16S rRNA-based microbial profiling in fresh-frozen and formalin-fixed paraffin-embedded colorectal cancer tissues

Sequencing-based studies have provided new insights on the role of the gut microbiota in human diseases, including colorectal cancer (CRC). Formalin-fixed, paraffin-embedded (FFPE) tissues represents the most widely available clinical material, albeit with several limitations. However, the impact of sample preservation on the microbiome composition of CRC has not been fully elucidated. In this study, we compared the microbial composition in 10 paired CRC tissues stored as fresh frozen (FF) and FFPE using 16S rRNA gene sequencing and RNA in situ hybridization (RNA-ISH). Analysis of diversity and composition showed distinct microbial profiles, with FF samples mainly enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Whereas, typical contaminant taxa, including Sphingomonadales and Rhodobacterales, were significantly increased in FFPE tissue samples. RNA-ISH confirmed the presence of tumor-associated bacteria, such as Fusobacterium and Bacteroides, and Propionibacterium previously described as both tumor-associated and contaminant taxa. An internal 16S rRNA amplicon-based quality control analysis showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. As larger studies using FFPE specimens are needed for retrospective microbiome characterization, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible analyses.