Figure 3. Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment.
(A) Fap2 is a Gal-GalNAc binding lectin. Hemagglutination by wild-type Fn and not by isogenic Fap2 inactivated mutants K50 and D22 in the absence (left) and in the presence (right) of 25 mM GalNAc.
(B) Representative image of FITC-labeled Fn (green) attachment to Hoechst-stained (blue) human colon adenocarcinoma sections in the absence (left) or presence (right) 300 mM GalNAc.
(C) Quantitation of fusobacterial binding (Fn/mm2) performed in (B). Each symbol represents the mean of three randomly selected fields per human section (n = 6). Mean ± SEM are shown; *p = 0.015, Wilcoxon signed-rank test.
(D) Representative image of Cy3-labeled Fn (red) and Cy5-labeled Fap2-inactivated isogenic mutant K50 (green) to a Hoechst-stained (blue) human colon adenocarcinoma section.
(E) Quantitation of fusobacterial binding (Fn/mm2) to TMA of human colon adenocarcinoma, adenoma, and normal tissue. Each symbol represents the mean of three randomly selected fields per human tissue core. Mean ± SEM are shown; ****p < 0.0001, Bonferroni-corrected Wilcoxon test.
(F) Attachment of FITC-labeled (green) Fn (left) or of Fap2-inactivated isogenic mutant D22 (right) to Hoechst-stained (blue) representative human colon adenocarcinoma sections.
(G) Quantitation of fusobacterial binding (Fn/mm2) described in (F). Each symbol represents the mean of three randomly selected fields per human section (n = 6). Mean ± SEM are shown; *p = 0.0119, one-tailed Mann-Whitney test.
(H) Fn colocalization with Gal-GalNAc in human CRC. Human colorectal adenocarcinoma sections were stained with Hoechst (blue) and incubated with Alexa Fluor 647-conjugated PNA (red) and FITC-labeled Fn (green). Dashed line indicates the CRC-adjacent normal tissue border. Representative image (left). Magnification of the inset CRC region is shown in the middle, and the inset adjacent to normal tissue is shown on the right.
See also Figure S1.