Bacterial lipopolysaccharide mediates the interplay between tumor microbiome and immune microenvironment in breast cancer: development of a prognostic risk score

The immunosuppressive microenvironment of breast cancer drives a ‘cold’ tumor phenotype characterized by limited immune cell infiltration and poor response to immunotherapy. While intratumoral microbiota and circulating lipopolysaccharide (LPS) have the potential to remodel immunity, the specific mechanisms and prognostic impact of LPS-driven immunomodulation in breast cancer remain unclear. We utilized the Comparative Toxicogenomics Database (CTD) to systematically identify LPS-related genes. Prognosis-associated LPS-related genes (PALRGs) were identified via Cox regression analyses. Subsequently, we applied LASSO-Cox regression to construct an LPS-related risk score (LPSRS) and evaluated its predictive performance and associations with immune microenvironment characteristics in TCGA-BRCA (n = 1,196) and GEO (n = 525) cohorts. For further validation, we recruited an independent clinical cohort of 56 breast cancer patients and confirmed differential expression of LPSRS genes and tumor microenvironment (TME) heterogeneity between high- and low-risk groups. We integrated RNA sequencing with 2bRAD-M sequencing to explore potential interactions between the tumor microbiome and host gene expression. Finally, we conducted mechanistic validation using 4T1 tumor-bearing mice, with treatment groups receiving either Pseudomonas. putida or purified LPS. We identified 26 PALRGs and constructed an 8-gene LPSRS signature as Risk Score which demonstrated strong prognostic performance (C-index = 0.750, 95% CI 0.712–0.788) across multiple independent cohorts and our clinical cohort. Notably, low-risk patients exhibited significant Pseudomonas enrichment (median abundance: 4.3% vs. 2.4%, P = 0.04). Correlation analysis revealed significant associations between Pseudomonas abundance and transcriptome ( P = 6.36 × 10 –6 ). In mechanistic validation experiments, Pseudomonas -treated mice showed an significant reduction in tumor volume ( P = 0.0009), accompanied by a 2- fold increase in M1 macrophage polarization ( P < 0.0001), confirming the beneficial remodeling of the TME. Intratumoral microbiota-related LPS is associated with microbiome-immunity crosstalk in breast cancer. LPSRS serves as a promising prognostic marker with therapeutic insights.