Characterising the Lung Microbiome in Lung Squamous Cell Carcinoma Stepwise Carcinogenesis using 16S rRNA Gene Sequencing and Host Bacteria Co-culture Infection Model

Abstract

Lung squamous cell carcinoma (LUSC) develops through stepwise carcinogenesis, yet the role of the airway microbiome in this process remains poorly defined. This study aimed to characterise the microbial composition and diversity across the histological continuum of LUSC and to evaluate the functional impact of Staphylococcus aureus on human bronchial epithelial cells (HBECs). A total of 66 tissue samples from 66 patients were analysed using targeted 16S rRNA V3–V4 sequencing. Co culture assays involving HBECs and Staphylococcus aureus were conducted to assess the impact of bacterial exposure on HBEC viability, using 96- and 48-well plate formats to monitor responses over multiple time points. The airway microbiome exhibited progressive dysbiosis, with declining alpha diversity and compositional shifts from normal bronchial epithelium to invasive LUSC. LUSC samples showed enrichment of Staphylococcus aureus, Bacillus subtilis, and Limosilactobacillus fermentum, while progressive carcinoma in situ (CIS) lesions were characterised by increased abundance of Staphylococcus hominis and Sphingomonas glacialis. Regressive lesions, by contrast, maintained more stable microbial communities similar to their matched normal tissues. Host factors, including age, sex, smoking status, and pack-years, did not significantly influence microbial diversity or composition. Co-culture experiments demonstrated a clear time- and dose-dependent reduction in HBEC viability following S. aureus exposure, particularly evident at 24 hours. These findings reveal a stepwise restructuring of the airway microbiome along the histological continuum of LUSC and highlight the potential of pathogenic taxa to drive epithelial injury. Together, they provide mechanistic insights into the ecological and functional dynamics underlying early carcinogenic processes in LUSC