Aged neutrophils form mitochondria-dependent vital NETs to promote breast cancer lung metastasis

BACKGROUND: Neutrophils-linked premetastatic niche plays a key role in tumor metastasis, but not much is known about the heterogeneity and diverse role of neutrophils in niche formation. Our study focuses on the existence and biological function of a rarely delved subset of neutrophils, named as tumor-associated aged neutrophils (Naged, CXCR4(+)CD62L(low)), involved in premetastatic niche formation during breast cancer metastasis. METHODS: We explored the distributions of Naged in 206 patients and mice models (4T1 and MMTV-PyMT) by flow cytometry. The ability of Naged to form neutrophil extracellular traps (NETs) and promote tumor metastasis in patients and mice was determined by polychromatic immunohistochemistry, scanning electron microscopy and real-time video detection. Furthermore, the differences among tumor-associated Naged, Non-Naged and inflammation-associated aged neutrophils were compared by transcriptome, the biological characteristics of Naged were comprehensively analyzed from the perspectives of morphology, the metabolic capacity and mitochondrial function were investigated by Seahorse, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) and transmission electron microscopy (TEM). Finally, 120 patients’ sample were applied to confirm the acceleration of Naged formation through secreted NAMPT, and the importance of blocking this pathway in mice was evaluated. RESULTS: We find that Naged accumulate in the lung premetastatic niche at early stage of breast tumorigenesis in multiple mice models and also exist in peripheral blood and metastatic lung of patients with breast cancer. Naged exhibit distinct cell marker and morphological feature of oversegmented nuclei. Further transcriptome reveals that Naged are completely different from those of Non-Aged or inflammation-associated aged neutrophils and illustrates that the key transcription factor SIRT1 in Naged is the core to maintain their lifespan via mitophagy for their function. The responsible mechanism is that SIRT1 can induce the opening of mitochondrial permeability transition pore channels to release mitochondrial DNA and lead to the mitochondria-dependent vital NETs formation, rather than traditional Cit-Histone H3 dependent fatal-NETs. Further mechanically investigation found tumor derived NAMPT could induce Naged formation. Additionally, therapeutic interventions of Naged and its formation-linked pathways could effectively decrease breast cancer lung metastasis. CONCLUSIONS: Naged exerts a vital role in breast cancer lung metastasis, and strategies targeting SIRT1-Naged-NETs axis show promise for translational application.