Monotherapy with a tumor-targeting mutant of Salmonella typhimurium cures orthotopic metastatic mouse models of human prostate cancer

Abstract

Bacterial infection occasionally has a marked therapeutic effect on malignancies, as noted as early as the 19th century. Recently, there have been attempts to develop cancer treatment by using tumor-targeting bacteria. These treatments were developed to deliver therapeutic molecules specifically to tumors. Researchers used anaerobic microorganisms that preferentially grew in necrotic tumor areas. However, the resulting tumor killing was, at best, limited. We have developed a far more effective bacterial cancer therapy by targeting viable tumor tissue by using Salmonella typhimurium leu-arg auxotrophs. Although these bacteria grow in viable as well as necrotic areas of tumors, the nutritional auxo trophy severely restricts growth in normal tissue. In the current study, we measured the antitumor efficacy of the S. typhimurium A1-R mutant, which is auxotrophic for leu-arg and has increased antitumor virulence selected by tumor passage. A1-R was used to treat metastatic PC-3 human prostate tumors that had been orthotopically implanted in nude mice. GFP was used to image tumor and metastatic growth. Of the 10 mice with the PC-3 tumors that were injected weekly with S. typhimurium A1-R, 7 were alive and well at the time the last untreated mouse died. Four A1-R-treated mice remain alive and well 6 months after implantation. Ten additional nontumor-bearing mice were injected weekly to determine the toxicity of S. typhimurium A1-R. No toxic effects were observed. The approach described here, where bacterial monotherapy effectively treats metastatic prostate tumors, is a significant improvement over previous bacterial tumor-therapy strategies that require combination with toxic chemotherapy.

Fig. 1.

Real-time imaging of S. typhimurium A1-R-induced cell death of PC-3 human prostate cancer cells in vitro. Dual-color PC-3 human prostate tumor cells, labeled with RFP in the cytoplasm and GFP in the nucleus, were grown in 24-well tissue culture plates to a density of ≈10⁴ cells per well. A1-R bacteria expressing GFP were grown in LB and added to the tumor cells (1 × 10⁵ cfu per well). After 1 h incubation at 37°C, the cells were rinsed and cultured in medium containing gentamycin sulfate (20 μg/ml) to kill external but not internal bacteria. Cells started to die by 60 min after infection. By 200 min, the RFP cytoplasm completely lysed and dispersed, and nuclei fragmented.

Fig. 2.

Time course of tumor progression after two treatments with S. typhimurium A1-R. Mice orthotopically implanted with PC-3 human prostate tumors were given A1-R at 15 and 27 days after tumor implantation. The tumor-bearing mice were imaged at the indicated time points and compared with untreated control.

Fig. 3.

Efficacy of weekly S. typhimurium A1-R treatment on PC-3 primary and metastatic tumor growth. Mice were given S. typhimurium A1-R weekly i.v. Primary and metastatic tumor size was measured by GFP imaging at day 30 after tumor transplantation. (A) GFP areas of A1-R-treated and untreated tumors were compared at day 30. The tumor size in the treated group was ≈75% smaller than the untreated group (n = 10; P = 0.04). (B) Imaging of tumor growth in eight mice of the untreated group at day 30. (C) Imaging of tumor growth of 10 mice in the A1-R-treated group at day 30.

Fig. 4.

Time course of tumor growth and regression of A1-R-treated orthotopic PC-3 in nude mice. The mice were implanted orthotopically with PC-3 tumors expressing GFP. Approximately 2 weeks after implantation, the tumors were externally imageable macroscopically. Ten mice were given S. typhimurium A1-R weekly i.v. Ten mice served as untreated controls. Primary and metastatic tumor size was measured by GFP imaging at different time points after tumor transplantation. (A) Whole-body images of an A1-R-treated mouse. (B) Whole-body images of an untreated mouse.

Fig. 5.

Survival efficacy of S. typhimurium A1-R. (A) Survival efficacy of S. typhimurium A1-R with weekly treatment (n = 10). (B) Survival efficacy of S. typhimurium A1-R with twice-only treatment (n = 10).

Fig. 6.

Toxicity of S. typhimurium A1-R. Nontumor-bearing mice were tested for the possible toxicity of S. typhimurium A1-R. The mice were injected i.v. weekly with the bacteria (5 × 10⁷ cfu). The survival time and body weight were compared with the untreated mice. A1-R-infected animals treated with repeated weekly injections of S. typhimurium A1-R survived as long as untreated mice with no significant body weight change.