Preliminary study of neutrophils and neutrophil extracellular traps (NETs) in canine mammary tumors

Neutrophils play a complex role in cancer biology, can contributing to tumor progression and immune defense. Neutrophil extracellular traps (NETs) have emerged as key modulators within the tumor microenvironment. Herein, the association between molecular classification, histological grade, necrosis, tumor-infiltrating neutrophils, and NETs was assessed in 19 canine mammary malignant tumors. Immunohistochemistry using citrullinated histone-3 (cith3) and myeloperoxidase (MPO) antibodies were used to detect NETs. A fading and re- staining method was applied on the same sections. NETs were scored based on the presence of cith3 positive areas and compared with tumor grade. The neutrophil score numerically increased as the tumor grade increased. The NET score was slightly higher in grade I carcinomas compared to carcinomas with other grades. On contrary, the necrosis score was also higher in grade II and III tumors than grade I tumors. A low but non-significant negative correlation existed between tumor grade and NET score (r =-0.219). No statistically significant associations between the tumor markers (ER, PR, HER2) and molecular subtypes with tumor grade, NET score, neutrophil count, and necrosis. In this study, the presence of NETs in canine malignant mammary tumor of different histological subtypes and grades was reported. Preliminary evidence was gathered that NETs are negatively correlated with tumor grade, suggesting their potential role in prognostication.