Oncology/Endocrine
Monotherapy with a Tumor-Targeting Mutant of S. typhimurium Inhibits Liver Metastasis in a Mouse Model of Pancreatic Cancer

https://doi.org/10.1016/j.jss.2009.02.023Get rights and content

Cancer of the exocrine pancreas is the fourth leading cause of cancer deaths in the United States. Currently, surgical resection is the only hope for cure. The majority of patients present with locally-advanced or metastatic disease. The most common site for distant metastasis is the liver. We report here a modified auxotrophic strain of S. typhimurium that can target and inhibit the growth of liver metastasis in a mouse model of pancreatic cancer. This strain of S. typhimurium is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support from tumor tissue. To increase tumor targeting ability and tumor killing efficacy, this strain was further modified by re-isolation from a tumor growing in a nude mouse and termed A1-R. In the present study, we demonstrate the efficacy of locally- as well as systemically-administered A1-R on liver metastasis of pancreatic cancer. Mice treated with A1-R given locally via intrasplenic injection or systemically via tail vein injection had a much lower hepatic and splenic tumor burden compared with control mice. Systemic treatment with intravenous A1-R also increased survival time. All results were statistically significant. This study suggests the clinical potential of bacterial treatment of a critical metastatic target of pancreatic cancer.

Introduction

Coley observed more than a century ago that some cancer patients were cured of their tumors following postoperative bacterial infection [1]. In the middle part of the century, Malmgren et al. showed that anaerobic bacteria had the ability to survive and replicate in necrotic tumor tissue with low oxygen content [2]. Several approaches aimed at utilizing bacteria for cancer therapy have subsequently been described 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. Bifidobacterium longum has been shown to selectively grow in hypoxic regions of tumors following intravenous administration. This effect was demonstrated in 7,12-dimethylbenzanthracene-induced rat mammary tumors by Yazawa et al. 14, 15. Vogelstein's laboratory created a strain of Clostridium novyi, an obligate anaerobe, which was depleted of its lethal toxin [16]. This strain of C. novyi was termed C. novyi NT. Following intravenous administration, the C. novyi NT spores germinated in the avascular regions of tumors in mice, causing damage to the surrounding viable tumor [16]. Combined with conventional chemotherapy or radiotherapy, intravenous C. novyi NT spores caused extensive tumor damage within 24 h [16].

Following attenuation by purine and other auxotrophic mutations, the facultative anaerobe S. typhimurium was used for cancer therapy 11, 17, 18. These genetically-modified bacteria replicated in tumors to levels more than 1000-fold greater than in normal tissue [11]. S. typhimurium was further modified genetically by disrupting the msbB gene to reduce the incidence of septic shock [11]. The msbB mutant of S. typhimurium has been tested in a Phase I clinical trial on metastatic melanoma and metastatic renal cell carcinoma patients [19]. To raise the therapeutic index, S. typhimurium was further attenuated by deletion of the purI and msbB genes [19]. The new strain of S. typhimurium, termed VNP20009, could then be safely administered to patients [19]. More studies are needed to completely characterize the safety and efficacy of the bacteria and to improve its therapeutic index.

Mengesha et al. utilized S. typhimurium as a vector for gene delivery by developing a hypoxia-inducible promoter (HIP-1) to limit gene expression to hypoxic tumors. HIP-1 was able to drive gene expression in bacteria residing in human tumor xenografts implanted in mice [20]. Genes linked to HIP-1 showed selective expression in tumors [20]. Yu et al. used green fluorescent protein (GFP)-labeled bacteria to visualize tumor-targeting abilities of 3 pathogens: Vibrio cholerae, S. typhimurium and Listeria monocytogenes 21, 22.

We initially developed a strain of S. typhimurium, termed A1, which selectively grew in tumors in mouse models of human cancer. In contrast, normal tissue rapidly cleared infecting bacteria, even in immunodeficient athymic mice. S. typhimurium A1 is auxotrophic (leu/arg-dependent), but receives sufficient support from tumor tissue. In vivo, the bacteria were previously shown to cause PC-3 human prostate cancer growth inhibition and regression in subcutaneous xenografts in nude mice [23]. To increase the tumor-targeting capability of S. typhimurium A1, the strain was reisolated after infection of a human colon tumor growing in nude mice. We previously showed that the tumor-isolated strain, termed A1-R, had increased tumor targeting ability both in vitro and in vivo. We also showed previously that A1-R demonstrated efficacy in the treatment of mouse models of orthotopic human breast cancer [24] and orthotopic mouse models of human prostate cancer [25]. In the present study, we utilized a mouse model of pancreatic cancer liver metastasis to evaluate the therapeutic efficacy of S. typhimurium A1-R.

