IL-32 drives PR3-dependent NET formation to promote portal vein tumor thrombosis and radioresistance in HCC

IL-32 is a pro-inflammatory cytokine associated with chronic inflammation. Neutrophil extracellular traps (NETs) contribute to thrombosis, tumor invasion, and metastasis. This study investigated whether IL-32 derived from hepatocellular carcinoma (HCC) cells induces NET formation, promotes portal vein tumor thrombus (PVTT), and contributes to radiotherapy resistance.

Immunofluorescence was used to assess co-localization of IL-32 and NET markers in tumor tissues. Mass cytometry analyzed immune cell composition in the tumor microenvironment. Neutrophils from healthy donors were used to evaluate NET formation. PAD4⁻/⁻ mice and DNase I treatment were used to assess NET function in vivo.

IL-32 and NETs were significantly elevated and positively correlated in HCC, particularly in patients with PVTT. Tumor-derived IL-32 induced robust NET formation in a dose-dependent manner, which was markedly attenuated by PR3 inhibition, identifying PR3 as a critical mediator. In HCC and PVTT tissues, IL-32 co-localized with NET markers and was associated with increased neutrophil infiltration. In vivo, NET depletion significantly suppressed intrahepatic metastasis. Notably, radiotherapy further enhanced NET formation, and elevated IL-32-NET levels were associated with radioresistance in patients.

Tumor-derived IL-32 drives PR3-dependent NET formation, promoting PVTT progression and contributing to radiotherapy resistance in HCC. Targeting the IL-32-PR3-NET axis may represent a potential therapeutic strategy.