A tumor-resident bacterium, Comamonas testosteroni, promotes cervical cancer progression through estrogen metabolic reprogramming

The intratumoral microbiome has emerged as an important regulator of tumor progression and immune signaling; however, its role in cervical cancer remains largely undefined. Here, we profiled intratumoral microbial communities using 16 S rRNA sequencing across fresh-frozen tumors, matched adjacent tissues, and formalin-fixed paraffin-embedded (FFPE) specimens. Microbial presence and cellular localization were validated by Single-cell Analysis of Host–Microbiome Interactions (SAHMI) and fluorescence in situ hybridization. Intratumoral microorganisms were consistently detected across tissue types and pathological subtypes. While alpha diversity showed no significant differences, beta diversity analysis revealed distinct compositional clustering in tumor tissues, particularly in adenosquamous carcinoma. Microbiota were predominantly localized within epithelial and immune cells. Notably, increased microbial alpha diversity was associated with poorer disease-free survival, and specific taxa correlated with clinical outcomes. Among these, Comamonas testosteroni was selected for functional investigation due to its relative abundance and prognostic relevance. In vivo, both live bacteria and bacterial conditioned medium (BCM) significantly promoted tumor growth. In vitro, BCM enhanced cervical cancer cell proliferation, migration, and invasion. Mechanistically, C. testosteroni metabolized estrogen, leading to reduced estrogen availability and suppression of M1 macrophage polarization, thereby modulating the tumor immune microenvironment. Collectively, our findings identify C. testosteroni as a tumor-promoting intratumoral bacterium that drives cervical cancer progression through estrogen metabolism–mediated immune modulation, highlighting a previously unrecognized microbiome–metabolism–immune signaling axis.