(Bacterial) Cancer Therapy on Therapy Development Because It Enables Precise Tuning and Limitless Functional Combinations

Nature Reviews Cancer | AOP, published online 14 October 2010; doi:10.1038/nrc2934 PersPectIves

using light5,31,32, magnetic resonance imaging INNOVATION (MRI)33 and positron emission tomography (PET)34–36. Finally, and most importantly, the Engineering the perfect ease of genetically manipulating bacteria is the feature that will have the greatest effect (bacterial) cancer therapy on therapy development because it enables precise tuning and limitless functional combinations. Neil S. Forbes Once fully implemented and tested, the Abstract | Bacterial therapies possess many unique mechanisms for treating cancer unique capabilities of bacterial therapies will change the way cancer is treated. The that are unachievable with standard methods. Bacteria can specifically target manufacture of drugs in tumours would tumours, actively penetrate tissue, are easily detected and can controllably induce beneficially shift temporal drug concentra- cytotoxicity. Over the past decade, Salmonella, Clostridium and other genera have tion profiles compared with intravenous been shown to control tumour growth and promote survival in animal models. In administration (FIG. 2). Because bacteria can this Innovation article I propose that synthetic biology techniques can be used to migrate and accumulate far from the vasculature, more of the therapeutic would be solve many of the key challenges that are associated with bacterial therapies, such present in distal regions for longer periods as toxicity, stability and efficiency, and can be used to tune their beneficial features, of time compared with small molecules allowing the engineering of ‘perfect’ cancer therapies. that diffuse only passively. Intratumoural production would be more toxic to can- Bacteria have unique capabilities that make about where and when drugs are admin- cer tissue and less toxic to normal tissue. them well-suited to be ‘perfect’ anticancer istered. Finally, the ability to be externally This inversion of drug localization would agents. Because their genetics can be easily detected would provide crucial information eliminate tumours from the inside out, manipulated, bacteria can be engineered about the state of the tumour, the success of and would have the simultaneous effects of to overcome the limitations that hamper localization and the efficacy of treatment. increasing efficacy and decreasing damage current cancer therapies. Many treatments, Bacteria can be viewed as these perfect to normal tissue. including chemotherapy and radiation, are robot therapies because they have bio- To date, many different bacterial strate- toxic to normal tissue and cannot completely logical mechanisms to carry out all of the gies have been implemented in animal destroy all cancer cells1. Three major causes ideal functions mentioned above (FIG. 1b). models (TABLES 1,2) and some human trials of these problems are incomplete tumour Over the past century, many genera of have been carried out (TABLE 3). Using these targeting, inadequate tissue penetration and bacteria have been shown to preferen- strategies, many researchers have observed limited toxicity to all cancer cells1–3. These tially accumulate in tumours, including experimental success, with reduced tumour drawbacks prevent effectual treatment and Salmonella4, Escherichia5, Clostridium6,7 and volume and increased survival and treat- are associated with increased morbidity Bifidobacterium8. Caulobacter 9, Listeria10,11, ment of metastatic disease (TABLE 1). Success and mortality. Proteus12 and Streptococcus13 have also has also been shown in treating multiple Using a top-down engineering approach, been investigated as anticancer agents. tumour sites (TABLE 1); the most notable is the ideal cancer therapy can be envisioned: For propulsion and sensing, bacteria have pancreatic cancer13,37, for which new targeted it would be tiny programmable ‘robot facto- flagella that enable tissue penetration14 and treatments could dramatically improve the ries’ (FIG. 1a) that specifically target tumours, chemotactic receptors that direct chemotaxis poor current prognosis of less than 25% are selectively cytotoxic to cancer cells, are towards molecular signals in the tumour 5-year survival. Since the mid-1990s, the self-propelled, are responsive to external microenvironment15,16. For example, the number of published bacterial therapy signals, can sense the local environment and TAR receptor detects aspartate secreted papers has increased, with a doubling time are externally detectable. Specific targeting by viable cancer cells, and the TRG recep- of 2.5 years (FIG. 1c). This rapid rise has been would allow the use of more toxic molecules tor promotes migration towards ribose in driven almost entirely by the increasing use without systemic effects. Self-propulsion necrotic tissue16. Selective cytotoxicity can of Salmonella as a delivery vector (FIG. 1c). would enable penetration into tumour be engineered by transfection with genes for This Innovation article describes many of regions that are inaccessible to passive therapeutic molecules, including toxins17–19, the advances that have fuelled this enthusi- therapies. Responsiveness to external sig- cytokines20,21, tumour antigens22 and apop- asm, including specific bacterial targeting of nals would enable the precise control of the tosis-inducing factors23–27. External control tumours; intratumoral penetration; native location and timing of cytotoxicity. Sensing can be achieved using gene promoter strate- bacterial cytotoxicity; expression of anti- the local environment would allow ‘smart’, gies that respond to small molecules17,28,29 or cancer agents; gene-triggering strategies; responsive therapies that can make decisions radiation23,26,27,30. Bacteria can be detected and detection of bacterial therapies.

