Integrated 2bRAD-M and 16S rRNA sequencing reveals intratumoral microbiota dysbiosis and clinical associations in gastric cancer

Gastric cancer (GC) is a malignant tumor with high morbidity and mortality in China. The intratumoral microbiota, a key component of the tumor microenvironment (TME), has been implicated in GC onset and progression. Sixty paired GC and adjacent normal tissue samples were collected. We integrated 2bRAD-M and 16S rRNA sequencing, together with 16S rRNA fluorescence in situ hybridization (FISH), lipopolysaccharide (LPS) immunohistochemistry (IHC), and bioinformatics analysis, to investigate microbial differences between the two tissue types and their associations with clinical features. The abundance of Firmicutes was markedly elevated in GC tissues, whereas the abundance of Proteobacteria and other phyla decreased. At the genus and species levels, Lactobacillus and Streptococcus pneumoniae were enriched in GC tissues, whereas Helicobacter pylori was reduced. Microbial abundance correlated with clinical features such as TNM stage and carcinoembryonic antigen (CEA) levels. Functional prediction revealed that microbial pathways related to metabolism and proliferation were significantly upregulated in GC tissues. This study confirms the presence of intratumoral microbiota dysbiosis in patients with GC and reveals associations between specific microbial taxa, functional pathways, and clinical characteristics. These findings suggest that the intratumoral microbiota may serve as a potential biomarker for GC and provide a basis for future therapeutic strategies, although further validation in larger prospective cohorts and experimental studies is needed.