Breast Cancer Vol. 13 No.

1 January 2006

Review Article
Genetically Engineered Bifidobacterium as a Drug Delivery System for Systemic Therapy of Metastatic Breast Cancer Patients
Minoru Fujimori
Division of Breast and Endocrine Surgery, Shinshu University School of Medicine, Japan. A fundamental obstacle in systemic therapy for metastatic breast cancer patients is specific targeting of therapy directly to a solid tumor. Hypoxic or necrotic regions are characteristic of solid tumors in many murine and human tumors, including the majority of primary tumors of the breast. A strain of anaerobic bacteria such as Bifidobacterium or Clostridium selectively localizes to and proliferates in solid tumors after systemic application. Another approach uses attenuated Salmonella strains that need tumor-specific nutrients to selectively proliferate and is a potential gene delivery system. We constructed a plasmid, pBLES100-S-eCD, which included the cytosine deaminase gene. Transfected Bifidobacterium longum produced cytosine deaminase in the hypoxic tumor. Enzyme/pro-drug therapy was confirmed to be effective for systemic administration. Breast Cancer 13:27-31, 2006. Key words: Genetically engineered bacteria, Tumor targeting, Hypoxia

Advanced or recurrent breast cancer is basically a generalized disease, so whole-body therapy is key post of the overall treatment strategy. One of the major limitations of conventional chemotherapy for breast cancer patients is the toxicity associated with the lack of specificity of drugs for tumor cells. Hypoxic or necrotic regions are characteristic of solid tumors in many murine and human tumors, including the majority of primary tumors of the breast and uterine cervix 1-3). Tissue oxygen electrode measurements taken in cancer patients show a median range of oxygen partial pressure of 10 to 30 mm Hg in tumors, with a significant proportion of readings below 2.5 mm Hg, whereas those in normal tissues range from 24 to 66 mm Hg 4). Cancer gene therapy approaches to solid tumor treatment have been limited by the ability of the delivery vectors to achieve specific high-level expression within tumor tissues or the tumor environment following systemic administration. Accordingly, gene therapy for solid tumors that exploits and targets gene expression in hypoxic tumor cells is currently being investigated 5). It is known that certain species of anaerobic bacteria,
Reprint requests to Minoru Fujimori, Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. E-mail: minoru1@hsp.md.shinshu-u.ac.jp

including the genera Clostridium and Bifidobacterium, can selectively germinate and grow in the hypoxic regions of solid tumors after intravenous injection 6, 7). To be able to exploit the potential of these bacteria for cancer gene therapy, detailed knowledge is required about such basic biological phenomena as cellular metabolism, gene expression, protein secretion and genetics. However, little is known about the genetic properties of the genus Bifidobacterium, mainly due to the lack of efficient and reproducible systems for genetic transfer and adequate selectable markers. In recent years, a convenient system for reproducible genetic transformation of strains of the genus Bifidobacterium has been developed 8, 9). Bifidobacterium longum (B. longum) can selectively germinate and grow in the hypoxic regions of solid tumors after intravenous injection 10). We proposed a new approach involving the genetically engineered B. longum for enzyme-prodrug therapy. We chose to use the combination of cytosine deaminase and 5-fluorocytosine (5FC) in initial studies of the feasibility of this strategy. The cellular toxicities of 5FC result from its deamination by the enzyme cytosine deaminase to give 5-fluorouracil (5FU).

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The Genus Bifidobacterium for Drug Delivery System

Fig 1Organ distribution of B. longum 105-A and 108-A after single i.v. administration of 6 10 6 cfu into tumor-bearing mice. Each point represents the mean of the number of bacilli per gram of tissue of six to eight mice.

