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-seq 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 9 breast cancer patients (3 luminal B and 6 triple-negative) and 6 non-malignant donors, sequenced, and analyzed to profile 84 neutrophils from triple-negative breast cancer (TNBC) and 128 control neutrophils. TNBC neutrophils displayed significant upregulation of migration-associated genes (–log₁₀ adjusted p ≈ 3) and respiratory complex genes (p < 1 × 10⁻⁹) compared with controls. Furthermore, TNBC and luminal breast cancer neutrophils shared highly similar transcriptomic profiles, both clearly distinct from those of healthy donors. These findings demonstrate that breast cancer induces a shared transcriptional reprogramming of circulating neutrophils across subtypes, suggesting systemic immune modulation linked to tumor presence. 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.