Bacterial targeting of tumours and protection from clearance by the could also reduce the usefulness of repeated One of the major advantages of bacterial ther- immune system42. These mechanisms enable dosing with bacteria, which is a limitation apies for cancer is the ability to specifically Salmonella to accumulate in tumours at that does not affect bacteria that are delivered target tumours. The mechanisms of bacterial ratios greater than 1000/1 compared with as spores47. accumulation in tumours differ depending organs that are rich in reticuloendothelial In in vitro tumour models, Salmonella on oxygen tolerance. Obligate anaerobes (for cells (such as the liver and spleen) and identify and penetrate tumours by detecting example, Clostridium and Bifidobacterium) Salmonella accumulate at even greater ratios and chemotaxing towards small molecule cannot survive in oxygen, and injected in other organs40,43–45. gradients of serine, aspartate and ribose15,16. bacterial spores can only germinate in the when injected systemically, Salmonella In addition, the growth rate of Salmonella is anoxic regions of tumours38,49. completely attach to the walls of tumour vasculature greater in in vitro tumours when dying cells deoxygenated tissue is unique to tumours with a low but measurable frequency are present15, a phenomenon which is also and is not present in most other organs of the (~0.035% of bacteria in the blood)40. In addi- observed in animal tumour models40,41,46. body. Obligate anaerobes are therefore highly tion, the number of bacteria that adheres The importance of this mechanism for pro- effective at accumulating in the large hypoxic is dependent on blood velocity, suggesting moting accumulation is supported by the regions of tumours14. This absolute specificity that haemodynamics have an important increased tumour specificity of auxotrophic was demonstrated by Malmgren et al.7 who role in the initial interaction of bacteria Salmonella that require leucine and arga- injected Clostridium into tumour-bearing with tumours40. Similarly, the accumula- nine, which are nutrients derived from dying mice and showed that only the mice with tion of Salmonella is associated with an tumour tissue31,48. tumours died from this infection. influx of blood into tumours, caused by Because tumours are immune-privileged Facultative anaerobes (for example, an immunologically induced rise in the environments49, bacteria can replicate unim- Salmonella and Escherichia) use a more com- blood concentration of tumour necrosis peded by the macrophage and neutrophil plex set of mechanisms to target tumours. factor-α (TnFα)41. This mechanism would clearance mechanisms that normally serve Five interacting mechanisms are thought be reduced for attenuated msbB– strains that to eliminate them50. In this way, the immune to control the accumulation of facultative elicit much lower (~10%) TnFα levels46. system has a complicated role in bacterio- anaerobes in tumours: entrapment of bacte- The production of TnFα immediately after lytic therapy: it provides a mechanism to ria in the chaotic vasculature of tumours40; injection therefore has contradictory effects: guide bacterial accumulation, but also flooding into tumours following inflamma- it promotes accumulation in tumours but is impedes dispersion and efficacy. The inter- tion41; chemotaxis towards compounds pro- also the primary cause of bacterial toxicity action between bacteria and the immune duced by tumours15,16; preferential growth owing to septic shock46. This dependence on system also works in reverse: many bacterial in tumour-specific microenvironments15,31; an immune response to promote targeting therapies sensitize the immune system to induce tumour clearance51,52. abRobot factory Bacterial cell Attractor molecule Intratumoral penetration Tumour Environmental Flagellum Chemotactic receptor targeting sensor Intratumoral targeting is an essential char- + Propeller + acteristic of an optimized cancer therapy + (FIG. 1). compared with normal tissue, ReporterAgent tumours have chaotic vasculature and large intercapillary distances, which impede the delivery of therapeutic molecules3,53. This Externally Reporter reduces therapeutic efficacy by creating cel- detectable signal