UNIVERSITY OF GONDAR COLLEGE OF MEDICINE AND HEALTH SCIENCES SCHOOL OF BIOMEDICAL AND LABORATORY SCIENCES DEPARTMENT OF MEDICAL MICROBIOLOGY TITLE OF SEMINAR

UNIVERSITY OF GONDAR
COLLEGE OF MEDICINE AND HEALTH SCIENCES
SCHOOL OF BIOMEDICAL AND LABORATORY SCIENCES
DEPARTMENT OF MEDICAL MICROBIOLOGY
TITLE OF SEMINAR: BACTERIA IN CANCER THERAPY
BY: MICHAEL GETIE
ADVISORS: Dr. BELAY TESSEMA (MSc, PhD)
Mr. WONDWOSSEN ABEBE (MSc)
A SEMINAR PAPER SUBMITTED TO DEPARTMENT OF MEDICAL MICROBIOLOGY, SCHOOL OF BIOMEDICAL AND LABORATORY SCINCE, UNIVERSTITY OF GONDAR FOR FULFILLMENT OF THE REQUIREMENT FOR SEMINAR I ON CURRENT TOPICS IN MEDICAL MICROBIOLOGY COURSE (MBIO711)

MAY, 2018
GONDAR, ETHIOPIA
AcknowledgmentsI would like to express my special thanks to my Advisors Dr. Belay Tessema and Mr. Wondwossen Abebe for their constructive advice, guidance, and encouragement given for me during the preparation of this seminar. I would also like to extend my special thanks to the Department of Medical Microbiology, School of Biomedical and Laboratory Sciences, College of Medicine and Health, University of Gondar for giving me the opportunity to conduct this seminar work.
Table of contents TOC o “1-3” h z u HYPERLINK l “_Toc514823849” Acknowledgments PAGEREF _Toc514823849 h I
Table of contents PAGEREF _Toc514823850 h IIList of Abbreviations PAGEREF _Toc514823851 h IIIList of figures and tables PAGEREF _Toc514823852 h IVAbstract PAGEREF _Toc514823853 h V1. Introduction PAGEREF _Toc514823854 h 12. Features of Bacteria Provide Benefits for Cancer Treatment PAGEREF _Toc514823855 h 32.1. Sensing the tumor microenvironment PAGEREF _Toc514823856 h 32.2. Motility PAGEREF _Toc514823857 h 32.3. Active delivery PAGEREF _Toc514823858 h 42.4. Controlled propagation PAGEREF _Toc514823859 h 42.5. Immune stimulation PAGEREF _Toc514823860 h 53. The benefits of bacterial cancer therapy PAGEREF _Toc514823861 h 53.1. Bacterial toxins PAGEREF _Toc514823862 h 63.1.1. Immunotoxins PAGEREF _Toc514823863 h 63.1.2. Escherichia coli toxins PAGEREF _Toc514823864 h 63.1.3. Type III secretion systems PAGEREF _Toc514823865 h 64. Role of bacteria in cancer gene therapy PAGEREF _Toc514823866 h 73.1.Prodrug approach to cancer gene therapy PAGEREF _Toc514823867 h 73.2.Cytosine deaminase genes PAGEREF _Toc514823868 h 73.3.Escherichia coli purine nucleoside phosphorylase gene PAGEREF _Toc514823869 h 83.4.The Escherichia coli nitro reductase B gene PAGEREF _Toc514823870 h 83.5.The Escherichia coli guanine phosphoribosyl transferase gene PAGEREF _Toc514823871 h 85. Salmonella typhimurium as a treatment option PAGEREF _Toc514823872 h 86. Key features of S. typhimurium that make it suitable for cancer therapy PAGEREF _Toc514823873 h 97. Attenuated strains for cancer therapy PAGEREF _Toc514823874 h 103.6.VNP20009 PAGEREF _Toc514823875 h 113.7.A1-R PAGEREF _Toc514823876 h 113.8.CRC2631 PAGEREF _Toc514823877 h 123.9.Other strains, like, ?ppGpp PAGEREF _Toc514823878 h 127. Strategies for S. typhimurium-mediated cancer therapy PAGEREF _Toc514823879 h 123.10.Native cytotoxicity and combinational therapy PAGEREF _Toc514823880 h 123.11.Expressing Anticancer Agents PAGEREF _Toc514823881 h 133.12.Immunomodulatory molecules PAGEREF _Toc514823882 h 133.13.Salmonella as a Vaccine Vector PAGEREF _Toc514823883 h 138. References PAGEREF _Toc514823884 h 169. Declaration PAGEREF _Toc514823885 h 24
List of AbbreviationsCD: Cluster of Differentiation
CFU: Colony Forming Units
DC: Dendritic Cell(s)
Gb3: Globotriosyl Ceramide
GDEPT: Gene Directed Enzyme Prodrug Therapy
Hly A: ?-Hemolysin
HPV: Human Papilloma Virus
IFN: Interferon
LPS: Lipopolysaccharide
MHC: Major Histocompatibility Complex
mAb: monoclonal Antibody
PAMP: Pathogen-Associated Molecular Pattern
ppGpp: Guanosine tetraphosphate
PSA: Prostate Specific Antigen
Stx: Shiga toxin
TAAs: Tumor Associated Antigens
TAR: Transmembrane Aspartate Receptors
Th1: Helper T Cell
TLR: Toll-like receptor(s)
TNF: Tumor Necrotizing Factor
TRG: T cell Receptor Gamma
VT: VeroToxin
List of figures and tablesTable 1. Overview of candidate live attenuated bacteria strains for cancer treatment…………12
Table 2: Advantages of Salmonella as an anti-tumor agent…………………………………….18
Table 3. Candidate attenuated S. typhimurium strains for targeted cancer therapy…………….19
Table 4. Applications of S. typhimurium as a vaccine vector………………………………….23
