Neutrophil Senescence Induced by Tumor-Derived HMGB1 Promotes Hepatocellular Carcinoma Progression through Neutrophil Extracellular Trap-Mediated Suppression of CD8+ T Cell Immunity.

Peripheral blood and tumor tissues from HCC patients exhibited a marked accumulation of aged neutrophils compared to healthy controls.

• Clinical cohort comprised 105 HCC patients and 41 healthy controls.
• Two public datasets (GSE149614 and GSE189903) were also analyzed to assess aged neutrophil abundance.
• Accumulation of aged neutrophils was observed in both peripheral blood and tumor tissue compartments.
• Aged neutrophil abundance was inversely correlated with CD8+ T cell abundance in HCC patients.

Aged neutrophils released excessive neutrophil extracellular traps (NETs) via a caspase-3-dependent mechanism that impaired CD8+ T cell proliferation and activation.

• The mechanism of NET release from aged neutrophils was identified as caspase-3-dependent.
• NETs released by aged neutrophils directly suppressed CD8+ T cell proliferation and activation.
• In vitro cellular experiments were used to dissect this molecular mechanism.
• This finding distinguishes the NET-release pathway in aged neutrophils from other known NET-formation mechanisms.

Suppression of CD8+ T cell activity by aged neutrophil-derived NETs promoted malignant behaviors of HCC cells.

• HCC cell malignant behaviors promoted included enhanced proliferation, reduced apoptosis, and increased angiogenesis.
• These functional consequences were assessed through in vitro cellular experiments.
• The effect was mediated through CD8+ T cell dysfunction rather than direct neutrophil-to-tumor cell interaction.

In vivo accumulation of aged neutrophils accelerated HCC progression through CD8+ T cell dysfunction, with minimal impact on distant metastasis.

• Both in vitro and in vivo experiments were performed to evaluate aged neutrophil involvement in HCC progression.
• The pro-tumorigenic effect in vivo was primarily mediated via suppression of CD8+ T cell activity.
• Aged neutrophil accumulation had minimal impact on distant metastasis in vivo.
• This suggests the primary role of aged neutrophils in HCC is local tumor progression rather than metastatic dissemination.

HCC cell-derived HMGB1 induced neutrophil senescence via activation of the Toll-like receptor 4 (TLR4) signaling pathway.

• High-mobility group box 1 (HMGB1) secreted by HCC cells was identified as the driver of neutrophil senescence.
• HMGB1 signaled through TLR4 to induce the senescence phenotype in neutrophils.
• This mechanism was elucidated through in vitro molecular experiments.
• HMGB1-TLR4 signaling represents the upstream mechanistic link between tumor cells and neutrophil aging.

Integrating circulating aged neutrophil levels with alpha-fetoprotein (AFP) substantially improved early HCC detection.

• Circulating aged neutrophils were evaluated as a noninvasive biomarker in the clinical cohort of 105 HCC patients and 41 healthy controls.
• Combining aged neutrophil abundance with AFP improved early HCC detection beyond AFP alone.
• Aged neutrophils were assessed as a 'robust noninvasive candidate marker for both HCC detection and assessment of malignant risk.'
• The clinical utility was assessed in the context of both early detection and risk stratification.

The CD8+ T cell-to-aged neutrophil ratio demonstrated strong potential for malignant risk stratification in HCC patients.

• The ratio of CD8+ T cells to aged neutrophils was evaluated as a clinical parameter.
• This ratio showed strong potential for distinguishing malignant risk categories among HCC patients.
• The inverse correlation between aged neutrophil abundance and CD8+ T cell abundance underpins this ratio's clinical relevance.
• Risk stratification utility was explored using the clinical cohort data.