Microbial dynamics with CRC progression: a study of the mucosal microbiota at multiple sites in cancers, adenomatous polyps, and healthy controls

Abstract

Accumulating evidence has related the gut microbiota to colorectal cancer (CRC). Fusobacterium nucleatum has repeatedly been linked to colorectal tumorigenesis. The aim of this study was to investigate microbial composition in different sampling sites, in order to profile the microbial dynamics with CRC progression. Further, we characterized the tumor-associated F. nucleatum subspecies. Here, we conducted Illumina Miseq next-generation sequencing of the 16S rRNA V4 region in biopsy samples, to investigate microbiota alterations in cancer patients, patients with adenomatous polyp, and healthy controls in Norway. Further, Fusobacterium positive tumor biopsies were subjected to MinION nanopore sequencing of Fusobacterium-specific amplicons to characterize the Fusobacterium species and subspecies. We found enrichment of oral biofilm-associated bacteria, Fusobacterium, Gemella, Parvimonas, Granulicatella, Leptotrichia, Peptostreptococcus, Campylobacter, Selenomonas, Porphyromonas, and Prevotella in cancer patients compared to adenomatous polyp patients and control patients. Higher abundance of amplicon sequence variants (ASVs) classified as Phascolarctobacterium, Bacteroides vulgatus, Bacteroides plebeius, Bacteroides eggerthii, Tyzzerella, Desulfovibrio, Frisingicoccus, Eubacterium coprostanoligenes group, and Lachnospiraceae were identified in cancer and adenomatous polyp patients compared to healthy controls. F. nucleatum ssp. animalis was the dominating subspecies. F. nucleatum ssp. nucleatum, F. nucleatum ssp. vincentii, Fusobacterium pseudoperiodonticum, Fusobacterium necrophorum, and Fusobacterium gonidiaformans were identified in five samples. Several biofilm-associated bacteria were enriched at multiple sites in cancer patients. Another group of bacteria was enriched in both cancer and polyps, suggesting that they may have a role in polyp development and possibly early stages of CRC.

Keywords: 16S rRNA amplicon sequencing; Biofilm bacteria; Colorectal cancer; Fusobacterium species and subspecies; Tumor biopsies.

Fig. 1

Illustration of the sampling sites where the biopsies were collected from this study

Fig. 2

Schematic illustration of 16S rRNA amplicon sequencing workflow. Asterisk indicates that 10 samples from Library prep 1 were included in Library Prep 2 to control for batch effects due to run-to-run variation

Fig. 3

A Violin plots with box plots of Shannon bacterial alpha diversity index in mucosal biopsies from all sampling sites from all three groups. Pairwise Wilcoxon rank sum test revealed significant differences between cancer and healthy controls (p = 0.003) and polyp and healthy controls (p = 0.03). There is no significant difference between cancer and polyp (p = 0.23)). B Violin plots with box plots of Inverse Simpson diversity index in mucosal biopsies from all sampling sites all three groups. Pairwise Wilcoxon rank sum test revealed no significant differences between the groups (healthy controls vs polyp p = 0.11, healthy controls vs cancer p = 0.10, and polyp vs cancer p = 0.95). C Violin plots of Shannon diversity index in mucosal biopsies in cancer patients. Pairwise Wilcoxon rank sum test showed no significant differences between sampling sites in cancer patients. p-values are shown in Fig. 3C. AC, ascending colon; TU, tumor; HT, adjacent healthy tissue; CS, colon sigmoideum

Fig. 4

Illustration of comparisons performed in differential abundance analysis. AC, ascending colon; CS, colon sigmoideum; HT, adjacent healthy tissue; TU, tumor/polyp tissue. The results from differential analysis done in comparisons 1–3 are shown in Fig. 5 and Tables 2, 3, 4, and 5, respectively

Fig. 5

A Bray–Curtis bacterial beta diversity analysis of mucosal biopsies from all sampling sites from all three groups showed significant differences between the groups (p = 0.001); cancer patients (red), patients with adenomatous polyps (blue) and healthy controls (green). B Beta diversity analysis did not reveal any significant differences between mucosal biopsies of different sampling sites (p = 0.998); tumors and polyps (purple), adjacent healthy tissues (blue), colon sigmoideum (green) and ascending colon (red). Each point represents a biopsy

Fig. 6

The figure illustrates enriched and depleted microbial ASVs between cancer, adenomatous polyp, and healthy controls (p < 0.05). A Cancer group vs adenomatous polyp group. B Cancer group vs healthy controls. C Adenomatous polyp group vs healthy controls. Abundance of Fusobacterium, Gemella, Parvimonas, and Granulicatella were higher in the cancer group compared to both other groups. ASVs classified as Phascolarctobacterium, B. vulgatus, B. plebeius, B. eggerthii, Tyzzerella, Desulfovibrio, Frisingicoccus, and Eubacterium coprostanoligenes_group and two ASVs classified as Lachnospiraceae were identified with higher abundance in both cancer and adenomatous polyp groups compared to healthy controls

Fig. 7

A The dynamics of microbial taxa with CRC progression shown for genera that were significantly increased in cancer compared to polyps and controls. The figure shows the relative abundance of taxa that were present at different samplings sites in cancer, polyps, and controls. B The dynamics of microbial taxa with CRC progression shown for ASVs that were significantly increased both in cancer and polyps compared to controls. The figure shows the relative abundance of taxa at different sampling sites in cancer, polyps, and controls

Fig. 7

A The dynamics of microbial taxa with CRC progression shown for genera that were significantly increased in cancer compared to polyps and controls. The figure shows the relative abundance of taxa that were present at different samplings sites in cancer, polyps, and controls. B The dynamics of microbial taxa with CRC progression shown for ASVs that were significantly increased both in cancer and polyps compared to controls. The figure shows the relative abundance of taxa at different sampling sites in cancer, polyps, and controls

Fig. 8

Bar chart illustrates the distribution of Fusobacterium species and subspecies in the colorectal cancer tumors. Results were obtained using Fusobacterium specific amplicon sequencing with MinION and taxonomic classification by WIMP. Asterisk indicates uncertain results due to low sequencing depth