Engineered Methioninase-expressing Tumor-targeting Salmonella typhimurium A1-R Inhibits Syngeneic-Cancer Mouse Models by Depleting Tumor Methionine.

Abstract

Background/aim: We previously developed Salmonella typhimurium A1-R, which selectively targets and kills tumors. In the present study, we established recombinant methioninase (rMETase)-producing Salmonella typhimurium A1-R (A1-R-rMETase), by transfer of the Pseudomonas putida methioninase gene, to target methionine addiction of syngeneic-cancer mouse models.

Materials and methods: A plasmid containing the Pseudomonas putida methioninase gene was extracted from METase-producing recombinant E. coli and inserted into Salmonella typhimurium A1-R using electroporation. Lewis Lung Carcinoma (LLC) cells (10⁶) were injected subcutaneously in male C57BL/6 mice aged 4-6 weeks. We determined that 10⁸ Salmonella typhimurium A1-R-rMETase administered iv was a safe dosage in C57BL/6 mice and was used for efficacy studies on LLC tumors in C57BL/6 mice. Tumor size was measured with calipers three times per week for 3 weeks. On day 22, tumor methionine levels were measured using HPLC in the control mice injected with phosphate-buffered saline (PBS) and the mice injected with Salmonella typhimurium A1-R-rMETase.

Results: The mean LLC tumor size of each group on day 22 was as follows: PBS control: 741.5 mm³; mice injected with Salmonella typhimurium A1-R: 566.3 mm³ (p=0.370); and mice injected with Salmonella typhimurium A1-R-rMETase: 198.8 mm³ (p=0.0003 vs control and p=0.0117 vs. Salmonella A1-R). The mice injected with Salmonella typhimurium A1-R-rMETase showed a significantly lower mean tumor methionine level than mice injected with PBS (5.9 nM/mg protein vs 11.1 nM/mg protein, p=0.0095). Salmonella typhimurium A1-R-rMETase grew continuously in the tumors but not in the liver or spleen.

Conclusion: Tumor-targeting Salmonella typhimurium A1-R engineered to express the Pseudomonas putida methioninase gene, inhibited LLC tumor growth in a syngeneic mouse model and reduced the methionine level in the tumor. Salmonella typhimurium A1-R-rMETase combines the tumor targeting and killing capability of Salmonella typhimurium A1-R plus rMETase which targets the methionine addiction of cancer.