AbstractCancer remains one of the major challenges of the 21st century. The increasing numbers of cases are not accompanied by adequate progress in therapy. The standard methods of treatment often do not lead to the expected effects. Therefore, it is extremely important to find new, more effective treatments. One of the most promising research directions is using bacterial as anti-cancer threptic agent. Conventional anti-cancer therapies such as surgery, radiotherapy, and chemotherapy are effective in the treatment of solid tumors only to some extent. Because they are highly toxic, ineffectively target tumors, and poorly penetrate tumor tissue. Those bacteria mentioned above have unique capabilities that make them well-suited as ‘perfect’ anticancer agents. Because their genetics can be easily manipulated, bacteria can be engineered to overcome the limitations that hamper current cancer therapies. The ideal criteria for the selection of therapeutic bacteria are as follows: Nontoxic to the host; selective for a specific type of tumor; has the ability to penetrate deeply into the tumor where ordinary treatment does not reach; nonimmunogenic (does not trigger an immune response immediately but may be cleared by the host); harmless to normal tissue; able to be manipulated easily; and has a drug carrier that may be controlled. Several bacterial strains have been evaluated as cancer therapeutics so far, Salmonella typhimurium being one of the most promising. There are multiple Salmonella strains being developed for targeted chemotherapy delivery, notably VNP20009, A1-R, and CRC2631. S. typhimurium genes are easily manipulated; thus, diverse attenuated strains of S. typhimurium have been designed and engineered as tumor-targeting therapeutics or drug delivery vehicles that show both an excellent safety profile and therapeutic efficacy in mouse models. S. typhimurium and its derivatives have been used both as direct tumoricidal agents and as cancer vaccine vectors.
Keywords: Cancer, bacterial cancer therapy, tumor targeting, Salmonella typhimurium
1. IntroductionCancer remains one of the major challenges of the 21st century. The increasing numbers of cases are not accompanied by adequate progress in therapy. The standard methods of treatment often do not lead to the expected effects. Therefore, it is extremely important to find new, more effective treatments. One of the most promising research directions is using bacterial as anti-cancer threptic agent. At present, cancer has one of the highest morbidity and mortality rates worldwide, nationwide and State wide 1. Cancer has become the second ranking cause of death in USA. It is prognosticated that during the year 2017, more than 1.6 million cases will be registered, which means that more than 4600 cancer cases will be reported every day 2.

Conventional anti-cancer therapies such as surgery, radiotherapy, and chemotherapy are effective in the treatment of solid tumors only to some extent. Because they are highly toxic, ineffectively target tumors, and poorly penetrate tumor tissue. So alternative techniques are being developed to target tumors 3.

Bacteria-mediated cancer therapy was first introduced by William Coley in the mid-19th century, when Streptococcus pyogene was used to treat solid tumors 2. This approach has several advantages over conventional therapies such as chemotherapy: bacteria have the ability to specifically target tumors, they actively proliferate in a variety of malignant tumors, they are easy to manipulate at the genetic level, and they are inexpensive to produce 3. In sum, bacterial cancer therapy has made great strides in past decade and is now considered a tangible option for future cancer therapy. The attenuated bacterial strains of Salmonella typhimurium defective in the synthesis of Guanosine tetraphosphate have tumor-targeting ability and developed diverse bacterial cancer therapy strategies based on these strains 4–6.
Bacteria have unique capabilities that make them well-suited as ‘perfect’ anticancer agents. Because their genetics easily manipulated, bacteria can be engineered to overcome the limitations that hamper current cancer therapies. Many current treatments, including chemotherapy and radiation, are toxic to normal tissue and cannot completely destroy all cancer cells 7. Three major causes of these problems are incomplete tumor targeting, inadequate tissue penetration and limited toxicity to all cancer cells 8. These drawbacks prevent effectual treatment and are associated with increased morbidity and mortality.

