Intratumoral microbial heterogeneity in HCC reveals a potential therapeutic target

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related mortality worldwide, and its therapeutic challenges are largely influenced by the complexity of the tumor microenvironment (TME). This study aimed to characterize intratumoral microbial heterogeneity, explore its role within the TME, and identify potential antitumor mechanisms and novel microbial targets.

A total of 113 tissue samples, including HCC tumor tissues (n = 48), matched adjacent normal tissues (n = 48), and normal liver tissues from patients with hepatic hemangioma (n = 17), were collected from patients at the Second Hospital of Nanjing, Nanjing Hospital affiliated to Nanjing University of Chinese Medicine. Microbial profiling was performed using 2bRAD-M sequencing, followed by microbial subtyping based on TME features. Metabolomic characterization of microbial subtypes was conducted via ultra-performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Functional validation was performed in vitro and in vivo.

Indicator species analysis, differential abundance analysis, and correlation with clinicopathological features identified Ralstonia sp. as a potential antitumor candidate. Subsequent in vitro and in vivo experiments confirmed that Ralstonia sp. significantly inhibits HCC growth (all p <0.05), potentially through modulation of the glycerophospholipid metabolic pathway. RNA sequencing combined with CIBERSORT analysis further demonstrated that Ralstonia sp. reshapes the tumor immune microenvironment, particularly influencing immune cell infiltration and metabolic status.

This study characterized intratumoral microbial communities and their metabolic heterogeneity in HCC, demonstrating that Ralstonia sp. exerts potential antitumor effects by regulating glycerophospholipid metabolism and modulating immune cell infiltration. These findings highlight a novel microbial target for HCC treatment.

This study provides scientific evidence supporting the existence and biological relevance of intratumoral microbiota in hepatocellular carcinoma (HCC), identifying Ralstonia sp. as a potential antitumor bacterium. By integrating microbiome, metabolomics, and transcriptomic analyses with functional validation, the findings reveal that Ralstonia sp. modulates the tumor microenvironment by regulating glycerophospholipid metabolism and immune cell infiltration. These results are significant for cancer microbiome researchers, oncologists, and translational scientists, highlighting microbial heterogeneity as a previously underrecognized factor in HCC biology. Clinically, the study lays the groundwork for the development of microbiota-based therapeutic strategies or biomarkers, though further mechanistic and translational studies are warranted to evaluate feasibility and safety in human settings.