Engineered Salmonella expressing IL-18 as a therapeutic strategy for colorectal cancer

Technical Summary: Engineered Salmonella Expressing IL-18 as a Therapeutic Strategy for Colorectal Cancer

Problem Statement
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality globally, primarily due to therapeutic resistance, metastatic progression, and the persistence of immunosuppressive tumor microenvironments (TME). Conventional therapies often fail to penetrate hypoxic tumor regions or effectively modulate the complex oncogenic pathways driving proliferation and immune evasion. While immunotherapy offers promise, its efficacy is limited in microsatellite-stable and metastatic CRC. There is an urgent need for strategies that can target the TME, modulate key signaling pathways (such as Wnt/β-catenin, JAK/STAT, and NF-κB), and restore effective antitumor immunity.

Methodology
The study utilized an attenuated strain of Salmonella enterica serovar Typhimurium (SL3261), which lacks the aroA gene, rendering it auxotrophic for aromatic amino acids and safe for use as a vector. The researchers genetically engineered this strain to express Interleukin-18 (IL-18) in two forms:

1. Pro-IL-18: The inactive precursor form.
2. IL-18 (Bioactive):* A truncated form lacking the first 105 base pairs, representing the mature, active cytokine.

The IL-18 coding sequences were fused with Emerald Green Fluorescent Protein (EmGFP) for expression verification and placed under the control of the nirB promoter, which is activated under anaerobic conditions (mimicking the tumor core). The constructs were subcloned into the pAE plasmid and transformed into SL3261.

Experimental Models and Assays:

• Cell Lines: CT-26 (mouse) and HCT-8 (human) colorectal cancer cells.
• Organoids:
  • Murine: APTAK organoids (engineered with Apc, TP53, Tgfbr2 loss and Kras/Akt activation) and organoids derived from primary tumors, liver metastases, and peritoneal carcinomatosis.
  • Human: Organoids derived from patient biopsies (primary tumors and liver metastases).
• Treatment Modalities:
  • Supernatant Treatment: Bacterial culture supernatants containing secreted IL-18 were applied to cell lines and organoids.
  • Co-culture: Live engineered bacteria were co-cultured with organoids under anaerobic conditions to mimic in vivo colonization.
  • Recombinant Control: Direct treatment with recombinant IL-18 protein.
• Readouts:
  • Viability: MTT assays (cell lines) and CellTiter-Glo luminescent assays (organoids) at 24, 48, 72, and 96 hours.
  • Protein Expression: Fluorescence emission (EmGFP) and ELISA for IL-18 quantification.
  • Inflammatory Mediators: ELISA for chemokines CXCL1 and CXCL2; Griess assay for Nitric Oxide (NO) production.
  • Signaling Pathways: Western blot analysis of phosphorylated and total forms of STAT1, STAT3, β-catenin, GSK-3β, p38 MAPK, and NF-κB.

Key Results

1. Successful Expression and Secretion: The engineered strains (pro_SL3261 and bio_SL3261) successfully expressed and secreted IL-18, confirmed by EmGFP fluorescence and ELISA. Bioactive IL-18 supernatants showed higher concentrations than pro-IL-18 supernatants.
2. Cytotoxicity in Cell Lines and Organoids:
   • Treatment with bacterial supernatants significantly reduced viability in CT-26 and HCT-8 cell lines.
   • Organoid Sensitivity: Organoids exhibited pronounced sensitivity. Murine APTAK organoids showed ~70% viability reduction. Human CRC organoids were even more sensitive, showing near-complete loss of viability after 96 hours of exposure to bioactive IL-18.
   • Co-culture Superiority: Direct co-culture with live bacteria induced rapid and sustained cytotoxicity, with bio_SL3261 reducing organoid viability to <15% by 96 hours.
   • Recombinant vs. Bacterial: Recombinant IL-18 alone required high concentrations (7.0 µg/mL) to suppress growth and showed transient effects, whereas bacterial delivery provided sustained suppression at lower effective doses.
3. Modulation of Inflammatory Mediators:
   • Chemokines: While non-engineered SL3261 increased pro-tumorigenic chemokines CXCL1 and CXCL2, IL-18-expressing strains (especially bio_SL3261) significantly attenuated their secretion, bringing levels close to negative controls.
   • Nitric Oxide: Bioactive IL-18 consistently induced high levels of NO production across all models, a known mediator of cytotoxicity and oxidative stress.
4. Signaling Pathway Reprogramming:
   • JAK/STAT: IL-18 treatment suppressed phosphorylated STAT3 (pro-survival) and induced STAT1 (antitumor) in specific models (e.g., peritoneal carcinomatosis).
   • Wnt/β-catenin: Treatment reduced levels of non-phosphorylated and phosphorylated β-catenin while enhancing GSK-3β activation, indicating suppression of the Wnt pathway.
   • NF-κB and p38: Cytoplasmic NF-κB was suppressed, particularly in APTAK organoids treated with bioactive IL-18. p38 MAPK activation was detectable but limited.

Key Contributions and Significance
The paper demonstrates that attenuated Salmonella Typhimurium SL3261 serves as an effective "microfactory" for the localized delivery of IL-18 within the colorectal tumor microenvironment. The study establishes that:

• Bioactive Form is Critical: The therapeutic efficacy is strictly dependent on the bioactive form of IL-18, which outperforms the precursor form across all models.
• Synergistic Mechanism: The antitumor effect is not solely due to the cytokine; it involves a synergy between bacterial factors (inducing innate immune responses) and the specific immunomodulatory effects of IL-18.
• Mechanistic Insight: The therapy functions by reprogramming the TME from a pro-tumorigenic state to a pro-inflammatory, antitumor state. This is achieved by suppressing oncogenic drivers (Wnt/β-catenin, STAT3) and inducing cytotoxic effectors (NO) while reducing pro-metastatic chemokines (CXCL1/CXCL2).
• Translational Potential: The use of patient-derived and metastatic organoids confirms that this strategy retains efficacy against aggressive, metastatic CRC phenotypes that are typically resistant to standard therapies.

The authors conclude that this bacterial-mediated delivery system overcomes limitations of systemic cytokine administration (such as rapid degradation and toxicity) and offers a potent, multifaceted immunotherapeutic strategy for advanced colorectal cancer.