Using a top-down engineering approach, the ideal cancer therapy can be envisioned: it would be tiny programmable robot factories that specifically target tumors, are selectively cytotoxic to cancer cells, are self-propelled, are responsive to external signals, can sense the local environmental and are externally detectable. Specific targeting would permit the use of more toxic molecules without systemic effects. Self-propulsion would enable penetration into tumor regions that are inaccessible to passive therapies. Responsiveness to external signals would enable precise control of the location and timing of cytotoxicity. Sensing the local environment would permit “smart,” responsive therapies that can make decisions about where and when drugs are administered. Finally, the ability to be externally detected would provide critical information about the state of the tumor, the success of localization and the efficacy of treatment. The ideal criteria for the selection of therapeutic bacteria are as follows: Nontoxic to the host; selective for a specific type of tumor; has the ability to penetrate deeply into the tumor where ordinary treatment does not reach; nonimmunogenic (does not trigger an immune response immediately but may be cleared by the host); harmless to normal tissue; able to be manipulated easily 9,10.

Bacteria can be viewed as these perfect robot therapies because they have biological mechanisms to perform all of the ideal functions mentioned above. Over the last century, many genera of bacteria have been shown to preferentially accumulate in tumors, including Salmonella and Escherichia have also been investigated as anticancer agents 11,12.

