Oral delivery of tumor-targeting Salmonella exhibits promising therapeutic efficacy and low toxicity

Abstract

Tumor-targeting bacteria have been developed as powerful anticancer agents. Salmonella typhimurium VNP20009, a representative tumor-targeting strain, has been systemically administered as a single-agent therapy at doses of 1 x 10(6) to 3 x 10(6) colony-forming unit (cfu)/mouse, or in combination with other antitumor agents at doses of 1 x 10(4) to 2 x 10(5) cfu/mouse. Recently, we reported that oral delivery of VNP20009 at the dose of 1 x 10(9) cfu/mouse induced significant anticancer effects comparable to that induced by systemic administration of this strain at 1 x 10(4) cfu/mouse. To further address the efficacy and safety of oral administration of bacteria, here we performed a systemically comparative analysis of anticancer efficacy and toxicity of VNP20009 administered: (i) orally at a dose of 1 x 10(9) cfu/mouse (VNP9-oral); (ii) intraperitoneally at a dose of 1 x 10(4) cfu/mouse (VNP4-i.p.); or (iii) intraperitoneally at a dose of 1 x 10(6) cfu/mouse in tumor-free and tumor-bearing murine models. The results showed that VNP9-oral, similar to VNP4-i.p., induced significant tumor growth inhibition whereas VNP6-i.p. induced better anticancer effect in the B16F10 melanoma model. Among three treatments, VNP9-oral induced the mildest and reversible toxicity whereas VNP6-i.p. resulted in the most serious and irreversible toxicities when compared to other two treatments. Moreover, the combination of VNP9-oral with a low dose of chemotherapeutics produced comparable antitumor effects but displayed significantly reduced toxicity when compared to VNP6-i.p. The findings demonstrated that oral administration, as a novel avenue in the application of bacteria, is highly safe and effective. Moreover, the present preclinical study should facilitate the optimization of bacterial therapies with improved anticancer efficacy and reduced adverse effects in future clinical trials.

Figure 1

Evaluation of antitumor effect and potential toxicity of systemic and oral infection of Salmonella. Antitumor effect (a) and body weight (b) of VNP9‐oral, VNP4‐i.p., and VNP6‐i.p. as a single agent therapy in a murine melanoma model. B16F10 melanoma–bearing mice were infected either orally with 10⁹ colony‐forming units (cfu)/mouse VNP20009 or intraperitoneally with 10⁶ or 10⁴ cfu/mouse bacteria. Tumor volume and body weight were determined over time. () P < 0.01 and *P < 0.001. (c,e) Antitumor effect of combination therapy of VNP9‐oral and 5‐fluorouracil (5‐FU) or cisplatin when compared to VNP6‐i.p. Lewis lung carcinoma‐bearing mice were treated as described in Materials and Methods section. (d,f) Body weight. () P < 0.05, () P < 0.01, and *P < 0.001, combination group versus VNP6‐i.p.

Figure 2

Bacteria titer in spleens (a) and spleen weight (b). Tumor‐free mice were infected with Salmonella as indicated. Bacteria replication in the spleen (a) and the weight of the spleen (b) were determined at 20 h, 10, 21, and 42 days post infection. Each bar represents the mean ± SD of five mice per group. () P < 0.05, () P < 0.01, and *P < 0.001 when compared to VNP6‐i.p.

Figure 3

Subpopulation analysis of immune cells in the spleen. Tumor‐free mice were infected with Salmonella and spleens were collected post infection for subpopulation analysis of immune cells as described in Materials and Methods section. (a) Macrophages; (b) granulocytes; (c) CD4⁺ T cells; (d) CD8⁺ T cells, and (e) B cells. Each bar represents the mean ± SD of three mice per group. () P < 0.05 and () P < 0.01 when compared to controls.

Figure 4

Bacterial replication and inflammatory response in livers. Tumor‐free mice were infected and related assays were performed at 20 h, 10, 21, and 42 days post infection. (a) Bacteria titer in livers; (b) infection foci within livers in hematoxylin–eosin (H&E) stained sections are indicated with arrows (magnification, ×40). (c) The number of infection foci per field. Each bar represents the mean ± SD of five mice per group. () P < 0.01 and *P < 0.001 when compared to VNP6‐i.p. [Correction added after online publication 30 September 2009.]

Figure 5

Liver function. Tumor‐free mice were infected and serum samples were collected at 20 h, 10, 21, and 42 days post infection for alanine aminotransferase (ALT) (a) and aspartate aminotransferase (AST) (b) assays. Each bar represents the mean ± SD of five mice per group. () P < 0.05, () P < 0.01, and *P < 0.001 when compared to VNP6‐i.p.

Figure 6

Induction of inflammatory cytokines after bacterial infection. Tumor‐free mice were infected and serum samples were collected at 0 h, 2 h, 6 h, and 3 days post infection for tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, and IL‐1β analysis. (a) TNF‐α, (b) IL‐6, and (c) IL‐1β. Each bar represents the mean ± SD of four mice per group. () P < 0.05, () P < 0.01, and *P < 0.001 when compared to controls.

Figure 7

Body weight. Tumor‐free mice were infected with bacteria, and body weight was determined at 6, 12, 18, and 28 days post infection (n = 10). () P < 0.01 for VNP6‐i.p. versus control, VNP9‐oral, or VNP4‐i.p.