Cytotoxic protein for Molecular lular regions that have low drug concentra- molecule visualization signal 1,3 Anticancer tions and reduced nutrient supply . low c protein levels of oxygen and glucose create quiescent 40 cells that are unresponsive to chemothera- s Total peutics that are designed to target rapidly Salmonella 30 growing cells. Proper intratumoral targeting Clostridium Escherichia enables direct drug delivery to these distal, 20 Bifidobacterium unresponsive cells that are far from the tumour vasculature (FIG. 2). In this way, the Number of paper 10 metabolic heterogeneity of tumours is both a blessing and a curse: molecular gradients 0 reduce therapeutic efficacy but also create 1960 1970 1980 1990 2000 2010 unique environments that can be targeted. Year Motility is the key feature of bacterial Figure 1 | Bacteria are the optimal robot factory cancer therapies. a | the perfect cancer therapy Nature Reviews | Cancer therapies that enables intratumoral targeting. could be imagined as a ‘robot factory’ that could carry out six important functions: target tumours, produce cytotoxic molecules, self-propel, respond to triggering signals, sense the local environment Bacteria can actively swim away from the and produce externally detectable signals. b | Bacteria have biological mechanisms to carry out these vasculature and penetrate deep into tumour functions: gene translation machinery to produce anticancer proteins (dark blue); flagella to chemo- tissue (FIG. 2). Because bacteria are complex tax; specific gene promoter regions to respond to molecular signals (red squares); chemotactic recep- living organisms that can acquire energy tors (blue); and machinery to produce detectable molecules (red). c | the number of papers describing from their environment, their transport is bacterial anticancer therapies has grown exponentially (dashed black line) since the mid-1990s. not entropically limited. This contrasts with

the concentration of passive molecules, Bacterial Chemotherapy which drops as a function of distance from therapeutic vasculature. Because bacteria are self- Bacterium molecule Blood vessel ‘Active’ propelled, their density can be higher far bacterial from the vascular source. It has been shown therapy that bacteria that can disperse throughout tumour tissue have a greater ability to regress tumours14. Salmonella have also been shown to chemotax towards molecules that are produced by dying tumour tissue15,16. Salmonella contain chemoreceptors that sense small ‘Passive’ molecules in the local environment. For chemotherapy example, using knockout models, it has been shown that the aspartate receptor intiates chemotaxis towards viable tumour tissue; Tumour System the serine receptor induces tissue penetration; and the ribose receptor directs Distal Proximal migration towards necrotic tissue16. In addition to intrinsic motility, the host tion immune system has a crucial role in preventing bacterial dissemination throughout entra tumours. neutrophils have been shown Conc to prevent bacteria from spreading from Time Time Time necrotic to viable tumour tissue50. This Chemotherapy Bacterial therapy containment is one possible reason that attenuated Salmonella had limited success in Figure 2 | The transport properties of bacterial therapies produce preferable drug concentra- reducing tumour growth in human trials54–56. tion profiles. When injected systemically, bacteria (blue syringe; brown bacteria)Natur specificallye Reviews | Canc accuer- depleting host neutrophils increases tumour mulate in tumours and migrate to distal regions far from the vasculature (shaded cells). these distal bacterial densities and enables spread regions are typically hypoxic and hypoglycaemic and contain quiescent and necrotic cells. Once trig- throughout viable tumour tissue50. gered (small arrows), bacteria begin to produce therapeutic molecules (blue ovals) that diffuse (large arrow) into viable tissue. systemically injected, passive chemotherapeutic molecules (red squares) Native bacterial cytotoxicity diffuse into tumour tissue from blood vessels (large arrow). the concentration of bacterially produced molecules (blue lines on graphs) is greatest in distal tumour regions and remains constant as long