Neutrophils matter: new clinical insights on their role in the progression of metastatic breast cancer

Background: Metastatic breast cancer (BC) remains a significant clinical challenge, needing innovative strategies to improve disease management. Increased neutrophil levels have been observed in both peripheral blood and tumor tissues of patients with different types of cancer, often being associated with poor clinical outcomes. These findings suggest a crucial role for neutrophils in tumor progression, raising interest in neutrophil-based therapies. However, the functional and phenotypic heterogeneity of neutrophils complicates their therapeutic targeting. This study aims to investigate the clinical impact of immunosuppressive, protumor low-density neutrophils (LDN) in metastatic BC, comparing them with normal high-density neutrophils (HDN) to better understand their role in disease progression. Methods: LDN and HDN subpopulations were isolated from the blood of 151 BC patients (72 metastatic, 79 non-metastatic) using density gradient centrifugation. Their frequency, phenotype, and function were analyzed by flow cytometry and in vitro experiments. Correlations between LDN levels and clinical data from metastatic BC patients were evaluated, alongside individual longitudinal assessments. Results: LDN accumulated significantly in the blood of BC patients, particularly in those with metastatic disease. Elevated LDN levels in these patients were associated with faster disease progression and reduced life expectancy, regardless of metastatic site. Longitudinal analysis revealed that higher LDN percentages often correlated with adverse clinical events, whereas lower levels of LDN were linked to stable disease. Functionally, LDN exhibited protumor properties, including elevated expressions of PD-L1 and MMP-9, contributing to immunosuppression and metastasis. Unlike HDN, which demonstrated cytotoxicity against tumor cells, LDN failed to reduce BC cell line viability in 3D co-cultures. Notably, BC cell lines exposed to LDN-conditioned medium showed increased invasive capacity and proliferation. This LDN-conditioned medium also favoured a myeloid anti-inflammatory phenotype while leading to impaired activation of T-cells, likely due to the arginase inhibitory effect. Conclusion: Our results highlighted LDN and their secreted factors as major drivers of BC progression and increased aggressiveness. These findings suggest that incorporating LDN assessment into clinical practice could aid in identifying high-risk patients and enable more personalized treatment approaches. Furthermore, our data strengthen the relevance of targeting specific neutrophil subsets or their functions to improve metastatic BC patient outcomes.