Alterations in gut microbiome in age- and diet-induced colorectal cancer (CRC) progression in a preclinical model.

223 Background: CRC is the third leading cause of cancer related deaths worldwide with age and diet among the strongest risk factors. Emerging evidence suggests gut microbiome plays important role in CRC and is highly impacted by the exposome primarily the food intake. Since diet and age plays an important role in gut microbiome diversity, the present study aimed to investigate the alterations in the gut microbiome in young versus old mice harboring CRC allografts and fed with different diets. Methods: Two cohorts of C57BL/6 including young (n = 9, age = 6 weeks) and old (n = 9, age = 20-24 months) mice were implanted with 1x10 6 MSI (microsatellite instable) CRC MC38 tumor cells. Mice in both cohorts were randomized into three different diet groups: normal diet (ND; standard chow), high-fat (HF) and calorie-restricted (CR: 30% reduction in total calories). Mice were housed individually under standard laboratory conditions. Mice fecal samples were collected and subjected to DNA isolation (ZymoBIOMICS-96 MagBead DNA Kit), followed by metagenomic shotgun and whole genome sequencing (ZymoBIOMICSe and Illumina NovaSeq, respectively). P ≤ 0.05 were considered as statistically significant. Results: Fecal microbiome analysis showed that old microbiome has higher alpha diversity compared to the young mice. No statistically significant differences in were found in microbiome diversities between the diet groups. Pairwise permanova results showed statistically significant differences between microbiomes of old vs. young groups ( P : 0.001), CR vs. HF groups ( P : 0.012) and HF vs. ND groups ( P : 0.021). ANCOM-BC analysis determined the differentiating features and microbial functional pathways in the young mice compared to the old mice group and in different diet groups with relative abundance differences of larger than log 10 2. Bacteroides thetaiotaomicron ( P : 1.43E-39) and Parabacteroides goldsteinii ( P : 2.20E-23) were enriched in young and old groups, respectively. Akkermansia muciniphila ( P : 0.008) was significantly enriched in ND compared to CR group. Lactococcus lactis ( P : 9.94E-160) and Lachnospiraceae bacterium A4 ( P : 4.95E-10) were significantly enriched in HF compared to ND diet groups and vice-versa, respectively. Among the functional pathways, CMP-legionaminate biosynthesis I ( P : 2.38E-13) and L-arginine biosynthesis IV (archaebacteria) ( P : 4.77E-11) were the most significantly enriched in young and old groups, respectively. Conclusions: Our findings highlight the differential diversity and microbial features of gut microbiome in age- and diet-induced CRC progression. Collectively, alteration in diet and age of the host lead to changes in gut microbiota which has a direct impact on activation of specific signaling pathways, metabolism and local and systemic immune responses, which may in turn affect chemosensitivity and outcome in CRC