Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults

Abstract

In this study we used stool profiling to identify intestinal bacteria and metabolites that are differentially represented in humans with colorectal cancer (CRC) compared to healthy controls to identify how microbial functions may influence CRC development. Stool samples were collected from healthy adults (n = 10) and colorectal cancer patients (n = 11) prior to colon resection surgery at the University of Colorado Health-Poudre Valley Hospital in Fort Collins, CO. The V4 region of the 16s rRNA gene was pyrosequenced and both short chain fatty acids and global stool metabolites were extracted and analyzed utilizing Gas Chromatography-Mass Spectrometry (GC-MS). There were no significant differences in the overall microbial community structure associated with the disease state, but several bacterial genera, particularly butyrate-producing species, were under-represented in the CRC samples, while a mucin-degrading species, Akkermansia muciniphila, was about 4-fold higher in CRC (p<0.01). Proportionately higher amounts of butyrate were seen in stool of healthy individuals while relative concentrations of acetate were higher in stools of CRC patients. GC-MS profiling revealed higher concentrations of amino acids in stool samples from CRC patients and higher poly and monounsaturated fatty acids and ursodeoxycholic acid, a conjugated bile acid in stool samples from healthy adults (p<0.01). Correlative analysis between the combined datasets revealed some potential relationships between stool metabolites and certain bacterial species. These associations could provide insight into microbial functions occurring in a cancer environment and will help direct future mechanistic studies. Using integrated "omics" approaches may prove a useful tool in identifying functional groups of gastrointestinal bacteria and their associated metabolites as novel therapeutic and chemopreventive targets.

Figure 1. Using the 3% genetic distance, we observed no clustering of samples according to total stool microbial communities based on disease status of the sample donor using either the unweighted measure Jaccard similarity (A) or the weighted Theta_YC distance (B).

Figure 2. Phyla-level microbial classification of bacteria from individual stool samples.

H sample numbers indicate samples from healthy adults while the C designation signifies samples from colon cancer patients.

Figure 3. The relative proportion of bacterially-produced short chain fatty acids (SCFA) differed significantly between stool of healthy adults and individuals with CRC.

Acetic acid, valeric acid, isobutyric acid, and isovaleric acid concentrations were proportionately higher while the anti-proliferative SCFA, butyric acid was significantly lower.

Figure 4. OPLS-DA scores plot generated from global GC-MS profiles differentiate stool metabolites from CRC patients and healthy adults.

Figure 5. A heat map showing Pearson’s correlations between groups of metabolites and bacterial genera/species that significantly differed between CRC patients and healthy adults.

Green boxes indicate positive associations and red boxes indicate negative associations.