Emerging strategies for combating Fusobacterium nucleatum in colorectal cancer treatment: Systematic review, improvements and future challenges

**Background:** Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide, with over 1.85 million cases and 850,000 deaths annually. The gut microbiota, particularly Fusobacterium nucleatum, plays a critical role in CRC pathogenesis. F. nucleatum is a Gram-negative anaerobe that promotes tumorigenesis through various mechanisms, including adherence via Fap2 and FadA adhesins, activation of TLR4/PAK1/β-catenin signaling, and induction of chemoresistance (e.g., to 5-fluorouracil) via BIRC3 upregulation and autophagy networks. It also contributes to immune evasion by inhibiting T-cell activity and promoting M2 macrophage polarization. F. nucleatum biofilms are prevalent in proximal CRC (89% vs. 12% distal) and enhance carcinogenic potential. Traditional antibiotics are limited due to gut microbiota disruption and resistance development. This review systematically evaluates non-antibiotic strategies to inhibit F. nucleatum and its biofilms for CRC treatment.

**Methods:** The authors conducted a systematic review of the literature, categorizing anti-F. nucleatum strategies into eight groups: natural extracts, inorganic chemicals, organic chemicals (photosensitizers, antimicrobial peptides, other compounds), polymers, inorganic-organic hybrid materials, bacteriophages, probiotics, and vaccines. For each category, the review summarizes the active compounds, mechanisms of action, and in vitro/in vivo efficacy, with a focus on studies published up to 2024. Key data points include minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), colony-forming unit (CFU) reductions, and biofilm disruption.

**Key Results:** - **Natural extracts:** Curcumin-cinnamaldehyde hybrids (CCHs) showed MIC of 9 μg/mL and MBC of 19 μg/mL against F. nucleatum. Pterostilbene (PTS) complexed with HP-β-CD had MIC of 0.02 mg/mL and MBC of 0.04 mg/mL, causing protein and nucleic acid leakage. Nano-defensins (fenugreek defensin on chitosan) had MIC of 10.6 μg/mL vs. 66 μg/mL for free defensin. - **Fatty acids:** Lauric acid (LA) in PAMAM-Pt-LA@HA and PG-Pt-LA/CB[7] nanosystems effectively eliminated intratumoral F. nucleatum, reducing 16S rRNA expression and NF-κB expression to 25.9%, and decreasing TNF-α and IL-6 levels in vivo. - **Inorganic chemicals:** Polycationic silver nanoclusters (pAgNCs) had initial MIC of 3.75 μg/mL, increasing to 13.5 μg/mL after 12 cycles (vs. kanamycin >500 μg/mL after 15 cycles). Ce-doped ZIF-8 NPs reduced CFUs by nearly two orders of magnitude. - **Organic chemicals:** Photodynamic therapy (PDT) with riboflavin/H2O2 reduced F. nucleatum counts by ≥4 log10 after 1 min. UCNPs@TiO2 under NIR irradiation decreased CFU counts and metabolic activity of 4-day biofilms. L-Lysine had MIC of 100 mM and MBC of 200 mM. Nal-P-113 at 20 μg/mL inhibited biofilm formation. G12R peptide had MIC of 25 μM vs. 200 μM for AmyI-1-18. - **Polymers:** Quaternary ammonium PAMAM-AZO@CP[5]A (Q-P-A@CP[5]A) eradicated about 70% of intratumoral F. nucleatum in vivo and induced apoptosis in HT29 cells co-cultured with F. nucleatum. - **Inorganic-organic hybrids:** CuTCPP-Fe2O3 showed 99.57±0.21% antibacterial efficacy against F. nucleatum vs. 85.51±1.78% for CuTCPP alone. CeCyan-Cu5.4O with cyanobacteria improved PDT under hypoxia. - **Bacteriophages:** Phage P2 selectively lysed F. nucleatum and reversed chemoresistance in CT26, HCT116, and HT29 cells. Phage FNU1 disrupted existing biofilms, with most cells showing damaged membranes. - **Probiotics:** Live probiotic pills (B. animalis CP-9, L. paracasei ET-66, L. salivarius AP-32) reduced F. nucleatum survival to 5.77%. L. gasseri HHuMIN D supernatant inhibited F. nucleatum by 89% at 5% dilution. - **Vaccines:** AhpC/alum immunization protected mice from F. nucleatum infection in 74.8% of cases (vs. 53.6% for AhpC alone). High anti-AhpC titer (1:1 dilution) reduced bacterial survival to 2.1%.

**Clinical Implications:** The review highlights that targeted elimination of F. nucleatum in CRC can potentially overcome chemoresistance, reduce inflammation, and improve treatment outcomes. However, challenges remain, including in vivo bioavailability of natural products, selectivity of photosensitizers and metal ions, stability of antimicrobial peptides, and delivery of probiotics and bacteriophages to the colon. Future directions include developing dual-function systems that simultaneously target cancer cells and F. nucleatum, improving intracellular bacterial eradication, and enhancing biocompatibility through smart carriers. The authors emphasize the need for further in vivo studies and clinical trials to translate these strategies into effective CRC therapies.