Abstract 187: Loss of neutrophil MHC class II expression and IRF8 suppression define a dystemic pre-metastatic reprogramming axis in breast cancer.

Abstract Neutrophils have recently gained attention as important contributors to the pre-metastatic niche. However, the systemic immune remodeling of neutrophils during breast cancer metastasis remains incompletely characterized. In this study, we investigated neutrophil-associated changes in both clinical breast cancer patients and experimental metastatic models. We retrospectively analyzed peripheral blood test results from 659 M0 breast cancer patients (2022-2025, SNUH) and 64 de novo M1 patients (2012-2024), all sampled within 15 days of initial presentation. Metastatic (M1) patients showed significantly elevated neutrophil counts, reduced lymphocyte counts, and higher neutrophil-to-lymphocyte ratio (NLR) compared to non-metastatic patients, suggesting systemic alterations in immune cell composition. To further examine the mechanistic basis of these findings, we orthotopically implanted metastatic 4T1 or non-metastatic EMT6 breast cancer cells into BALB/c mice and isolated circulating neutrophils at a pre-metastatic timepoint (3 weeks post-implantation) for RNA sequencing. Neutrophils from 4T1-bearing mice demonstrated increased expression of immune-modulatory and tumor-associated genes, including Lbp, Ltf, Prok2, Lipg, S100a8, and S100a9. In contrast, multiple genes encoding MHC class II molecules (H2-DMb2, H2-Ea, H2-Ab1, Cd74) were markedly downregulated. Importantly, the transcription factor Irf8, which regulates neutrophil and antigen-presenting cell differentiation, was significantly suppressed. Flow cytometric analysis confirmed these transcriptomic changes at the protein level. While neutrophils from PBS- or EMT6-bearing mice maintained weak MHC class II expression, neutrophils from 4T1-bearing mice showed a substantial population lacking MHC class II. qPCR of sorted MHC class II-negative neutrophils further revealed reduced expression of Irf8, Cd74, and other antigen-presentation-related genes. Extending these findings to early metastatic sites, multicolor FACS analysis of lung and liver tissues demonstrated both increased neutrophil accumulation and reduced MHC class II surface expression in 4T1-bearing mice, even before overt metastasis. Together, these results indicate that metastatic breast cancer is associated with: (1) increased circulating neutrophils and NLR, (2) IRF8 downregulation with reduced antigen-presentation programs, (3) systemic expansion of MHC class II-low neutrophils in blood and pre-metastatic organs. Notably, these changes arise prior to detectable metastatic colonization, suggesting a potential role in shaping a metastasis-permissive immune environment. These findings support the possibility that restoring IRF8-dependent antigen-presentation programs in neutrophils could represent a novel therapeutic strategy to limit metastatic progression in breast cancer. Citation Format: Woohang Heo, Jinah Lim, Youngwoong Kwak, Yujeong Her, Hamin Jeong, Ji Su Kim, Sieun Yang, Min-Ju Sung, Jeongwon Song, Hyeong-Gon Moon. Loss of neutrophil MHC class II expression and IRF8 suppression define a dystemic pre-metastatic reprogramming axis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 187.