Selective Growth of Bifidobacterium longum in Tumor Tissues

Male C57BL/6 mice (6 to 8 weeks old) and female Sprague-Dawley rats (6 weeks old) were used in a study of the effect of B. longum on tumor growth. Approximately 5 10 5 tumor cells (Lewis lung cancer and B16-F10 melanoma) were inoculated into the right thigh muscle of mice, and the resulting solid tumors were obtained 2 weeks later. For autochthonous tumors, rats were given 10 mg of 7,12-dimethylbenz[a]-anthracene (DMBA) by weekly intragastric gavage for 2 weeks. At 23 weeks after the first dose of DMBA, 89% of rats developed mammary tumors. B. longum was then injected into the tail vein of the animals (6 10 6/ mouse; 2 10 8 cfu/rat). The mice were sacrificed at 24, 48, 96 or 168 h after injection of B. longum, and the tumors and normal tissues were excised and homogenized. The diluted tissue homogenates were then cultured under anaerobic conditions. On day 3 of culture, the number of colonies per dish and the number of two strains of B. longum organisms (B. longum 105-A and B. longum 108-A)
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per gram of tissue after intravenous administration of bacilli into mice bearing Lewis lung cancer were determined. At 168 h, tumors had approximately 6 10 4 cfu/g of tumor tissue, regardless of the bacterial strain. In contrast, the number of B. longum 105-A and B. longum 108-A bacilli in normal tissues, such as the liver, spleen, kidney and lung, decreased immediately after injection and were below detectable levels after 168 h and 96 h, respectively (Fig 1). Colonies only developed on the agar plates inoculated with tumor tissues (Fig 2)10). Similar results were also obtained in DMBA-induced mammary tumors in rats. The detectability of bacilli in the tumors following a single administration of transformed 2 10 8 B. longum 105-A was examined in relation to the tumor size. B. longum was detected in all tumors with a volume size of 62.5 mm3 11). Mammary carcinomas developing in SpragueDawley rats after DMBA administration represent a classic model of autologous carcinogenesis 12, 13). Metastasized or disseminated lesions as well as primary disease should be amenable to this treatment as long as regions of hypoxia are present. Furthermore, detection of a primary tumor locus

Breast Cancer Vol. 13 No. 1 January 2006

Fig 3Molecular structure of pBLES100-S-eCD.

Fig 2Comparison of the number of genetically engineered B. longum 105-A in both tumors and normal tissues from mice after 168 hours.

CD:cytosine deaminase gene B.longum

Transform Cancer cells

5FU
5FC :Nontoxic

:Toxic
Anticancer effect

Fig 4 Schematic representation of Bifidobacterial Selective Targeting - Cytosine Deaminase (BEST-CD) therapy.

or of a metastatic focus when tumor diameters were more than about 0.5 cm (tumor volume 62.5 mm3), as shown in our results, may be diagnostically feasible with the transformation of a suitable marker gene. These results suggested that the only requirement for success of this tumor targeting therapy strategy should be the presence of hypoxia in the treated tumors.

Bifidobacterial Selective Targeting - Cytosine Deaminase (BEST-CD) Therapy

B. longum is an effective hypoxic tumor-specif-

ic vector and potentially may be utilize for enzymeprodrug therapy. Enzyme-prodrug therapy is being developed as treatment for cancer and other pathological conditions. To measure the feasibility of this strategy, cytosine deaminase (CD) and 5fluorocytosine (5-FC) were chosen for the initial studies. To treatment of solid tumors by systemic administration of 5-FU has some disadvantages. First, it is necessary to administer large doses of 5-FU to the whole body to distribute a sufficiently dense concentration of 5-FU to the tumor. Second, various side effects accompany massive doses of 5-FU. 5-FC is a less toxic prodrug than 5-FU, and therefore it is possible to administer larger doses
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The Genus Bifidobacterium for Drug Delivery System

of 5-FC. If CD, which converts 5-FC into 5-FU, exists only in the tumor tissue, it might be possible to obtain high-concentration of 5-FU without simultaneous distribution among normal tissues. A plasmid, pBLES100-S-eCD, was constructed to adapt enzyme-prodrug therapy to the Bifidobacterial tumor-specific gene delivery system, which included the HU gene promoter and the gene encoding the Cytosine deaminase of E. coli into the B. longum-E. coli shuttle vector pBLES100 (Fig 3). It has been proven that HU gene that encodes a histon like DNA binding protein highly express in B. longum14). We proposed a new approach involving the genetically engineered B. longum for enzyme-prodrug therapy to use the combination of cytosine deaminase and 5-fluorocytosine (5FC) (Fig 4)15). This strategy has been named as Bifidobacterial Selective Targeting - Cytosine Deaminase (BEST-CD) therapy. Some bacterial vectors for cancer gene therapy have been reported. In such cases, bacteria proliferate between tumor cells and thus the therapeutic gene is not introduced into the tumor cell, but rather the therapeutic protein is produced in the tumor. This mechanism differentiates enzyme-prodrug therapy from what is called gene therapy, which uses virus and liposome vectors. Candidate bacterial vectors for gene introduction include species of Bifidobacterium, Clostridium and Salmonella 16-20). Engineered Clostridium acetobutylicum has been reported as particularly useful for enzyme-prodrug therapy 21). However, Clostridium and Salmonella are pathogenic 22, 23). In contrast, Bifidobacterium is a normal bacterial flora in the intestine, and is the nonpathogenic. In addition, the results of antigenicity tests of B. longum in guinea pigs have suggested that systemic administration of B. longum is as safe as injection of saline. Moreover, we have observed no adverse effects of intravenous administration of B. longum in dogs and cynomolgus monkeys (data not shown). It is our belief that BEST-CD therapy can be use to tumor targeting therapy of metastatic breast cancer patients with systemic disease.

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