Studies of tumor microenvironments have shown that regions of hypoxia and necrosis are present to varying degrees. Currently, there are two explanations for these observations: tumor cells grow too fast and angiogenesis is out paced by tumor growth and vascularization of tumors is poor and heterogeneous, resulting in an insufficient and inconsistent oxygen supply for a tumor 13,14. As a result, an anaerobic environment can develop in tumors, and this microenvironment provides suitable conditions for the colonization of facultative and obligate anaerobes 15. Alternatively, although some bacteria have the ability to suppress tumor growth, they are not directly cytotoxic to tumor cells. For these bacteria, genetic modifications could be made to make them effective antitumor vectors. Moreover, these bacteria could be combined with chemotherapeutic agents to provide cancer treatment 16.
S. typhimurium can grow under both aerobic and anaerobic conditions and so can colonize both large and small tumors. To increase therapeutic efficacy, bacterial therapy strategies were developed in combination with radiotherapy 17, and chemotherapy 18 or designed such that the bacteria delivered anticancer molecules 19.
2. Features of Bacteria Provide Benefits for Cancer TreatmentSuccessful delivery of the danger signal into the tumor tissue is possible due to a number of bacterial mechanisms that can be therapeutically relevant. Bacteria-based treatments can benefit from microbial metabolism, motility and sensitivity to address the key issues in cancer therapy: low specificity towards cancer tissue and insufficient penetration of the tumor, both of which are limiting to currently used treatment modalities. Cancer cells form a complex and heterogeneous system that is poorly accessible to chemotherapeutic drugs 20, while motile microbial organisms are able to cross biological barriers, act against hemodynamic gradients and preferentially accumulate in the tumor tissue. In contrast to passively-diffusing therapeutics, which produce relatively large drug concentrations in the bloodstream and relatively low drug concentrations in the tumor, bacteria offer unique mechanisms that can facilitate site-specific treatment, highly focused on the tumor and safe to other tissues. This can be possible due to several features of bacteria-based therapeutics:
2.1. Sensing the tumor microenvironmentTumor tissue has a complex and heterogenic metabolism that makes it particularly resistant to systemic treatment. The natural ability of bacteria to receive signals via chemoreceptors can be used to effectively target this unique microenvironment. Oxygen concentration is one of the most important signals for anaerobic bacteria and is of particular interest in anticancer therapy since hypoxia is unique microenvironment formed by the tumor tissue. Moreover, auxotrophic bacterial strains that rely on the uptake of certain metabolites can recognize the tumor microenvironment as a source of nutrients. This phenomenon can facilitate specific accumulation of bacteria in the tumor. One of the most effective examples is the use of engineered auxotrophic strains of S. typhimurium that can only germinate in oxygen-free tumor regions 21.
2.2. Motility
The bacterial microorganism is not only capable of detecting chemo attractants but can also actively follow chemical gradients. This contrasts with passive therapeutics that simply diffuse into tissues from the circulation. Bacteria are able to penetrate deep into the tumor tissue and perform specific actions, e.g. express proteins or transfer genes, to tumor cells localized remotely from the vasculature. This feature can also allow bacteria to cross physiological barriers and accumulate in cellular regions that are either distant and inaccessible for passive therapeutics or quiescent and unresponsive to chemo- therapy. For example, motile strains of Salmonella were shown to effectively penetrate tumor tissue in vitro 22. In addition to motility, the host immune system plays a critical role in preventing bacterial dissemination throughout tumors. Neutrophils have been shown to prevent bacteria from spreading from necrotic into viable tumor tissue. Depleting host neutrophils increases tumor bacterial densities and enables spread throughout viable tumor tissue 23. S. typhimurium specifically accumulate and proliferate in tumor tissues, resulting in bacterial numbers that are over 1,000-fold higher (as high as 1010 cfu/g tissue) than those in normal tissues such as liver and spleen tissue 24. Bacterial colonization of tumor tissues deprives cancer cells of nutrients and activates antitumor immunity, leading to tumor cell death 25.
2.3. Active deliveryUnlike chemical or biological molecules, microorganisms are metabolically active and are able to perform specific metabolic tasks at the tumor site. These include the production of cytotoxic agents (e.g. bacterial toxin), expression of immunomodulatory molecules (e.g. cytokine) or enzymatic conversion of a prodrug into an active therapeutic. Strains derived from intracellular pathogens can infect tumor cells and deliver specific proteins or genes to the tumor tissue 26. Nevertheless, the intertumoral action is not always necessary as bacteria can also express tumor-related antigens to stimulate systemic anticancer immune responses. A Listeria monocytogenes-based cancer treatment that has recently entered clinical development is an example of this approach, where the bacterium delivers tumor antigens directly to the antigen presenting cells 27. 2.4. Controlled propagationPreferential growth in tumor tissue exhibited by many bacterial species is not exclusive to bacteria; experimental oncolytic viral therapies are based on a similar principle. However, once administered to the patient viral vectors are beyond external control. In contrast, bacterial therapeutics are susceptible to antibiotic treatment and therefore fully manageable in the clinical setting therapy can be stopped at the onset of adverse effects or when the bacteria are no longer needed. In fact, the use of live biotherapeutics that contain antibiotic resistance genes in clinical trials is not recommended by the regulatory agencies 28. 2.5. Immune stimulationTumors are immunosuppressive in nature and escape immune surveillance by limiting the maturation and infiltration of immune cells 25. Bacterial vectors augment the anti-tumor immune response not only because of their cargo but also due to their own potent immunostimulatory activity. A growing tumor creates an immunosuppressive environment and establishes immune escape mechanisms that limit the maturation of dendritic cells (DCs) as well as the priming and migration of specific T cells into the tumor. Bacteria provide a strong danger signal to the immune system. The specific conserved bacterial structures such as components of the cell wall or unmethylated CpG sites in bacterial deoxyribonucleic acid (DNA) constitute so called pathogen-associated molecular patterns (PAMPs) that are recognized by Toll like receptors expressed by innate immune cells. PAMPs activate innate and initiate adaptive immune responses. The induction of Tumor necrosis factor, Interferon gamma and Interleukin (IL12) results in the recruitment and activation of DCs which upon migration to the lymph nodes efficiently present tumor antigens to T cells may 26. It has been shown that indeed microorganisms colonizing tumors and promoting an inflammatory reaction in the tumor microenvironment potentiate the anti-tumor host response. The unique features of bacterial therapeutics create the opportunity for novel anticancer strategies, that combine tumor related molecular gradients, natural bacterial features and genetic engineering. Bacteria meet all the requirements for an ideal tumor-targeting agent and might become a novel tool in the anticancer toolbox 27.

3. The benefits of bacterial cancer therapyOne of the major advantages of bacterial therapies for cancer is the ability to specifically target tumors. The mechanisms of bacterial accumulation in tumors differ depending on oxygen tolerance. Facultative anaerobes (Escherichia coli, Salmonella and Shigella felxineri) use a more complex set of mechanisms to target tumors. Five interacting mechanisms are thought to control the accumulation of facultative anaerobes in tumors: entrapment of bacteria in the chaotic vasculature of tumors, flooding into tumors following inflammation, chemotaxis toward compounds produced by tumors, preferential growth in tumor-specific microenvironments and protection from clearance by the immune system 29-31.

