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CURATOR
A pinboard by
Madhura Bhave

PhD Student, Nanyang Technological University, Singapore

PINBOARD SUMMARY

An innovative form of bacterial therapy using non-viable derivatives of Clostridium sporogenes

Despite the advancement in science, traditional cancer treatments such as chemotherapy and radiation therapy are still limited in their efficacy by features of the tumor microenvironment, such as hypoxia and multi-drug resistance. Bacterial cancer therapy has garnered a lot of interest in recent times as it has the potential to overcome both these factors. However, most bacterial therapy uses live bacteria or spores, which poses the risk infection and toxicity. To address these limitations, we developed two non-viable derivatives of a non-pathogenic and non-toxic bacterium, Clostridium sporogenes. These derivatives, heat-inactivated bacteria and conditioned media containing secreted bacterial products, were tested for their anti-cancer efficacy in vitro on novel 3-dimensional spheroid model of colorectal cancer cells. After the two derivatives were found to effectively inhibit the growth of the cancer cells in the 3D spheroid model, they were tested in vivo in immunocompetent mice bearing colorectal tumors. It was found that the derivatives inhibit the growth of tumors in the mice and also improve the survival rates. Notably, the heat-inactivated bacteria were found to stimulate the immune system such that the treated mice rejected future tumor establishment. My work in this field disrupts the conventional approach to treating cancer, and has contributed to creating a whole host of new possibilities in the fight against cancer. I hope to continue working on understanding the role of the immune system in response to the heat-inactivated bacteria treatment and elucidating the mechanism of the inhibitive effect of both the derivatives.

11 ITEMS PINNED

Escherichia coli Nissle 1917 facilitates tumor detection by positron emission tomography and optical imaging.

Abstract: Bacteria-based tumor-targeted therapy is a modality of growing interest in anticancer strategies. Imaging bacteria specifically targeting and replicating within tumors using radiotracer techniques and optical imaging can provide confirmation of successful colonization of malignant tissue.The uptake of radiolabeled pyrimidine nucleoside analogues and [18F]FDG by Escherichia coli Nissle 1917 (EcN) was assessed both in vitro and in vivo. The targeting of EcN to 4T1 breast tumors was monitored by positron emission tomography (PET) and optical imaging. The accumulation of radiotracer in the tumors was correlated with the number of bacteria. Optical imaging based on bioluminescence was done using EcN bacteria that encode luciferase genes under the control of an l-arabinose-inducible P(BAD) promoter system.We showed that EcN can be detected using radiolabeled pyrimidine nucleoside analogues, [18F]FDG and PET. Importantly, this imaging paradigm does not require transformation of the bacterium with a reporter gene. Imaging with [18F]FDG provided lower contrast than [18F]FEAU due to high FDG accumulation in control (nontreated) tumors and surrounding tissues. A linear correlation was shown between the number of viable bacteria in tumors and the accumulation of [18F]FEAU, but not [18F]FDG. The presence of EcN was also confirmed by bioluminescence imaging.EcN can be imaged by PET, based on the expression of endogenous E. coli thymidine kinase, and this imaging paradigm could be translated to patient studies for the detection of solid tumors. Bioluminescence imaging provides a low-cost alternative to PET imaging in small animals.

Pub.: 29 Mar '08, Pinned: 19 Nov '17

Salmonella promoters preferentially activated inside tumors.

