC patients.