3.1. Bacterial toxins3.1.1. ImmunotoxinsImmunotoxins constitute a new modality for the treatment of cancer, since they target cells displaying specific surface-receptors or antigens. Immunotoxins contain a ligand such as a growth factor, monoclonal antibody (mAb), or fragment of an antibody which is connected to a protein toxin. After the ligand subunit binds to the surface of the target cell, the molecule internalizes and the toxin kills the cell. Immunotoxins have been produced to target hematological malignancies and solid tumors via a wide variety of growth factor receptors and antigens 32.
3.1.2. Escherichia coli toxinsEscherichia coli has several toxins and the shiga toxin (Stx) family contains two types called Stx1: VT1 or Shigalike toxin: SLT1) and Stx2 (VT2, SLT2); both of which are encoded by bacteriophages. VT1 has been studied for anticancer effect because it binds to specific receptors on the surface of some types of malignant tumor cells and kills them by inhibiting protein synthesis. It is active only against tumor cell lines that express the VT1 receptor, globotriosyl ceramide-Gb3 (also called CD77). After binding occurs, the receptive cell is penetrated and killed. Select Therapeutics is developing a technology based on expression of verotoxin receptors by human dendritic cells 33. 3.1.3. Type III secretion systemsVarious Gram-negative pathogens use a novel protein secretion system as a basic virulence mechanism. This is called the Type III secretion system and can inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes 34.
Type III secretion systems allow Yersinia spp, Salmonella spp, Shigella spp, and enteropathogenic E. coli adhering at the surface of a eukaryotic cell to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to destroy or subvert the target cell 35. The presence of Type III secretion systems exclusively in bacteria with a pathogenic potential may provide a unique target for the development of therapeutic agents that may spare the normal flora. Type III secretion system may also be used for the delivery of heterologous proteins as therapeutic agents 36.

4. Role of bacteria in cancer gene therapyThe use of genetically modified bacteria for selective destruction of tumors, and bacterial gene-directed enzyme prodrug therapy have shown promising potential. E. coli genes and enzymes have become part of well-known prodrug approaches to cancer. Prodrugs are chemicals that are pharmacodynamically and toxicologically inert but which can be converted in vivo to highly active species 37. Prodrug approach to cancer gene therapyThis strategy overcomes the unacceptable side effects of bacterial therapy and uses anaerobic bacteria that have been transformed with an enzyme that can convert a non-toxic prodrug into a toxic drug. With the proliferation of the bacteria in the necrotic and hypoxic areas of the tumor, the enzyme is expressed solely in the tumor. Thus, a systemically applied prodrug is metabolized to the toxic drug only in the tumor. Two classes of prodrugs that are relevant to bacteria are 5-fluorocytosine activated by bacterial cytosine deaminase and CB-1954 activated by bacterial nitro reductase. Suicide gene therapy, also referred to as the bystander effect, is based on introducing a drug sensitivity gene into target cells, which are then killed by the drug at doses that are not detrimental to normal cells. Most suicide genes currently under investigation mediate sensitivity by encoding viral or bacterial enzymes that convert inactive forms of a drug into toxic metabolites capable of inhibiting nucleic acid synthesis. The ultimate success of GDEPT will depend on the ability to achieve efficient gene delivery to, and expression in, target cells whilst minimizing expression in other tissues 38. Cytosine deaminase genes Cytosine deaminase (CD) is an enzyme present in several bacteria and fungi but not in mammalian cells, catalyses the hydrolytic deamination of cytosine into uracil. It can also deaminate the non-toxic analogue 5-fluorocytosine to toxic antimetabolite 5-fluorouracil – an agent frequently used in cancer chemotherapy. Introduction of CD genes into cultures of human colorectal carcinoma cells induces marked sensitivity to 5-fluorocytosine 39. Attenuated VNP20009 expressing E. coli cytosine deaminase (CD) have been injected directly into the tumors of cancer patients; this enzyme converts 5-fluorocytosine (5-FC), an antifungal agent with limited systemic toxicity, into 5-fluorouracil (5-FU), a cytotoxic anticancer drug commonly used in the clinic to treat head and neck, gastric, colorectal, pancreatic, and breast cancers 40.

Escherichia coli purine nucleoside phosphorylase geneAnother strategy to increase the ‘bystander killing’ is to transfect the cells with E. coli purine nucleoside phosphorylase gene (PNP) and subsequently treat with non-toxic deoxyadenosine analogue containing a toxic adenine analogue. E. coli PNP is capable of catalyzing the conversion of several non-toxic deoxyadenosine analogues to highly toxic analogues. A strategy using E. coli PNP to create highly toxic, membrane-permeant compounds that kill both replicating and non-replicating cells is feasible in vivo, further supporting the development of this cancer gene therapy approach 41. Enzymatic proteins expressed by engineered S. typhimurium can convert nontoxic prodrugs into toxic anticancer drugs in cancer tissues, thereby minimizing systemic toxicity 42.