Abstract: Salmonella enterica and avirulent derivatives prefer solid tumors over normal tissue in animal models. The identification of endogenous Salmonella promoters that are preferentially activated in tumors could further our understanding of this phenomenon. Toward this goal, a random library of S. enterica typhimurium 14028 genomic DNA was cloned upstream of a promoterless gene encoding the green fluorescent protein (GFP) TurboGFP. A population of Salmonella containing this library was injected i.v. into tumor-free nude mice and into human PC3 prostate tumors growing subcutaneously in nude mice. After 2 days, fluorescence-activated cell sorting was used to enrich for bacterial clones expressing GFP from spleens or tumors. The resulting libraries were hybridized to an oligonucleotide tiling array of the Salmonella genome. Eighty-six intergenic regions were found to be enriched in tumor samples but not in spleen. Twenty of these candidate promoters were also detected in the sequences of 100 random clones from a library enriched for expression in bacteria growing in tumors. Three candidate promoter clones were individually tested in vivo, and enhanced GFP expression in bacteria growing in tumor relative to spleen was confirmed. Two of the three clones (pflE and ansB promoter regions) are known to be induced in hypoxic conditions that pertain to many tumors. For many of the other candidate promoters preferentially induced in bacteria growing in tumors, regulatory mechanisms may not be related to hypoxia. The expression of therapeutics in Salmonella under the regulation of one or more promoters that are activated preferentially in tumors has the potential to improve the targeting of drug delivery.

Pub.: 19 Jun '08, Pinned: 19 Nov '17

Bacterial delivery of siRNAs: a new approach to solid tumor therapy.

Abstract: RNAi is a powerful research tool for specific gene silencing and may also lead to promising novel therapeutic strategies. However, the development of RNAi-based therapies has been slow due to the lack of targeted delivery methods. The biggest challenge in the use of siRNA-based therapies is the delivery to target cells. There are many additional obstacles to in vivo delivery of siRNAs, such as degradation by endogenous enzymes and interaction with blood components leading to nonspecific uptake into cells, which govern biodistribution and availability of siRNA in the body. Naked unmodified synthetic siRNA including plasmid-carried-shRNA-expression constructs cannot penetrate cellular membranes, and therefore, systemic application is unlikely to be successful. The success of gene therapy by siRNAs relies on the development of safe, economical, and efficacious in vivo delivery systems into the target cells. Attenuated Salmonella have been employed recently as vectors to deliver silencing hairpin RNA (shRNA) expression plasmids into mammalian cells. This approach has achieved gene silencing in vitro and in vivo. The facultative anaerobic, invasive Salmonella have a natural tropism for solid tumors including metastatic tumors. Genetically modified, attenuated Salmonella have been used recently both as potential antitumor agents by themselves, and to deliver specific tumoricidal therapies. This chapter describes the use of attenuated bacteria as tumor-targeting delivery systems for cancer therapy.

Pub.: 24 Mar '09, Pinned: 19 Nov '17

Inflammation and immunity in the tumor environment.

Abstract: The relationship between inflammation, innate immunity and cancer is widely accepted. Cancer-associated inflammation includes infiltrating leukocytes, cytokines, chemokines, growth factors, lipid messengers and matrix-degrading enzymes. Tumor-associated macrophages and lymphocyte subpopulations are major components of the leukocyte infiltrate in most tumors. However, the cytokine and chemokine expression profile of the tumor microenvironment may be more relevant than its specific immune cell content. Apart from inflammatory cells, tumor stroma consists of new blood vessels and connective tissue. Many factors produced by tumor cells promote tumor angiogenesis and generation of extracellular matrix. Investigations regarding the link between inflammation and cancer are vital for identifying cell or protein targets for cancer prevention and therapy. Based on the relation between inflammation and cancer, different forms of immunotherapy have been developed. In a mouse model, we investigated the potential of Streptococcus pyogenes to achieve a bacteria-related immune response against tumor cells followed by tumor regression. As a model of pancreatic carcinoma, the aggressively growing and poorly immunogenic Panc02 tumor model was chosen. Our findings showed that a local application of bacteria mediates complete tumor regression. Future investigations should focus on the optimization of immunotherapeutic approaches that incorporate live bacteria or bacterial components.

Pub.: 23 Nov '10, Pinned: 19 Nov '17

Bacterial-mediated knockdown of tumor resistance to an oncolytic virus enhances therapy.

Abstract: Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.

Pub.: 27 Feb '14, Pinned: 19 Nov '17

Impediments to enhancement of CPT-11 anticancer activity by E. coli directed beta-glucuronidase therapy.

Abstract: CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.

Pub.: 18 Feb '15, Pinned: 19 Nov '17