Abstract 3052: Fusobacterial-dominant colorectal cancer biofilms are associated with high levels of the polyamine N1,N12-diacetylspermine
Authors: Drewes Julia L. , Queen Jessica , Domingue Jada C. , Wensel Caroline R. , Cai Yuping , Wanyiri Jane , Roslani April C. , Vadivelu Jamuna , Johnson Caroline H. , Sears Cynthia L. 
Published: June 2022

Abstract
Background: Mucus-invasive colonic bacterial biofilms are dense, polymicrobial communities that are in direct contact with the epithelium. Prior data from our group has demonstrated that these invasive biofilms are prevalent in colorectal cancer, are directly tumorigenic in germ-free and specific pathogen-free ApcMin/+ mouse models, and are associated with enrichment of the polyamine N1,N12-diactylspermine (DAS) in patient samples. However, whether the composition of these biofilms impacts their pro-tumorigenic behavior and/or metabolism is presently unknown.
Methods: Carnoy’s-fixed CRC resections from 115 patients from the University of Malaya in Malaysia were stained with the all-bacterial EUB338 probe and screened for mucus-invasive biofilms (BF). All BF+ samples were subsequently stained with oligonucleotide probes targeting Bacteroidetes, Fusobacteria, Lachnospiraceae, and γ/β-Proteobacteria. Imaging was performed on a Zeiss780 confocal microscope with linear unmixing. Untargeted metabolomics was performed on 5-10 samples of each biofilm type using a Waters UPLC Xevo GS-XS quadrupole time-of-flight (QTOF) mass spectrometer. Five Fusobacterium nucleatum (Fn) strains derived from CRC cohorts were singly gavaged into germ-free ApcMin/+ mice and colons were harvested for tumors 11 wk later.
Results: Seventy-nine of 115 Malaysian tumors (69%) were BF+ using EUB338 staining. As with our prior US CRC cohort, BFs were more prevalent on the tumors proximal to the hepatic flexure (35/39, 90%) vs. those that were distal (44/76, 58%) (Chi-square p &lt; 0.001). Of the BF+ tumors (BF+T), 55% were polymicrobial (predominantly Bacteroidetes/Lachnospiraceae), 40% were polymicrobial with fusobacterial blooms, and 5% were Proteobacteria dominant. Untargeted metabolomics of BF- healthy biopsies, BF-T, and BF+T with various BF composition subtypes revealed a gradient in levels of the polyamine DAS, with BF+T with fusobacterial blooms having the highest levels (p = 0.055 vs. Proteo-dominant BF+T, p = 0.043 vs. polymicrobial BF+T, p = 0.015 vs. BF-T, p &lt; 0.001 vs. BF- healthy biopsies), suggesting that fusobacteria may drive proliferation and polyamine production in CRC tissues. However, inoculation of 5 different CRC-derived Fn strains including a strain isolated from a Malaysia CRC patient did not promote tumorigenesis in germ-free ApcMin/+ mice.
Conclusions: Fusobacterial-dominant biofilms are prevalent in a Malaysian CRC cohort and are associated with high levels of the polyamine N1,N12-diacetylspermine. However, F. nucleatum strains were not tumorigenic in germ-free ApcMin/+ mice, suggesting that the association between Fn, DAS, and tumorigenesis may require other bacteria in order to confer a procarcinogenic state.
Citation Format: Julia L. Drewes, Jessica Queen, Jada C. Domingue, Caroline R. Wensel, Yuping Cai, Jane Wanyiri, April C. Roslani, Jamuna Vadivelu, Caroline H. Johnson, Cynthia L. Sears. Fusobacterial-dominant colorectal cancer biofilms are associated with high levels of the polyamine N1,N12-diacetylspermine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3052.