Impact of engineered probiotic expressing FomA-targeted antimicrobial peptides on intestinal tumorigenesis in a mouse model of <i>Fusobacterium nucleatum</i> –associated colorectal cancer.

Abstract

e15157 Background: Fusobacterium nucleatum ( Fn ) is one of the most enriched bacteria in colorectal adenomas and adenocarcinomas, where it promotes tumor progression, suppresses anti-tumor immunity, and contributes to chemoresistance. Current antibiotic approaches to eliminate Fn cause collateral damage to beneficial gut microbiota and diminish therapeutic efficacy. To eliminate Fn directly, we engineered Lactococcus lactis MG1363 to express guided antimicrobial peptides (gAMPs) comprising Cathelin-related peptide CPSC5 linked to a Statherin-derived guide peptide (YQPVPE) that specifically binds FomA, an outer membrane porin and key virulence factor of Fn . Building on our previous in vitro findings, which demonstrated that gAMPs selectively inhibited F n growth and biofilm formation, this study sought to address efficacy in vivo. Methods: We tested our engineered probiotic in the Apc^Min/+^ mouse model. We pre-treated 32 male and female mice with streptomycin (2 mg/mL/d x7d) followed by DSS (1.5% x7d) and colonized with the 7_1 Fn strain (1 × 10 9 cells/ 200 µl x1d), which has shown to promote CRC in animal models. Results: Preliminary results demonstrate striking tumor suppression across all L. lactis treatment groups compared to untreated controls. Control mice exhibited 100% tumor incidence (4/4 mice) with 32 total tumors. Treatment with empty vector L. lactis alone significantly reduced tumor incidence to 33% (2/6 mice) with only 4 total tumors, consistent with prior reports of L. lactis immune stimulatory properties, while unguided AMP- L. lactis showed 83% tumor incidence (5/6 mice) with 22 total tumors. Most strikingly, gAMP- L. lactis treatment achieved the greatest tumor suppression, reducing incidence to just 17% (1/6 mice; p&lt;0.05 vs. control) with only a single tumor observed across all treated animals. These data demonstrate that FomA-targeted gAMP delivery provides superior efficacy over both empty vector probiotic and unguided AMP approaches. Conclusions: Ongoing studies are examining the effects of gAMP-probiotic treatment further, on gut microbiome composition and function, anti-tumor immune responses, expression of oncogenic signaling genes, and Fn eradication. Overall, these data suggest the therapeutic potential of engineered probiotics secreting pathogen-targeted antimicrobial peptides as a precision approach to prevent and treat cancer.