The gut microbial metabolite formate exacerbates colorectal cancer progression.

The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression.

Fitness for purpose of stabilized stool samples for bile acid metabolite analyses.

Biobanks and cohort studies are increasingly utilizing chemical stabilizers to collect and store stool samples for downstream DNA-based microbiome analyses. While stabilizers permit ambient-temperature collection and storage of samples for gut microbiome studies, the use of the same sample type for downstream metabolomics assays has not been explored. Microbiome-metabolomics analysis of fecal samples is increasingly getting attention to further elucidate the mechanisms by which the gut microbiota influences the host. In this study, we evaluated fitness-for-purpose of OMNIgene-GUT-collected stool samples for downstream metabolomics assays in the scope of fecal bile acids (BA) quantification. Biocrates Bile Acids Kit was used for the quantification of BA from eight healthy donors' samples collected in (1) OMNIgene-GUT kit and (2) snap frozen in -80 °C in duplicates. A highly selective reversed phase LC-MS/MS analysis method in negative ion multiple reaction monitoring (MRM) detection mode was applied to determine the BA concentrations in each sample.Total fecal BA levels were detectable in OMNIgene-GUT-collected samples (range: 29.9-903.7 pmol/mg). Paired t-test confirmed that there was a significant difference in the total BAs between the OMNIgene-GUT and snap frozen samples (p < 0.05). Extractions from snap frozen samples resulted in higher concentrations of total BAs (range: 243.7-1136.2 pmol/mg). Qualitative differences between individual donors' BA profiles were detectable using the two sample collection methods. No significant difference was found in the relative concentrations of primary (CA, CDCA) or secondary (DCA, LCA, UDCA) unconjugated BAs to the total BA concentrations in OMNIgene-GUT-collected samples as compared with the snap frozen samples (Wilcoxon-Mann-Whitney test, p > 0.05). Passing-Bablok method comparison and correlation analyis showed a high degree of correlation in the relative concentrations of CA, CDCA, DCA and LCA between OMNIgene-GUT and snap frozen samples. For these four bile acids, the two methods are comparable at an acceptability bias of 30%. We conclude that the OMNIgene-GUT-collected stool samples are fit-for-purpose for downstream fecal bile acids analysis.

Method Validation for Extraction of DNA from Human Stool Samples for Downstream Microbiome Analysis.

Background: A formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A previously optimized stool processing protocol was validated for fitness-for-purpose for downstream microbiome analysis. Materials and Methods: DNA extraction from human stool was validated with various collection tubes, stabilizing solutions and storage conditions in terms of fitness-for-purpose for downstream microbiome analysis, robustness, and sample stability. Acceptance criteria were based on accurate identification of a reference material, homogeneity of extracted samples, and sample stability in a 2-year period. Results: The automated DNA extraction using the chemagic™ Magnetic Separation Module I (MSM I) extracted 8 out of 8 bacteria in the ZymoBIOMICS® Microbial Community Standard. Seven tested stabilizing solutions (OMNIgene®â€¢GUT, RNAlater®, AquaStool™, RNAssist, PerkinElmer SEB lysis buffer, and DNA Genotek's CP-150) were all compatible with the chemagic MSM I and showed no significant difference in microbiome alpha diversity and no significant difference in the overall microbiome composition compared to the baseline snap-frozen stool sample. None of the stabilizing solutions showed intensive polymerase chain reaction (PCR) inhibition in the SPUD assay. However, when we take into account more stringent criteria which include a higher double-stranded DNA yield, higher DNA purity, and absence of PCR inhibition, we recommend the use of OMNIgene•GUT, RNAlater, or AquaStool as alternatives to rapid freezing of samples. The highest sample homogeneity was achieved with RNAlater- and OMNIgene•GUT -stabilized samples. Sample stability after a 2-year storage in -80°C was seen with OMNIgene•GUT -stabilized samples. Conclusions: We validated a combination of a stool processing method with various collection methods, suitable for downstream microbiome applications. Sample collection, storage conditions and DNA extraction methods can influence the microbiome profile results. Laboratories and biobanks should ensure that these conditions are systematically recorded in the scope of accreditation.