as Many successful experiments have shown expression of these proteins continues. the concentration of chemotherapeutic molecules is greatest that the natural toxicity of bacteria is suf- in systemic blood and drops as it is cleared by the liver or kidneys. Based on these profiles, bacterially ficient to regress tumours (TABLE 1). native produced molecules will be more cytotoxic (shown by dashed lines on the graph) in the distal regions bacterial cytotoxicity is caused by sensitiza- of tumours and less systemically toxic. the profile of passive molecules is less favourable, with more tion of the immune system and competition systemic toxicity and less efficacy deep in tissue. for nutrients42. Although some organisms naturally produce toxins, these are typically removed to prevent pathogenicity14. Much Clostridium, an obligate anaerobe, could was seen in 4 of the 35 animals65. There early work on bacterial therapies relied on regress tumours in mice (TABLE 1). There was is also potential that Salmonella could be their natural toxicity because direct genetic sufficient enthusiasm to initiate a small clini- delivered orally to reduce toxicity. Following modification of bacteria was not possible. cal trial, and oncolysis was observed in three oral administration in mice, Salmonella The ability of bacteria to regress tumours out of five patients following injection with preferentially accumulated in tumours and has been recognized since the early 1800s57. Clostridium butyricum63 (TABLE 3). maintained its anticancer effects66 with very In the time before strict antiseptic tech- More recently, Salmonella has been low toxicity67. Oral delivery might be dif- nique, tumour regression was occasionally tested for its anticancer properties4,46 ferent in humans, in which bacterial escape observed following severe bacterial infec- and, similar to Clostridium, Salmonella is from the gut to the circulation occurs less tion57. This observation led to the develop- naturally cytotoxic and has been shown to often than in mice68. ment of coley’s toxin, a bacterial extract that regress tumours when administered alone stimulates a general immune response57–59. (TABLE 1). Immunosensitization is one of the Expression of anticancer agents Because of this early success, this approach key mechanisms of Salmonella cytotoxicity; Another advantage of bacterial anticancer persists in many contemporary strategies20,60, accumulation of Salmonella choleraesuis agents is that they can be genetically modi- which are similarly designed to stimulate in tumours induces neutrophil infiltration fied to increase their effectiveness. Many immune responses (TABLE 2). The idea that and antitumour immune responses64. when strategies have been used (TABLES 1,2) and living bacteria could be anticancer therapeu- investigated in human trials, Salmonella two major mechanisms have been studied: tic agents was first advanced in the middle of with a modified lipid A (strain vnP200009) the direct expression of proteins that have the twentieth century6,7. The increased avail- was found to be non-toxic and tumour physiological activities against tumours ability of antibiotics and the discovery that colonization was observed55. In dogs admin- and the transfer of eukaryotic expression tumours contain anoxic regions61 spurred istered with vnP200009, tumour coloniza- vectors into infected cancer cells. For both multiple investigations6,62 that showed that tion was also observed and a complete cure of these mechanisms, three categories of

anticancer agents have been investigated: bacterial protein that is readily transported FASl specifically induces apoptosis in cells cytotoxic agents that directly kill cancer cells, to the bacterial surface and secreted with- that possess the FAS receptor24. TnFα and cytokines that stimulate immune cells to kill out modification17,18. Multiple groups have TRAIl have been shown to be cytotoxic cancer cells and tumour antigens that sensi- shown that treating mice with Escherichia towards colon, breast, lung, prostate, renal, tize the immune system against cancer cells. coli or Salmonella typhimurium that express ovarian, bladder, glioma and pancreatic Prodrug strategies have been reviewed previ- clyA reduces tumour