The Escherichia coli nitro reductase B geneThis gene encodes nitroreductase B enzyme and has been used with prodrug CB-1954 in an HSV-tk system 43. Transfection of mammalian cells with E. coli nitro reductase gene, using a recombinant retroviral vector, has been demonstrated to confer increased sensitivity to CB-1954 up to 770-fold. Other prodrugs such as nitrofurazone (increase of sensitivity 97-fold) also deserve investigation. The gene for an E. coli nitro reductase was introduced into a strain of C. beijerinckii known to activate the nontoxic prodrug CB-1954 to a toxic anticancer drug 44. Nitro reductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB-1954, by a factor of 22. These findings strongly suggest that obligate anaerobic bacteria such as clostridia, because of their selective growth in the hypoxic regions of solid tumors, can be utilized as highly specific gene delivery vectors for cancer therapy.

The Escherichia coli guanine phosphoribosyl transferase geneThis gene encodes xanthine-guanine phosphoribosyl transferase, a bacterial enzyme responsible for transfer of ribose phosphate to xanthine and certain xanthine analogues. This gene has been shown to sensitize rat glioma cells to killing by 6-thioxanthine or 6-thioguanine. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the gpt gene to provide chemo sensitivity against 6-thioguanine or 6-thioxanthine 45. 5. Salmonella typhimurium as a treatment optionSalmonella has historically been used most successfully as anti-cancer microbes 46. These organisms have been used directly as therapeutics, as well as delivery vehicles for anti-cancer therapeutics. However, recent advances in tumor targeting with attenuated S. typhimurium have selected this pathogen as the bacterium of choice for cancer therapy development resulting in it being employed in human trials. Moreover, unlike the strictly anaerobic Clostridium which must be delivered in spore form, S. typhimurium is motile, easily genetically manipulated, and grows as a facultative anaerobe in the presence or absence of oxygen 47.
The anoxic environment often found in tumors makes the tumor site an attractive niche for S. typhimurium growth. Through the use of their flagella, Salmonella migrate towards the tumor, attracted by the high concentrations of nutrients available within the tumor microenvironment 48. The metabolism of S. typhimurium has been exploited to attenuate the strains used in cancer therapy, manipulating the bacteria so that they grow only at tumor sites. Metabolic genes have been removed from S. typhimurium, such as purI in the mutant strain VNP20009, rendering the mutant bacterium auxotrophic for certain compounds that are found in very high concentrations at tumor sites, in this case purines 49. Such genetic engineering, forces migration of S. typhimurium towards tumors in order to survive and results in the bacteria accumulating at the tumor site at over a 1,000 times higher levels than in normal tissue 16. Migration towards the tumor site is based on the ability of S. typhimurium to sense nutrients using a number of receptors that are located at the bacterial poles on the outer membrane of the bacteria. Two notable receptors have been characterized; transmembrane aspartate receptors (TAR) receptor, which detects aspartate secreted by cancerous tissues and the T cell receptor gamma (TRG) receptor, which aids in migration towards ribose found in necrotic tissues. Further manipulation of this ability to chemotaxis towards tumors may lead to the development of bacteria that are directed toward specific regions within tumors. Towards this end, it was discovered that the aspartate receptor controls migration towards tumors, the serine receptor initiates penetration, and the ribose/galactose receptor directs Salmonella into necrotic regions 50. Therefore, knocking out a particular receptor may help direct Salmonella to particular region within the tumor. Other alterations, such as truncation of lipid A 49, reduces the immunogenicity of the bacteria, which in turn, reduces the risk of an adverse inflammatory reaction and possible toxic shock.
6. Key features of S. typhimurium that make it suitable for cancer therapySalmonella have unique properties that can overcome limitations: (1) the ability to sense and target tumors, (2) preferential growth in a tumor-specific microenvironment, (3) good intertumoral penetration, (4) low cytotoxicity and immunogenicity, and (5) versatile programmability 9. Salmonellae can be engineered as part of an active therapeutic approach to cancer and have multiple advantages over conventional therapies.
Table 2: Advantages of Salmonella as an anti-tumor agent
Feature Underlying Reason References
Systemic administration VNP20009 can be delivered intravenously or by direct injection into a tumor 51
Tumor specificity Salmonella can accumulate at levels 1,000-fold higher in tumors as opposed to normal tissues reducing the risk of toxic side effects of proteins or compounds delivered systemically. 52
Replication competent Salmonella replicates to an effective dose within the target tumor 53
Broad tumor specificity Salmonella targets a broad range of solid tumors, including melanoma, lung, colon, breast, renal, hepatic, and prostate tumors 54
Delivery capacity Salmonella is metabolically active and can continuously produce a protein of interest during infection of the tumor 55
Antibiotic sensitivity Salmonella can be easily removed following treatment with antibiotics Native Cytotoxicity Ability of the bacteria to produce virulence factors that leads to cytotoxicity and attract immune cells to the tumors helps in further tumor regression 56
7. Attenuated strains for cancer therapyS. typhimurium (Salmonella enterica serovar Typhimurium) is the most widely studied engineered bacterial cancer therapy and has been used for a variety of applications, reaching as far as clinical trials. As a facultative anaerobe, it can grow in both the hypoxic core of tumors as well as the non-hypoxic regions. The most prominent strains of S. typhimurium, all genetically attenuated for safety, are VNP20009 and A1-R, and recently other strains have been investigated such as SL7207 and CRC2631 57,16. Various S. typhimurium mutant strains have been summarized in table 3. Different strategies have been used to engineer bacteria to reduce cytotoxic effects in normal organs and increase specific colonization of tumors.

