Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.

Inflammation is a key feature in breast cancer progression, with neutrophil extracellular traps (NETs) playing an important role. NETs are DNA-based structures released by neutrophils that can promote tumor adhesion, invasion, and immune evasion. Another crucial mechanism is the inflammasome, a multiprotein complex that drives inflammation through cytokine release. Both mechanisms are present in tumors and may act synergistically. In this study, we evaluated how isolated NETs modulate the NLRP3 inflammasome in a human breast cancer model. Exposure of MDA-MB-231 cells to NETs increased the expression of NLRP3, CASP1, and IL1B. Blocking IL-1R with Anakinra reduced IL1B expression, while inhibition of the P2X7 receptor with A740003 decreased NLRP3 and IL1B. ELISA confirmed that NETs stimulate IL-1β release, which was reduced by MCC950, Anakinra, and A740003. Functionally, NETs accelerated tumor cell migration, and this effect was inhibited by MCC950 and Anakinra. Bioinformatics analysis of TCGA breast cancer samples showed differential inflammasome gene expression among subtypes and a positive correlation between inflammasome components and NET-related genes. These findings highlight the interplay between inflammatory and immune mechanisms in breast cancer progression and may support the development of new therapeutic strategies.