SPP1+ neutrophil and exhausted CD8⁺ T cells infiltration predicting the early recurrence of hepatocellular carcinoma patients after R0 resection.

[BACKGROUND] Early recurrence of liver cancer is significantly associated with poor prognosis. Further dissection of the cellular heterogeneity in the tumor microenvironment (TME) of patients at high risk of early recurrence after liver cancer surgery is highly necessary.

[METHODS] This study integrated clinical data, single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and bulk RNA-seq data to reveal and verify a TME characteristic associated with high risk of early recurrence after liver cancer surgery.

[RESULTS] Clinical analysis of 402 patients identified neutrophil-to-lymphocyte ratio ≥3, alpha-fetoprotein ≥400 ng/mL, and microvascular invasion as independent predictors of early recurrence. scRNA-seq of 28 hepatocellular carcinoma (HCC) samples revealed nine neutrophil subtypes, with SPP1⁺ neutrophils (enriched in TNFα/NF-κB, hypoxia, and TGF-β pathways) and exhausted CD8⁺ T cells (expressing PD-1 and CTLA-4) significantly infiltrating the TME of patients with early recurrence. These findings were corroborated in an independent validation cohort comprising scRNA-seq data from 17 HCC tissues and 7 matched peripheral blood samples. Spatial analysis demonstrated their co-localization at tumor margins, forming an immunosuppressive barrier. Ligand-receptor interaction analysis highlighted SPP1-CD44, TGFB1-TGFBR, and TNF-TNFRSF axes as key mediators of neutrophil-T cell crosstalk, inducing T cell dysfunction. Validation in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma cohort comprising 365 patients confirmed that high infiltration of SPP1⁺ neutrophils and exhausted CD8⁺ T cells was significantly correlated with shorter recurrence-free (P < 0.0001) and overall survival (P < 0.0001).

[CONCLUSIONS] This study shows that the infiltration of SPP1⁺ neutrophils and exhausted CD8⁺ T cells is associated with a high risk of early recurrence in HCC. SPP1⁺ neutrophils may serve as key mediator of immune suppression through spatial interactions with exhausted CD8⁺ T cells, suggesting that they could be potential therapeutic targets to mitigate early recurrence in HCC.