growth17–19. tumours23,71. when systemically adminis- ously69,70 and are not discussed here. Three of the cytotoxic agents are mem- tered as protein drugs, all three members bers of the TnFα family: FAS ligand (FASl), of this family have two deficiencies that are Cytotoxic agents. Bacterial toxins are the TnF-related apoptosis-inducing ligand overcome by bacterial delivery: hepatotox- most obvious cytotoxic agents because these (TRAIl) and TnFα23–27. These proteins icity and a short circulatory half-life23,25–27. genes are native to bacterial physiology. selectively induce apoptosis through death Producing these proteins in situ would cytolysin A (clyA; also known as HlyE) receptor pathways, which activate caspase 8 maintain a higher continual concentration is a bacterial toxin that functions by form- and caspase 3, an important apoptotic medi- in tumours compared with delivery to the ing pores in mammalian cell membranes ator23. All three are selectively cytotoxic to circulatory system (FIG. 2), and would reduce and inducing apoptosis18,19. clyA is a native cancer cells compared with normal cells23,24. the systemic toxicity that is associated with their administration as small molecules. FASl is also immunologically active: it table 1 | Efficacy of bacterial therapies and strategies in animal models attracts tumour-rejecting granulocytes, Category Genus refs induces interleukin-23 (Il-23) production Strategies showing tumour regression and/or increased survival by dendritic cells and stimulates the prolif- Native bacterial toxicity Bifidobacterium 8 eration of T cells — three mechanisms that might culminate in the specific killing of Caulobacter 9 cancer cells24. Clostridium 6,62,115–117 Escherichia 118 Cytokines. Bacteria can also be engineered Listeria 10,11 to deliver specific cytokines that have antitumour effects (TABLE 2). cytokines induce Proteus 12 immune cells to clear tumours by stimulat- Salmonella 4,37,46,47,64–67,119–122 ing multiple mechanisms, such as immune Streptococcus 13 cell activation, proliferation and migration. when administered as a small molecule, combination with other therapies Clostridium 14,107–110 Il-2 activates the cytolytic function of natu- Escherichia 18 ral killer and lymphokine-activated killer Salmonella 52,123–125 cells72 and promotes lymphocyte prolifera- 73 Agents with control of expression Salmonella 19,23 tion . Similar to Il-2, Il-18 (also known as interferon-γ (IFnγ)-inducing factor) expression of anticancer agents Escherichia 18 induces T cell and natural killer cell prolif- Salmonella 20–22,24,74–79,81,82,126 eration and enhances their production of Gene transfer Salmonella 44,51,88–95 cytokines74. Il-18 also suppresses angio- 74 rNA interference Salmonella 96 genesis by inhibiting fibroblast growth . ccl21 controls the migration of immune Prodrug cleavage Clostridium 48,127–129 cells and may prevent tumour-induced Salmonella 130–134 immunosuppression21. lIGHT (also known Strategies that reduced metastatic burden or prevented metastasis formation as TnFSF14) is a TnF family cytokine that is Native bacterial toxicity Salmonella 121,135,136 homologous to lymphotoxin, which induces dendritic cell growth20. expression of anticancer agents Escherichia 18 Il-2 is the most extensively studied bac- Salmonella 20,21,24,74–76,93 terially delivered cytokine72,73,75–80. Reports Sites targeted showing tumour regression or increased survival describing Il-2 delivery by Salmonella were Breast cancer Salmonella 48,131 the first to suggest that this genus could be effectively used as an anticancer agent73,80. colon cancer Salmonella 131 Oral administration of Salmonella express- Hepatocellular carcinoma Salmonella 64 ing Il-2 has been shown to function prophy- 79 Melanoma Salmonella 130,131 lactically and prevent tumour formation . despite multiple anticancer effects, Il-2 and Neuroblastoma Salmonella 78 Il-18 have had limited success as chemo- Pancreatic cancer Salmonella 37 therapeutics because of severe systemic Streptococcus 13 toxicity72–74. Similar to the TnFα family Prostate cancer Salmonella 96 agents, local production of these cytokines in tumours would limit toxicity while stimu- spinal cord glioma Salmonella 122 lating tumour infiltration by lymphocytes72.

