Distinct gut microbiome patterns associate with consensus molecular subtypes of colorectal cancer

Abstract

Colorectal cancer (CRC) is a heterogeneous disease and recent advances in subtype classification have successfully stratified the disease using molecular profiling. The contribution of bacterial species to CRC development is increasingly acknowledged, and here, we sought to analyse CRC microbiomes and relate them to tumour consensus molecular subtypes (CMS), in order to better understand the relationship between bacterial species and the molecular mechanisms associated with CRC subtypes. We classified 34 tumours into CRC subtypes using RNA-sequencing derived gene expression and determined relative abundances of bacterial taxonomic groups using 16S rRNA amplicon metabarcoding. 16S rRNA analysis showed enrichment of Fusobacteria and Bacteroidetes, and decreased levels of Firmicutes and Proteobacteria in CMS1. A more detailed analysis of bacterial taxa using non-human RNA-sequencing reads uncovered distinct bacterial communities associated with each molecular subtype. The most highly enriched species associated with CMS1 included Fusobacterium hwasookii and Porphyromonas gingivalis. CMS2 was enriched for Selenomas and Prevotella species, while CMS3 had few significant associations. Targeted quantitative PCR validated these findings and also showed an enrichment of Fusobacterium nucleatum, Parvimonas micra and Peptostreptococcus stomatis in CMS1. In this study, we have successfully associated individual bacterial species to CRC subtypes for the first time.

HARMONIES: A Hybrid Approach for Microbiome Networks Inference via Exploiting Sparsity

“…This study suggested that P. anaerobius directly interacted with colonic epithelial cells and also promoted CRC by modifying the tumor immune microenvironment. While the causal role of the species Parvimonas micra (P. micra) has not been biologically validated, multiple clinical studies reported an elevated level of P. micra in CRC patients Yu et al (2017); Purcell et al (2017); Dai et al (2018). Of interest, Parvimonas were closely associated with animal-based diets, which have previously been shown to be significantly associated with increased risk for CRC Chan et al (2011).…”

Molecular subtyping improves prognostication of Stage 2 colorectal cancer

“…RNA was extraction was carried out as detailed previously. [10] Briefly, RNA was extracted from < 20 mg of tissue using RNEasy Plus Mini Kit (Qiagen), including DNAse treatment, following tissue disruption using a Retsch Mixer Mill.…”

“…RNA-sequencing was carried out using the Illumina HiSeq 2500 V4 platform to produce 125bp paired end reads, as previously described. [10] In brief, library preparation, including ribosomal RNA depletion using RiboZero Gold, was carried out using Illumina TruSeq V2 reagents. The libraries were sequenced on 3 x 5 lanes of an Illumina HiSeq 2500 instrument.…”

“…[20] These criteria may have excluded many advanced-stage tumours, which were shown to be associated with CMS4. [8] Intra-tumoural heterogeneity may also affect the classification of CMS4 tumours, as the EMT-associated genes seen in CMS4 tumours may reflect upregulated genes derived from fibroblast and mesenchymal cells present in the stromal background rather than directly from the tumour itself, [9,[21][22][23] and several studies have suggested that the location and number of tumour biopsies can undermine the accuracy of CMS; [24][25][26] a limitation of this study is the use of a single tumour sample to carry out gene-expression profiling.…”

MinION Sequencing of colorectal cancer tumour microbiomes – a comparison with amplicon-based and RNA-Sequencing

“…Tissue samples were frozen in liquid nitrogen and stored at -80 o C post resection, and subsequently transferred to RNAlater ICE TM (Qiagen) and stored at -20 o C. As described previously [18], nucleic acid extraction was performed on < 20mg of tissue by a single operator in one batch to avoid variation in protocol. Tissue disruption was carried out using a Retch Mixer mill.…”