Table 3. Candidate attenuated S. typhimurium strains for targeted cancer therapy
Strains Genotype Descriptions
VNP20009 msb, purILipid A-modified to reduce septic shock induction; purine dependent
A1-R Leucine and arginine auxotrophsLeucine/ arginine dependent
ppGppre1A, spoTDefective in ppGpp synthesis; noninvasive to mammalian cells
SL3261 aroABlocked in aromatic synthesis
VNP20009VNP20009. The most intensively studied tumor-targeting Salmonella strain is VNP20009, which was derived from S. typhimurium ATCC14028. VNP20009 is a genetically attenuated strain developed at Yale University that possesses an excellent safety profile and is derived from S. typhimurium ATCC14028. This safety profile includes genetically attenuated virulence (a deletion in the purI gene), reduction of septic shock potential (a deletion in the msbB gene), and antibiotic susceptibility. This strain keeps its genetic and phenotypic stability after multiple generations both in vitro and in vivo 16. A1-RAnother S. typhimurium ATCC14028 tumor targeting strain that selectively grew in tumor xenografts (A1) was developed at University of California (San Diego) by treating S. typhimurium with nitrosoguanidine (NTG) to induce mutations and the resulting auxotrophic pool selected by ability to grow in successive tumor xenografts 58. This leucine and arginine auxotrophic strain grew more strongly in neoplastic tissues than in normal organs, and the colonizing bacteria could be re-isolated from tumor tissue 59. CRC2631 CRC2631 is a tumor-targeting Salmonella strain model developed at the Cancer Research Center (Columbia,MO) and is a candidate therapeutic derived from the S. typhimurium LT2 wild type 64. This strain was developed using archived Salmonella strains from the original Demerec collection of LT2 auxotrophs 60. These strains have been stored in agar stabs for more than four decades at room temperature and have generated dramatic genetic diversity including deletions, duplications, frameshifts, inversions, and transpositions 61,62. Other strains, like, ?ppGppThe three strains described above represent attenuated S. typhimurium, which has been well studied with respect to cancer. Three basic mechanisms are involved in the creation of mutant strains. First, modification of bacterial components to reduce the induction of inflammation, e.g., removal of lipid A from VNP20009 (msbB, purI) 63, second, creation of nutrient auxotrophs by depleting certain genes to enable the bacteria to survive and proliferate in tumor tissues (e.g., leucine and arginine auxotrophic A1-R (Leu, Arg) 64, third, creation of strains by inactivating or downregulating expression of endotoxin-related genes (including ?ppGpp (relA-, spot) 65, and SB824 (aroA, sptP) 66. To engineer such bacteria with better performance, we usually combine different strategies, for example, to yield the lipid A mutant VNP20009 that is defective in purine synthesis. Also, nutrient auxotrophs always show downregulated expression of endotoxin genes.

7. Strategies for S. typhimurium-mediated cancer therapyAttenuated S. typhimurium suppress various cancers in mouse models. Different strategies have been developed to increase their effectiveness, including combinational therapy with radiation or chemical drugs and genetic engineering of bacteria to express therapeutics such as cytotoxic proteins, cytokines, prodrug enzymes, regulators, and genetic materials used for DNA vaccine.

Native cytotoxicity and combinational therapyAttenuated S. typhimurium effectively inhibits tumor growth. Native bacterial cytotoxicity is mediated by activation of the host immune system and by depriving cancer cells of nutrients. Bacterial components (such as LPS, flagellin, and CpG) and signals/molecules released from damaged cancer cells activate the TLR and NLR signaling pathways, resulting in the production of proinflammatory cytokines (IL-1?, TNF-?, and IL-18), which mediate an antitumor immune response. In addition, rapidly proliferating bacteria deprive tumors of nutrients, resulting in cancer cell starvation and death 67.
Expressing Anticancer AgentsAttenuated S. typhimurium can be used as a vector to deliver and express tumor-specific cytotoxic agents to retard tumor growth. However, expression of toxic genes must be tightly regulated by inducible or tumor-specific promoters to avoid unintended damage in normal tissues 68. Cytolysin A (ClyA, HlyE) is a native bacterial toxin produced by E. coli, Salmonella Typhi, and Paratyphi A, and is cytotoxic to cultured mammalian cells due to its pore-forming activity 69,70.