Treatment with Salmonella that express the genes for cytotoxic and immunological systems, may produce stronger, more stable lIGHT or ccl21 has been shown to induce agents into cancer cells (TABLE 2). compared expression. However, the expression of the leukocyte and neutrophil infiltration and with direct expression, this approach has transfer genes might be harder to control87; it inhibit tumour growth20,21. benefits as well as drawbacks. Gene transfer, could be limited by poor transfer efficiency; which uses more permanent mammalian transferred genes may be heterogeneously Tumour-specific antigens and antibodies. The expression of tumour-specific table 2 | Bacterial strategies antigens is another bacterial strategy that Category Strategy refs uses the host immune system (TABLE 2). It functions by sensitizing immune cells and Gene triggering γ-irradiation with pRecA promoter 23,26,27,30 preventing the formation of tumours that l-arabinose with pBAD promoter 17,28,29 22,60,81,82 present those antigens . For example, Oxygen (FNr) with FF+20*, HIP-1, pfle and ansB promoters 19,101,103 RAF1 (also known as c-RAF) is a transcription factor that is upregulated in many salicylate with Xyls2-dependent Pm promoter 99 tumours22; prostate-specific antigen (PSA) combinations with Anti-vascular agents 14,123 60 other treatments is upregulated in many prostate tumours ; chemotherapeutic drugs 14,51,108,110 and nY-ESO-1 (also known as cTG1B) is a Heat shock proteins 125 germ cell protein often expressed by tumour cells82. To induce a more efficient immune Heavy metals 107 response, PSA has been fused to cholera radiation 18,109,124 toxin subunit B (ctxB), a mucosal adju- Imaging Bioluminescence 5,17,104–106 vant60. Alternatively, a nonspecific immune Fluorescence 5,31,32 response can be induced by the expression of a potent antigen, such as canine parvo- MrI 33,137 virus (cPv)81. To facilitate interaction Pet 34–36 with immune cells, different protein secre- Expressed anticancer agents tion systems have been used; for example, RAF1 and ctxB–PSA were fused to the cytotoxic agents cytolysin A 17–19 α-haemolysin secretion signal22,60 and cPv FAsL 24 was bound to OmpA, a membrane pro- tNFα 25–27 tein that forms outer membrane vesicles81. trAIL 23 Because these strategies rely on a systemic immune response, it is not necessary for cytokines ccL21 21 these antigens to be expressed in tumours82. IL-2 72,73,75–80 Also, because the response is retained by the IL-18 74 immune system, these bacterial therapies LIGHt 20 could be used for prevention or as treatment vaccines. Antigens and ctxB–PsA fusion protein 60 antibodies Alternatively, bacteria can be engineered cPv–OmpA fusion protein 81 to express single-chain antibodies to inhibit NY-esO-1 tumour antigen 82 proteins that are necessary for tumour cell rAF1 22 function. For example, Clostridium novyi has been modified to express single-chain single chain HIF1α antibodies 83 antibodies that bind the hypoxia inducible Genetic transfer factor 1α (HIF1α) antigen83. HIF1α is an cytotoxic and endostatin 44 important target because it is associated with anti-angiogenic resistance to radiotherapy and chemo therapy agents thrombospondin 1 51 and poor clinical outcome83. Preliminary trAIL and sMAc 88 studies have shown that bacterially produced cytokines and FLt3L 92 antibodies bind the HIF1α epitope83. growth factors GM-csF 90 Gene transfer. The ability of therapeutic IL-12 89–91 bacteria to transfer genetic material to tumour antigens AFP 95 mammalian cells was first reported in 1995, veGFr2 93,94 when it was shown that Shigellae could Gene silencing Stat3 96 transfer plasmid dnA into baby hamster (shrNA) kidney cells84. Soon after, it was shown that Bcl2 97 Salmonella could also be used for trans- AFP, α-fetoprotein; cPv, canine parvovirus; ctxB, cholera toxin subunit B; FAsL, FAs ligand; FNr, fumarate and kingdom dnA transfer85,86. These reports nitrate reduction; GM-csF, granulocyte/macrophage colony stimulating factor; HIF1α, hypoxia inducible factor 1α; IL, interleukin; MrI, magnetic resonance imaging; Pet, positron emission tomography; PsA, prostate- generated considerable enthusiasm for using specific antigen; Stat3, signal transducer and activator of transcription 3; tNFα, tumour necrosis factor-α; bacteria (specifically Salmonella) to transfer trAIL, tNF-related apoptosis-inducing ligand; veGFr2, vascular endothelial growth factor receptor 2.

