Metabolism links bacterial biofilms and colon carcinogenesis

Abstract

Bacterial biofilms in the colon alter the host tissue microenvironment. A role for biofilms in colon cancer metabolism has been suggested but to date has not been evaluated. Using metabolomics, we investigated the metabolic influence that microbial biofilms have on colon tissues and the related occurrence of cancer. Patient-matched colon cancers and histologically normal tissues, with or without biofilms, were examined. We show the upregulation of polyamine metabolites in tissues from cancer hosts with significant enhancement of N(1), N(12)-diacetylspermine in both biofilm-positive cancer and normal tissues. Antibiotic treatment, which cleared biofilms, decreased N(1), N(12)-diacetylspermine levels to those seen in biofilm-negative tissues, indicating that host cancer and bacterial biofilm structures contribute to the polyamine metabolite pool. These results show that colonic mucosal biofilms alter the cancer metabolome to produce a regulator of cellular proliferation and colon cancer growth potentially affecting cancer development and progression.

Figure 1. Untargeted metabolomics

(A) Cloud plot showing dysregulated features between normal tissues and patient-matched colon cancer tissues (n=30, two-tailed Wilcoxon test). Total ion chromatograms for each sample can be seen on the plot. (B) Relative abundance of N¹, N¹²-diacetylspermine (two-tailed Wilcoxon test, ****p<0.0001). (C) Comparison of biofilm negative cancers, n=11 (lower part of plot) to biofilm positive cancers n=8 (upper part of plot) (two-tailed Mann-Whitney test). (D) Relative abundance of N¹, N¹²-diacetylspermine (two-tailed Mann-Whitney test) **p<0.01. Tandem MS spectrum of N¹, N¹²-diacetylspermine in samples and standards is shown. See also Table S1 and Figure S1.

Figure 2. Biofilm effects on metabolites in colon tissues

(A) Scheme of polyamine metabolism. Polyamine oxidase (PAO), spermidine/spermine N1-acetyltransferase (SSAT), ornithine decarboxylase (ODC), histone acetyltransferase (HAT), spermidine synthase (SRM), spermine oxidase (SMO), spermine synthase (SMS). (B) Targeted metabolomics, concentrations of metabolites in cancers with (n=9) or without (n=10) biofilms (two-tailed Mann-Whitney test). Empty symbols indicate left-sided biofilm positive samples. Error bars are SEM, *p<0.05, n.s = not significant. (C) Nanostructure-initiator mass spectrometry imaging on biofilm positive normal and cancer tissue. Scale =100 μm. (D) Hematoxylin and eosin staining, *mucosal edge. Scale = 500 μm left, 200 μm right column. See also Figure S2.

Figure 3. Immunohistochemical analysis (IHC) of spermidine/spermine N¹-acetyltransferase (SSAT)

(A) SSAT IHC of cancer and paired normal tissues from patients with and without a biofilm (scale bar 50 μm). (B) Scoring of epithelial SSAT IHC in cancers and paired normal tissues with (n=9) and without (n=11) a biofilm. (C) HCT116 cells stimulated with N¹,N¹¹-bis(ethyl) norspermine with positive SSAT staining compared with unstimulated HCT116 cells lacking SSAT. (D) SSAT staining of cancer tissue with and without primary antibody. IHC 0-3 scoring system (E) with representative cytoplasmic staining intensities. Selected inserts are displayed in the bottom panel. All images were captured at 400x (scale bars 50 μm).

Figure 4. Antibiotic-treated cancers

(A) Fluorescent in situ hybridization of all bacteria (red). Right-sided normal and paired cancer tissue from non-antibiotic and antibiotic-treated patients, DAPI counterstain (scale 10 μm). (B) Microbial culture data from antibiotic-treated (n=8) (blue) and non-antibiotic-treated patients (n=8) (red). Absolute concentrations of metabolites in (C) right-sided cancers from antibiotic-treated patients (n=6) and cancers with biofilms (n=9) from non-antibiotic-treated patients. (D) Right-sided cancers from antibiotic-treated patients (n=6) and cancers without biofilms (n=10) from non-antibiotic-treated patients. Statistics; two-tailed Mann-Whitney test, error bars are SEM, *p<0.05, ***p<0.001, **p<0.0001, n.s = not significant. See also Figure S3.