Immunomodulatory molecules
Immunomodulatory molecules such as cytokines and chemokines are able to stimulate the host immune system to clear tumors. Attenuated S. typhimurium have been engineered to deliver immunocompetent cytokines such as IL-2, and IL-18 and CCL21 71. Tumor-specific cytokines produced by bacteria kill cancer cells by triggering the host immune system via upregulation of immune cell activation, proliferation, and migration. IL-2 is a signaling molecule that regulates lymphocyte activity. IL-2-induced tumor suppression correlates with reduced angiogenesis and increased necrosis within tumor tissues 72. IL-18 (also known as IFN-?-inducing factor) increases the cytolytic activity of T cells and NK cells, along with cytokine production. Furthermore, IL-18 upregulates MHC class I antigen expression and drives the differentiation of CD4+ helper T cells into Th1 cells and suppresses angiogenesis by inhibiting the proliferation of endothelial cells, thereby amplifying the antitumor effects mediated by NK cells, macrophages, and CD8+ T cells. CCL21 controls the migration of lymphocytes, dendritic cells, and NK cells 73.
Salmonella as a Vaccine VectorAttenuated S. typhimurium is widely used as a DNA vaccine vector. Bacteria-mediated delivery of cancer-specific antigens or antibodies, growth factor-targeting domains, and anti-apoptosis or tumor-associated macrophage-targeting proteins can stimulate the immune system, promote inflammation, and increase antigen presentation to T cells. Most DNA vaccines are orally administered and have the potential to inhibit or prevent tumor growth when given in multiple doses. Survivin, which is highly regulated and optimally expressed during the G2/M phase of the cell cycle, is essential for anti-apoptotic function. A DNA vaccine encoding survivin could induce CD8 T cell-mediated anticancer activity 74. Endoglin (CD105) is a coreceptor for the TGF-? receptor complex and is overexpressed on proliferating endothelial cells; thus, it is an attractive target for antiangiogenesis-mediated cancer therapy 75.
Table 4. Applications of S. typhimurium as a vaccine vector
Plasmid containing: Promoter Salmonella
strain Tumor References
Survivin epitopes-UbCMV SL7207 NXS2 neuroblastoma 76
Endoglin CMV SL7207 B16F10 melanoma
Renca renal
Carcinoma 77
8. Conclusion
In general, several studies on bacterial cancer therapy have been conducted. Bacterial cancer therapy is now considered a tangible option for future cancer therapy. The potential for bacterial therapy seems endless because of the bacteria has the ability to specifically target tumors, they actively proliferate in a variety of malignant tumors, they are easy to manipulate at the genetic level, and they are inexpensive to produce but some fundamental issues need to be reconciled before this kind of therapy moves into a clinic setting. Insufficient colonization of tumors appears to be the major obstacle identified in clinical trials, a problem that was not evident in animal models. Overcoming this challenge is one of the priorities in developing bacteria as cancer therapy agents. But the ease of genetic manipulation in S. typhimurium may prove to be the key in surmounting this hurdle. Toxicity of S. typhimurium is also problematic, with the bacteria too attenuated to destroy tumors once at the tumor site due to toxin removal, or poor expression of anti-cancer compounds, or the risks of toxicity are too great for injection of S. typhimurium into severely immunocompromised patients. Overcoming these limitations, particularly with respect to using bacterial proteins in therapy, are key to moving this aspect of bacterial cancer therapy forward. The arena of using bacteria as an anti-cancer agent is still new; further studies are imperative to scrutinize the clinical significance of bacteria-based cancer therapy. The findings presented in this review suggest that this promising cancer therapy needs to be optimized and developed further.

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9. DeclarationI, the undersigned, declare that this seminar is my own work and that all sources of material used for the seminar have been duly acknowledged.
Name: Michael Getie (BSc, MSc candidate)
Signature:__
Place of submission: Department of Medical Microbiology and School of Biomedical and Laboratory Sciences, CMHS, University of Gondar.

Date of Submission: May 23/2018
This seminar has been submitted for final submission with our approval as advisors.

Name Signature Dr. Belay Tessema (MSc, PhD)
Mr. Wondwossen Abebe (MSc) This seminar has been submitted for final submission with our approval as examiners.

Name Signature
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