Neutrophil-mediated NK cell dysfunction via Eomes and T-bet suppression promotes breast cancer liver metastasis

Metastatic disease is the primary cause of cancer-related mortality, and immune evasion is a critical barrier to effective clearance of disseminated tumor cells. Natural killer (NK) cells are cytotoxic lymphocytes that contribute to early immune surveillance of metastases, but their function is often impaired in solid tumors. Neutrophils, the most abundant circulating leukocytes, have emerged as context-dependent regulators of tumor progression and immune suppression. While neutrophil-NK cell crosstalk has been implicated in shaping anti-tumor immunity, the mechanisms by which neutrophils modulate NK cell function remain poorly defined. Using a syngeneic triple-negative breast cancer mouse model of liver metastasis, we investigated the immunoregulatory role of neutrophils in modulating NK cell activity. The impact of neutrophils isolated from breast cancer liver metastases, when co-cultured with NK cells, on the expression of key NK transcription factors, Eomes and T-bet was measured. In addition, the impact of neutrophil co-culture on NK-mediated tumor cell killing was also assessed. Finally, the expression of NK activating and inhibitory receptors were measured following exposure to neutrophils. We found that neutrophils accumulate in the metastatic liver and progressively outnumber NK cells during growth of the breast cancer liver metastases. Neutrophils isolated from metastasis-bearing mice suppressed NK cell cytotoxicity and induced downregulation of key regulators of NK cell maturation and effector function, Eomes and T-bet, through a contact-dependent mechanism. NK cells from metastasis-bearing mice exhibited hallmarks of functional exhaustion, including reduced activating receptor expression and elevated inhibitory receptor expression. These findings uncover a previously unrecognized component of neutrophil-mediated NK cell suppression in breast cancer liver metastasis, directly linking neutrophil-NK interaction with reduced Eomes and T-bet expression within NK cells and impaired NK cell-mediated tumor killing. This work advances our understanding of neutrophil-driven immune regulation in metastasis and provides mechanistic insight into the limited efficacy of NK cell-based immunotherapies in solid tumors. Targeting neutrophil-NK cell interactions may represent a promising therapeutic strategy to restore innate immune function and limit metastatic outgrowth.