Intratumoral bacteria interact with metabolites and genetic alterations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), which ranks globally as the third leading cause of cancer-related deaths, is highly prevalent, and most patients are diagnosed with advanced-stage cancer when treatments are largely ineffective. 1Thus, there is an urgent need for earlier diagnosis to improve HCC patient outcomes.Although many types of tumors have intratumor bacteria, the tumor microbiome is poorly characterized because of limitations in the technology for detection.Recently, Nejman et al. found that different tumor types have different microbiomes and that bacterial metabolism is closely associated with clinical features. 2They identified cancer type-specific microbial signatures for the microbiome from seven types of human tumors. 2Unique microbial reads and signatures were found in tissue and blood within and between most major cancer types for 33 types of cancer in The Cancer Genome Atlas, suggesting that the cancer microbiome might provide novel information for cancer diagnosis. 3While better sequencing technology has identified the intratumoral microbiome as an important component of the tumor microenvironment, 4,5 the microbes, their metabolites, and the underlying gene regulatory network in HCC are poorly characterized.To study the microbiome, we analyzed 47 pairs of HCC tissues and normal control liver tissues from The First Affiliated Hospital, College of Medicine, Zhejiang University (Supplementary Table.1).Differences in bacterial communities between samples for core and unique operational taxonomic units (OTUs) were determined by Venn diagram analysis using 16 S rDNA sequencing (Supplementary Fig. 1a).The microbial population structure differed between HCC and control tissues at the phylum, family level, and genus levels (Supplementary Fig. 1b-d).The α diversity of microbes in tumors was analyzed using the Wilcoxon rank-sum test (Fig. 1a), and Bray-Curtis-based principal component analysis (PCA) revealed that the overall microbial composition of HCC tissues deviated markedly from that of normal liver tissues (Fig. 1b).The top 20 microbial taxa with significant differences in relative abundance between cancer and paracancerous tissues are shown in Fig. 1c.We also identified statistically significant differences for representative sequences of the top 100 genera, obtained by multiple sequence alignment, as determined by ANOSIM analysis (analysis of similarities) (Supplementary Fig. 1e,f).The predominant taxa were further characterized by high dimensional class comparisons using linear discriminant analysis (LDA) of effect size (LEfSe) with LDA value distribution histogram and a cladogram shown in Supplementary Fig. 1g,h.Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified significant differences in microbial functions for Oscillospira, Mucispirillum, Helicobacter, Roseburia, Ruminococcus, and Anaerotruncus (Supplementary Fig. 1i).Collectively, these results identified the major differences in the microbiomes between HCC and normal liver tissues.The signature microbial environments in HCC patients suggested that variations in metabolites may be affected by the tumor microbiota.Liquid chromatography-mass spectrometry (LC-