Decoding circulating neutrophil phenotypes in triple-negative breast cancer via optimized single-cell RNA sequencing.

Neutrophils are emerging as key regulators of tumor biology, influencing cancer progression, immune evasion, and therapeutic response. However, their transcriptomic characterization remains challenging due to low RNA content, limiting confident discrimination from other leukocyte populations at the single-cell level. To address this, we developed a robust analytical framework for single-cell RNA sequencing data, calculating a neutrophil score for each cell based on two curated neutrophil marker gene panels and excluding cells expressing non-neutrophil marker genes.White blood cells were isolated from liquid biopsies of six triple-negative breast cancer (TNBC) patients and six non-malignant donors, sequenced, and analyzed to profile 84 neutrophils isolated from TNBC patients and 128 neutrophils isolated from control donors. TNBC neutrophils displayed significant upregulation of migration-associated genes (-log10 adjusted p ≈ 3) and respiratory complex genes (p < 1 × 10-9) compared with controls.These findings demonstrate significant transcriptomic differences between the TNBC and control neutrophils, revealing a part of the influence of the TNBC tumor on circulating immune cells. Moreover, this work provides a validated framework for precise neutrophil identification and functional characterization in single-cell studies and lays the foundation for dissecting their mechanistic roles in cancer progression.