br Inferred functional changes in GC associated
3.4. Inferred functional changes in GC-associated gastric mucosal microbiota
The functional content of the gastric microbiota was predicted by PiCRUSt based on closed-reference OTU picking. In our present study, 19 Clusters of Orthologous Groups (COG) functional categories were tested, which identified 6 differentially abundant COGs with a QFDR b 0.05 between normal and peritumoral microbiota, 8 between normal and tumoral microbiota, and 10 between peritumoral and tumoral mi-crobiota (Fig. 6a). Eleven COG categories, including carbohydrate trans-port and metabolism, coenzyme transport and metabolism, nucleotide transport and metabolism, and amino Mitomycin C transport and metabolism, exhibited the most significant differences among the three microhabi-tats (QFDR b 0.05, Fig. 6b; Benjamini–Hochberg FDR method). Among these differential COGs, nucleotide transport and metabolism, amino acid transport and metabolism and inorganic ion transport and metab-olism were significantly enriched in the tumoral microbiota. In addition, we also compared 64 Kyoto Encyclopedia of Genes and Genome (KEGG) pathways at level 2. At an FDR of 0.05, we identified more differentially abundant pathways between tumor microbiota and others (Fig. S15). Consistent with the significant alterations of HP-associated gastric microbiota, the KEGG pathways were changed between the HP+ and HP− groups in normal and peritumoral tissues (Fig. S16). Specifically, gastric acid secretion was significantly higher in the HP+ group of the tumoral microbiota. Together, these functional changes in the gastric microbiota may contribute to GC initiation and progression.
The GC tumor microenvironment is colonised with site-specific gas-tric microbiota that foster symbiotic interactions, which has been con-sidered as an important factor in GC initiation and progression. The normal microenvironment acts as a barrier to tumorigenesis, while the tumor microenvironment can induce and promote cancer. Cur-rently, considerable progress has been made towards understanding the altered composition and diversity of the gastric microbiota in gastric diseases, which have always considered the whole stomach as one hab-itat [4,5,7–11,15,16]. Tseng et al. found that GC tissue and neighbouring normal tissue had similar gastric microbiota in early-stage GC patients , while Li et al. reported that there was little difference in the gastric microbiota between antrum and body biopsy specimens . However, the stomach microhabitats are not uniform; rather, they vary consider-ably across sites within the stomach, at the same site over time, and with health status. The different nutritional compositions within the stomach microhabitats contribute to altered diversity and composition of the gastric microbiota. It is more difficult to obtain healthy stomach tissues as controls; therefore, our present, large-scale trial study screened those confirmed GC tumoral, and peritumoral tissue, and neighbouring normal tissue from the same GC patient to investigate dis-crepancies in the gastric microbiota. In contrast to previous studies, our self-control study found alterations of diversity and composition of the gastric microbiota in tumor and tumor-free microhabitats, which affect both bacteria-bacteria interactions as well as bacteria-host interactions
Fig. 4. Overall structure of the early-stage and late-stage gastric microbiota in the three stomach microhabitats. The α-diversity indices such as Shannon (a), Simpson (b), Chao 1 (c) and Heip evenness (d) were estimated by QIIME. Data are presented as mean ± standard deviation. Unpaired t-tests (two-tailed) were used to analyse variation among the three microhabitats in the same GC stage. Plots of principal coordinate analysis (PCoA) of the gastric microbiota in the early-stage and late-stage based on the unweighted UniFrac distance (e and f), weighted UniFrac distance (g and h) and Bray-Curtis distance (i and j). Propionibacterium acnes (k), Streptococcus anginosus (l), Prevotella copri (m) and Sphingomonas yabuuchiae (n) were different among the three microhabitats in the same GC stage. Data are presented as mean ± standard deviation. Mann-Whitney U tests were used to analyse variation among the three stomach microhabitats. *p b .05. r> that occur in stomach microhabitats during GC development. Previous studies on colorectal cancer have suggested that different bacterial spe-cies preferentially inhabit the tumor sites but not tumor-free sites [41,42]. The altered gastric microbiota influences inflammation and im-munity both locally at the mucosal level and systemically. Our previous study demonstrated that patients with GC had increased regulatory T cells (Tregs) in peripheral blood and carcinoma tissue . Disorders of the intimate interactions between the gastric microbiota in the tumor microenvironment and immune system may contribute to GC development by eliciting tumor-promoting immune responses.