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

The gastrointestinal tract harbors a large population of microorganisms that play a pivotal role in host immune response and metabolic functions. In recent years, several studies have attempted to uncover the relationships between resident microbiota and the development of gastrointestinal diseases, including colorectal cancer. The advent of next-generation sequencing technologies has enabled the characterization of microbial composition and alterations in patients with colorectal cancer. In this context, historical tissues stored in biobanks represent an invaluable resource for clinical genomic studies. However, the impact of tissue preservation on the microbiome architecture has not been completely explored. In this study, we compared the microbial composition in two groups of paired CRC tissues stored as fresh frozen (FF, 10 samples) and formalin-fixed, paraffin-embedded (FFPE, 10 samples) by using 16S rRNA gene sequencing (V3-V4 region). After sequencing on an Illumina MiSeq platform, DADA2 software was used for data processing. Analysis of alpha and beta diversity showed a significant difference between the two groups. In addition, analysis of relative abundances revealed systematic differences in terms of taxa distribution between FF and FFPE samples. We hypothesize that variations between matched pairs of FF and FFPE samples might derive from the fragmented nature of FFPE DNA. Our findings demonstrated that sample preservation influences the characterization of CRC biopsies, suggesting that microbial profiles of FF samples cannot be directly inferred from FFPE specimens and viceversa.