Section snippets

GFP Gene Transfection of S. typhimurium [23]

S. typhimurium (ATCC 14028) was grown at 37 °C to midlogarithmic phase in liquid LB and harvested at 4 °C. Bacteria (2.0 × 108) in 40 μL of 10% glycerol were mixed with 2 μL of pGFP vector (Clontech, Mountain View, CA) containing the hr-GFP gene (Stratagene, La Jolla, CA) and placed on ice for 5 min before electroporation with a gene pulser apparatus (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Electroporation was done at 1.8 kV.

Induction of Bacterial Mutations with Nitrosoguanidine (NTG) and Selection for Auxotrophs [23]

Freshly prepared NTG (1 mg/mL in sterile water)

Intrasplenic Injection of XPA-1 RFP P5 Human Pancreatic Cancer Cells Creates Liver Metastasis in Nude Mice

As described in the Materials and Methods section, a total of 50 nude mice were each given an intrasplenic injection of XPA-1 RFP P5B or P5A cell suspension. Post-procedural survival was 98%. There was one perioperative death. Tumor take in the livers of the untreated controls was 100% as seen at the conclusion of each experiment.

A1-R delivered Via Intrasplenic Injection Suppresses XPA-1 RFP P5B Tumor Growth in the Liver and Spleen

GFP-labeled S. typhimurium A1-R bacteria were delivered via operative intrasplenic injection as described above. The 4 treated mice received weekly intrasplenic S.

Discussion

From the results described above, it is clear that S. typhimurium A1-R is effective in inhibiting the growth and development of pancreatic cancer in nude mice. The most common metastatic site of pancreatic cancer is the liver. The present study showed that both intrasplenic and intravenous S. typhimurium A1-R had a dramatic effect on liver metastasis in our mouse model of pancreatic cancer. The intrasplenic delivery of bacteria seemed to achieve more consistent results. The greater variation in

Acknowledgments

This study was supported in part by the National Institutes of Health, National Cancer Institute grant numbers CA119841 and CA126023 and the U.S. Army Medical Research and Materiel Command, Department of Defense Prostate Cancer Research Program grant number W81XWH-06-1-0117.

References (29)

  • W.B. Coley

    Late results of the treatment of inoperable sarcoma by the mixed toxins of erysipelas and Bacillus prodigiosus

    Am J Med Sci

    (1906)
  • R.A. Malmgren et al.

    Localization of the vegetative form of Clostridium tetani in mouse tumors following intravenous spore administration

    Cancer Res

    (1955)
  • D. Gericke et al.

    Oncolysis by Clostridia. II. Experiments on a tumor spectrum with a variety of Clostridia in combination with heavy metal

    Cancer Res

    (1964)
  • J.R. Moese et al.

    Oncolysis by Clostridia. I. Activity of Clostridium Butyricum (M-55) and other nonpathogenic Clostridia against the Ehrlich Carcinoma

    Cancer Res

    (1964)
  • E.H. Thiele et al.

    Oncolysis by Clostridia III. Effects of Clostridia and chemotherapeutic agents on rodent tumors

    Cancer Res

    (1964)
  • Y. Kohwi et al.

    Antitumor effect of Bifidobacterium infantis in mice

    Gann

    (1978)
  • N.T. Kimura et al.

    Selective localization and growth of Bifidobacterium bifidum in mouse tumors following intravenous administration

    Cancer Res

    (1980)
  • M.E. Fox et al.

    Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered Clostridia

    Gene Ther

    (1996)
  • M.J. Lemmon et al.

    Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment

    Gene Ther

    (1997)
  • J.M. Brown et al.

    The unique physiology of solid tumors: Opportunities (and problems) for cancer therapy

    Cancer Res

    (1998)
  • K.B. Low et al.

    Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo

    Nat Biotechnol

    (1999)
  • C. Clairmont et al.

    Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium

    J Infect Dis

    (2000)
  • M. Sznol et al.

    Use of preferentially replicating bacteria for the treatment of cancer

    J Clin Invest

    (2000)
  • K. Yazawa et al.

    Bifidobacterium longum as a delivery system for cancer gene therapy: Selective localization and growth in hypoxic tumors

    Cancer Gene Ther

    (2000)
  • Cited by (0)

    This study was supported in part by the National Institutes of Health, National Cancer Institute grant numbers CA119841 and CA126023 and the U.S. Army Medical Research and Materiel Command, Department of Defense Prostate Cancer Research Program grant number W81XWH-06-1-0117.

    View full text