Cryoablation-induced neutrophil Ca2+ elevation and NET formation exacerbate immune escape in colorectal cancer liver metastasis

Abstract

Abstract Background Liver metastasis poses a significant barrier to effective immunotherapy in patients with colorectal cancer. Cryoablation has emerged as a vital supplementary therapeutic approach for these patients. However, its impact on the tumor microenvironment following the ablation of liver metastases remains unclear. Methods We acquired multi-omics time-series data at 1 day, 5 days, and 14 days post-cryoablation, based on tumor and peripheral blood samples from clinical patients, cell co-culture models, and a liver metastases mouse model built on the MC38 cell line in C57BL/6 J mice. This dataset included single-cell transcriptomic sequencing, bulk tissue transcriptomic sequencing, 4D-Label-Free proteomics, flow cytometry data, western blot data, and histological immunofluorescence staining of pathological specimens. Results We found that a neutrophil-related inflammatory state persisted for at least 14 days post-cryoablation. During this period, neutrophils underwent phenotypic changes, shifting from the N1 to the N2 type. Cryoablation also caused a significant increase in intracellular Ca2+ concentration in neutrophils, which triggered the formation of PAD4-dependent neutrophil extracellular traps (NETs), further promoting immune evasion. Moreover, animal studies demonstrated that depleting or inhibiting the CXCL2-CXCR2 signaling axis within neutrophils, or degrading NETs, could effectively restore the host’s anti-tumor immune response. Conclusions These findings underscore the critical role of neutrophils and their NETs in immune escape following cryoablation. Targeting the CXCL2-CXCR2-Ca2+-PAD4 axis could enhance the therapeutic response to PD-1 antibodies, providing a potential strategy to improve treatment outcomes for colorectal cancer with liver metastases. Graphical Abstract

Keywords

• Cryoablation
• Neutrophils
• Liver metastasis
• Immunotherapy