PhD Student, Nanyang Technological University, Singapore
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.
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
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
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
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
Abstract: Preliminary experiments showed that MC-1 magnetotactic bacteria (MTB) could be used for the delivery of therapeutic agents to tumoral lesions. Each bacterium can provide a significant thrust propulsion force generated by two flagella bundles exceeding 4pN. Furthermore, a chain of single-domain magnetosomes embedded in the cell allows computer directional control and tracking using a magnetic resonance imaging (MRI) system. Although these embedded functionalities suggest that MTB when under the influence of an external computer could be considered as biological microrobots with the potential of targeting tumors, little is known about their level of penetration in tumoral tissues. In this paper, in vitro experiments were performed to assess the capability of these bacteria to penetrate tumor tissue for the delivery of therapeutic agents. Multicellular tumor spheroids were used since they reproduce many properties of solid tumors. The results show the ability of these MTB when submitted to a directional magnetic field to penetrate inside a 3D multicellular tumor spheroid through openings present in the tissue.
Pub.: 26 Nov '10, Pinned: 19 Nov '17
Abstract: Acute bacterial infections have beneficial effects on tumor patients. To eliminate side effects evoked by viable microbes, we here assessed the immunotherapeutic potential of inactivated bacteria on colorectal carcinomas. Our In vitro results indicate a cell-specific direct cytotoxicity towards tumor cells presented by G1-arrest. Antitumoral activity was boosted in the presence of leukocytes. Long time stimulations revealed massive activation of NK cells even in complete autologous settings. In vivo, repetitive local treatment mediated tumor growth control. Evaluation of residual tumors identified increased infiltrates, with NK cells (CD49b(+), NKG2D(+)) being the main responding cell population. Substantial NK cell-mediated delay of tumor growth was also achieved in T-cell deficient mice xenografted with human colorectal carcinomas. Of note, local as well as systemic therapy mediated tumor growth control. These data highlight the potential of avitalized bacteria to especially activate the immune system's innate arm and they should be considered for future integrated immunotherapy.
Pub.: 06 Apr '11, Pinned: 19 Nov '17
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
Abstract: Genetically engineered bacteria have the potential to diagnose and treat a wide range of diseases linked to the gastrointestinal tract, or gut. Such engineered microbes will be less expensive and invasive than current diagnostics and more effective and safe than current therapeutics. Recent advances in synthetic biology have dramatically improved the reliability with which bacteria can be engineered with the sensors, genetic circuits, and output (actuator) genes necessary for diagnostic and therapeutic functions. However, to deploy such bacteria in vivo, researchers must identify appropriate gut-adapted strains and consider performance metrics such as sensor detection thresholds, circuit computation speed, growth rate effects, and the evolutionary stability of engineered genetic systems. Other recent reviews have focused on engineering bacteria to target cancer or genetically modifying the endogenous gut microbiota in situ. Here, we develop a standard approach for engineering "smart probiotics," which both diagnose and treat disease, as well as "diagnostic gut bacteria" and "drug factory probiotics," which perform only the former and latter function, respectively. We focus on the use of cutting-edge synthetic biology tools, gut-specific design considerations, and current and future engineering challenges.
Pub.: 21 Oct '17, Pinned: 19 Nov '17
Abstract: The idea of using killed microorganisms or their parts for a stimulation of immunity in the cancer immunotherapy is very old, but the question of interactions and binding of these preparations to tumor cells has not been addressed so far. The attachment of Zymosan A and both Gram-positive and Gram-negative bacteria to tumor cells was tested in in vivo experiments. This binding was accomplished by charge interactions, anchoring based on hydrophobic chains and covalent bonds and proved to be crucial for a strong immunotherapeutic effect. The establishment of conditions for simultaneous stimulation of both Toll-like and phagocytic receptors led to very strong synergy. It resulted in tumor shrinkage and its temporary or permanent elimination. The role of neutrophils in cancer immunotherapy was demonstrated and the mechanism of their action (frustrated phagocytosis) was proposed. Finally, therapeutic approaches applicable for safe human cancer immunotherapy are discussed. Heat killed Mycobacterium tuberculosis covalently attached to tumor cells seems to be promising tool for this therapy.
Pub.: 06 Aug '16, Pinned: 19 Nov '17
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
Abstract: Increasing the specificity of chemotherapy may improve the efficacy of cancer treatment. Toward this aim, we developed a strain of bacteria to express enzymes for selective prodrug activation and non-invasive imaging in tumors. beta-glucuronidase and the luxCDABE gene cluster were expressed in the DH5alpha strain of Escherichia coli to generate DH5alpha-lux/betaG. These bacteria emitted light for imaging and hydrolyzed the glucuronide prodrug 9ACG to the topoisomerase I inhibitor 9-aminocamptothecin (9AC). By optical imaging, colony-forming units (CFUs) and staining for betaG activity, we found that DH5alpha-lux/betaG preferentially localized and replicated within CL1-5 human lung tumors in mice. The intensity of luminescence, CFU and betaG activity increased with time, indicating bacterial replication occurred in tumors. In comparison with DH5alpha-lux/betaG, 9AC or 9ACG treatment, combined systemic administration of DH5alpha-lux/betaG followed by 9ACG prodrug treatment significantly (P<0.005) delayed the growth of CL1-5 tumors. Our results demonstrate that prodrug-activating bacteria may be useful for selective cancer chemotherapy.
Pub.: 29 Mar '08, Pinned: 19 Nov '17