Blautia coccoides JCM1395T Achieved Intratumoral Growth with Minimal Inflammation: Evidence for Live Bacterial Therapeutic Potential by an Optimized Sample Preparation and Colony PCR Method.

We demonstrate that Blautia coccoides JCM1395T has the potential to be used for tumor-targeted live bacterial therapeutics. Prior to studying its in vivo biodistribution, a sample preparation method for reliable quantitative analysis of bacteria in biological tissues was required. Gram-positive bacteria have a thick outer layer of peptidoglycans, which hindered the extraction of 16S rRNA genes for colony PCR. We developed the following method to solve the issue; the method we developed is as follows. The homogenates of the isolated tissue were seeded on agar medium, and bacteria were isolated as colonies. Each colony was heat-treated, crushed with glass beads, and further treated with restriction enzymes to cleave DNAs for colony PCR. With this method, Blautia coccoides JCM1395T and Bacteroides vulgatus JCM5826T were individually detected from tumors in mice intravenously receiving their mixture. Since this method is very simple and reproducible, and does not involve any genetic modification, it can be applied to exploring a wide range of bacterial species. We especially demonstrate that Blautia coccoides JCM1395T efficiently proliferate in tumors when intravenously injected into tumor-bearing mice. Furthermore, these bacteria showed minimal innate immunological responses, i.e., elevated serum tumor necrosis factor α and interleukin-6, similar to Bifidobacterium sp., which was previously studied as a therapeutic agent with a small immunostimulating effect.

Impact of tumoral structure and bacterial species on growth and biodistribution of live bacterial therapeutics in xenografted tumours.

Live bacterial therapeutics is gaining attention, especially for cancer therapy, because anaerobic bacteria selectively grow inside the solid tumours. However, the effect of tumour structure and bacterial characteristics on the pharmacokinetics of tumours is unclear; therefore, we aimed to elucidate the effects of tumour structure and types of bacteria on tumoral bacterial growth. Using six mouse xenograft models, including stroma-rich tumours similar to clinical tumours, and two models of live bacterial therapeutics, Salmonella typhimurium VNP20009 and Escherichia coli DH5α, we investigated bacterial growth and distribution in tumours after intravenous administration. Rapid growth of E. coli was observed in HCT116 and other tumours with few collagens, blood vessels not covered by mural cells, and a cancer cell area proliferated disorderly, whereas tumours with contrasting features, such as BxPC-3, showed lower bacterial growth and a limited intratumor distribution. Alternatively, Salmonella typhimurium VNP20009, when successfully proliferated (the probability was approximately 50%), grew to 108 colony forming units/g tissue even in BxPC-3 tumours, and its intratumor distribution was extensive. This study suggests that the development of new methods to modify tumour structure will be essential for the development of anti-tumour clinical therapies based on live bacterial therapeutics.

Biosorption-based 64Cu-labeling of bacteria for pharmacokinetic positron-emission tomography.

Biosorption-based bacterial 64Cu-labeling and its application in pharmacokinetic positron-emission tomography (PET) were investigated. Both gram-positive and gram-negative bacteria were efficiently labeled with [64Cu]Cu2+ ion in saline at room temperature within 5 min. The labeling ratio for Escherichia coli drastically decreased with trypsin pretreatment and the co-presence of excess Cu2+ ion, indicating the existence of specific Cu2+ binding sites on the E. coli cell surface. Washing with lysogeny broth medium was effective in purifying 64Cu-labeled E. coli for kinetic study; the labeling stability was approximately 90% in serum for 15 min. According to dynamic PET imaging in colon-26 tumor-bearing mice, 64Cu-labeled E. coli immediately disappeared from the blood circulation and primarily accumulated in the liver. In addition, transient pulmonary distribution was observed, being in a dose-dependently accelerated manner. Considering the simplicity and versatility of biosorption-based bacterial 64Cu-labeling without genetic modification, the early-phase pharmacokinetic PET with 64Cu-labeled bacteria is promising for assessing toxicological aspects of bacteria-mediated cancer therapy as well as a variety of bacterial pathogenicities in infectious diseases.