Recent advances in cancer therapeutics, such as targeted therapy and immunotherapy, have raised the hope for cures for many cancer types. However, there are still ongoing challenges to the pursuit of novel therapeutic approaches, including high toxicity to normal tissue and cells, difficulties in treating deep tumor tissue, and the possibility of drug resistance in tumor cells. The use of live tumor-targeting bacteria provides a unique therapeutic option that meets these challenges. Compared with most other therapeutics, tumor-targeting bacteria have versatile capabilities for suppressing cancer. Bacteria preferentially accumulate and proliferate within tumors, where they can initiate antitumor immune responses. Bacteria can be further programmed via simple genetic manipulation or sophisticated synthetic bioengineering to produce and deliver anticancer agents based on clinical needs. Therapeutic approaches using live tumor-targeting bacteria can be applied either as a monotherapy or in combination with other anticancer therapies to achieve better clinical outcomes. In this review, we introduce and summarize the potential benefits and challenges of this anticancer approach. We further discuss how live bacteria interact with tumor microenvironments to induce tumor regression. We also provide examples of different methods for engineering bacteria to improve efficacy and safety. Finally, we introduce past and ongoing clinical trials involving tumor-targeting bacteria.

Surface-exposed calreticulin (ecto-CRT) is a well-known "eat-me" signal exhibited by dying cells that contributes to their recognition and destruction by the immune system. We assessed the use of a CRT-specific binding peptide for imaging ecto-CRT during immunogenic cell death and its utility for early prediction of treatment response. Methods: A synthetic CRT-specific peptide, KLGFFKR (CRTpep), was labeled with fluorescein isothiocyanate or 18 F, and the characteristics of ecto-CRT were evaluated in a colon cancer cell line in vitro and in vivo. Results: In vitro flow cytometry, immunofluorescence staining, and in vivo small-animal PET imaging results showed that CRTpep detected preapoptotic cells treated with immunogenic drugs or radiation but not those treated with the nonimmunogenic drug or a nontherapeutic dose of immunogenic drug. Conclusion:The present results indicate that the CRT-specific peptide would enable the prediction of therapeutic response, thereby facilitating early decisions on continuation or discontinuation of immunogenic treatment.

Specific tags with defined amino acid residues are widely used to purify or probe target proteins. Interestingly, the tagging system occasionally results in an increase of the recombinant protein expression in vivo. Here, we systematically examined this phenomenon using a poly-histidine (His)-tag fused to N- or C-terminal region of green, red, and blue fluorescent proteins by quantification and uneven distribution in cytoplasm of Escherichia coli. This effect was further supported by the distinct over-expression of several unrelated proteins, such as esterase, neopullulanase, and chloramphenicol acetyltransferase, tagging with the same tag. These results suggest that a poly-His-tag placed at N-terminal region can induce over-expression of recombinant protein via subcellular uneven distribution in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.