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. 2020 Aug 7:10:400.
doi: 10.3389/fcimb.2020.00400. eCollection 2020.

Colon Cancer-Associated Fusobacterium nucleatum May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory System

Jawad Abed  1   2 , Naseem Maalouf  1 , Abigail L Manson  3 , Ashlee M Earl  3 , Lishay Parhi  1 , Johanna E M Emgård  1 , Michael Klutstein  1 , Shay Tayeb  4 , Gideon Almogy  5 , Karine A Atlan  6 , Stella Chaushu  2 , Eran Israeli  7 , Ofer Mandelboim  8 , Wendy S Garrett  3   9   10 , Gilad Bachrach  1
Affiliations

Colon Cancer-Associated Fusobacterium nucleatum May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory System

Jawad Abed et al. Front Cell Infect Microbiol. .

Abstract

Fusobacterium nucleatum is a common oral bacterium that is enriched in colorectal adenomas and adenocarcinomas (CRC). In humans, high fusobacterial CRC abundance is associated with chemoresistance and poor prognosis. In animal models, fusobacteria accelerate CRC progression. Targeting F. nucleatum may reduce fusobacteria cancer progression and therefore determining the origin of CRC F. nucleatum and the route by which it reaches colon tumors is of biologic and therapeutic importance. Arbitrarily primed PCR performed previously on matched same-patients CRC and saliva F. nucleatum isolates, suggested that CRC F. nucleatum may originate from the oral cavity. However, the origin of CRC fusobacteria as well as the route of their arrival to the tumor have not been well-established. Herein, we performed and analyzed whole genome sequencing of paired, same-patient oral, and CRC F. nucleatum isolates and confirmed that CRC-fusobacteria originate from the oral microbial reservoir. Oral fusobacteria may translocate to CRC by descending via the digestive tract or using the hematogenous route during frequent transient bacteremia caused by chewing, daily hygiene activities, or dental procedures. Using the orthotropic CT26 mouse model we previously showed that IV injected F. nucleatum colonize CRC. Here, we compared CRC colonization by gavage vs. intravenous inoculated F. nucleatum in the MC38 and CT26 mouse orthotropic CRC models. Under the tested conditions, hematogenous fusobacteria were more successful in CRC colonization than gavaged ones. Our results therefore provide evidence that the hematogenous route may be the preferred way by which oral fusobacteria reach colon tumors.

Keywords: CRC (colorectal cancer); Fusobacterium nucleatum; cancer; oral microbes; transient bacteremia.

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Figures

Figure 1
Figure 1
CRC fusobacteria are of oral origin. (A) Metadata, saliva, and CRC fusobacteria PCR and culture status of CRC patients. (B) The distribution of whole-genome ANI values for each 1,000 bp genomic window for each pairwise comparison of strains. Almost all windows resulted in 100.0% identity, with only a few outliers depicted here (see section Methods). Average ANI values were 100.00 ± 0.03% (T2 vs. O2), 100.00 ± 0.04% (JAoral001 vs. JACRC001), and 99.98 ± 0.38% (CRC003 vs. oral003). (C) Midpoint-rooted, SNP-based phylogeny of F. nucleatum strains reveals that T2 and O2 are highly related. A heat map illustrating the SNP matrix is shown. For the closely-related strains, the count of SNPs differing is shown in the red boxes. T2 and O2 differ by zero SNPs, and CRC003/ORAL003 differ by 183 SNPs and JAcrc001/JAoral001 differ by 2 SNPs.
Figure 2
Figure 2
The bloodstream is an efficient route of oral F. nucleatum for CRC enrichment. (A) MC38 mice CRC cells display high Gal-GalNAc levels. Representative images of MC38 cells and of human adenocarcinoma HT-29 cells unstained (left panels), or stained with FITC-labeled Gal-GalNAc-specific PNA (green, right panels). (B) Hemagglutination demonstrating Fap2-dependent Gal-GalNAc binding by matching oral and CRC isolates O2/T2 with Oral003/CRC003 and JAoral001/JAcrc001 in the absence (left) and in the presence (right) of 25 mM GalNAc. The F. nucleatum ATCC 23726 Fap2 inactivated mutant K50 was used for negative control. Non-hemagglutinated erythrocytes settle in the bottom of the round bottom well. (C) in vivo experimental scheme of the orthotopic rectal MC38-luc mouse CRC model. At day 9 post-tumor implantation mice were randomized to an oral or intravenous inoculation group. Oral inoculations were performed on day 9, 12, and 15. A single intravenous inoculation was performed on day 15. (D,E) CRC colonization by hematogenously or orally administered fusobacteria. (D) Abundance (CFU/gr tissue) and relative fusobacterial gDNA abundance (2−ΔCt) (E) in tumor (T) samples and in adjacent normal (N) colon samples from MC38 transplanted mice inoculated once with 5 × 106-1 × 107 intravenously (IV) or three times by oral gavage (Gavage) with 10~10 F. nucleatum O2 or Oral003. ****p < 0.0001, Bonferroni-corrected two-tailed Mann-Whitney test. ***p < 0.001 Bonferroni-corrected two-tailed Mann-Whitney test for gavage vs. IV, Bonferroni-corrected one-tailed Wilcoxon test for tumor vs. normal. **p < 0.01 Bonferroni-corrected one-tailed Wilcoxon test. n.s., not statistically significant. Each symbol represents data from individual mice. Error bars show Mean ± SEM.
Figure 3
Figure 3
Kinetics of F. nucleatum ATCC 23726 enrichment in the CT-26 orthotopic model. (A) Detection of implanted CT-26 cells stably transfected with the luciferase (luc) gene under the mucosa of the distal rectum of C57BL/6 wild-type mice. (B) Relative fusobacterial gDNA abundance (2−ΔCt) in tumor samples and in adjacent normal colon samples from CT26 transplanted BALB/cJ mice inoculated once with 5 × 107-1 × 108 intravenously (IV) or three times by oral gavage (Gavage) with 10~10 F. nucleatum ATCC 23726. *p < 0.05, Bonferroni-corrected two-tallied Wilcoxon test, ***p < 0.001, Bonferroni-corrected two-tailed Mann-Whitney test. (C) Abundance (CFU/gr tissue) and (D) relative fusobacterial gDNA abundance (2−ΔCt) in tumor samples collected 2, 6, 24, and 72 h after IV inoculation of CT26 transplanted BALB/cJ mice with 5 × 107-1 × 108 F. nucleatum ATCC 23726. *p = 0.02, ***p = 0.0001, ****p < 0.0001, Bonferroni-corrected two-tailed Mann-Whitney test. Error bars show Mean ± SEM.
Figure 4
Figure 4
Efficiency of CRC colonization by hematogenous fusobacteria is dose-dependent. (A) Relative fusobacterial gDNA abundance (2−ΔCt), and proportion CRC colonization (B) in tumor samples from MC38 transplanted mice intravenously inoculated daily three times with 5 × 103-1 × 104, 5 × 104-1 × 105 or with 5 × 106-1 × 107 F. nucleatum JAoral001. Each symbol represents one mouse. Bar show median. *p < 0.05, the proportion of mice with fusobacterial-colonized tumors increases significantly with the amount of bacteria injected, as determined by an exact one-sided Permutation Test implemented by StatXact* (p = 0.0442 with the scores set equal to the amount of bacteria, p = 0.04633 with the scores equal to the log).
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