distributed in tissues; and genes could also Antibodies against AFP, an embryonic and cytokines that are known to be toxic transfer to tissues other than those that they protein that is overexpressed in hepato- cannot be constitutively expressed. Tumour- are targeted towards. cellular carcinoma and is not present in specific antigens do not need to be expressed Many of the same strategies have been normal adult tissue, prevents the formation in tumours, and so the tight control of attempted with gene transfer as with direct of liver and colon tumours95. vEGFR2 is an the genes expressing these antigens is not expression: cytotoxic agents, cytokines and endothelial cell receptor that controls angio- necessary82. tumour antigens (TABLE 2). Two early reports genesis, and antibodies against vEGFR2 There are two categories of gene-triggering describe the transfer of the anti-angiogenic have been shown to prevent angiogenesis strategies: extracellular triggers and envi- genes endostatin44 and thrombospondin 1 and tumour growth in glioblastoma94 and ronmental sensors (TABLE 2). Three external (REF. 51), which kill tumours by preventing lung cancer93 models. triggers have been investigated: l-arabinose, new blood vessel formation and cutting salicylate and γ-irradiation (FIG. 3). The pBAd off the nutrient supply44. Although direct Gene silencing. A complementary strategy system uses the regulatory protein Arac to administration of endostatin to patients to the bacterial induction of gene expression respond to extracellular l-arabinose17,28,29 with cancer showed only minimal anti- is gene silencing. Silencing is achieved by and is very tightly regulated98. The sali- tumour activity, transfer of endostatin from transferring plasmids encoding small hair- cylate system is also tightly regulated and Salmonella reduced microvessel density, pin RnAs (shRnA) from Salmonella into its cascade amplifies gene expression, decreased vascular endothelial growth fac- cancer cells96,97. Gene-specific shRnAs are producing induction ratios of 20–150-fold tor (vEGF) expression and slowed tumour processed by the enzyme dicer into small in vitro99. Both l-arabinose and salicylate growth in mice44. Using a similar strategy to interfering RnAs (siRnAs) that induce the are suitable and non-toxic biological direct expression, the reduction of tumour degradation of target mRnAs96. To date, triggers. In mouse models, it has been growth was shown by transferring the two genes have been silenced using this shown that intravenous administration of genes encoding TRAIl and SMAc (also technique, signal transducer and activa- l-arabinose can activate gene expression in known as dIABlO) into tumour cells from tor of transcription 3 (Stat3)96 and Bcl2 colonized tumours29. Salmonella88. (REF. 97). Both factors inhibit apoptosis, The RecA mechanism uses γ-irradiation The antitumour effects of three cytokines and STAT3 promotes cancer cell growth. as a trigger of gene expression (FIG. 3) and is and growth factors have been explored by Overexpression of these factors has been based on the SOS dnA repair system23,26,27,30. bacterial gene transfer: Il-12 (REFS 89–91), associated with many tumour types, Irradiation has a major advantage over granulocyte/macrophage colony-stimulating including prostate cancer and malignant molecular triggers because it can directly factor (GM-cSF)90 and FlT3l92. Similar to melanoma96,97. Silencing of Stat3 has been penetrate tumour tissue and is not restricted bacterially expressed cytokines, these mol- shown to prevent prostate tumour and by diffusion limitations2. γ-irradiation ecules stimulate natural killer cells, T cells metastasis formation in mice97. causes dnA damage and activates RecA23, and dendritic cells89–91. In addition, Il-12 which promotes autoproteolysis of the induces IFnγ production and GM-cSF acti- Gene-triggering strategies repressor lexA. The lysis of lexA, a repres- vates neutrophils and macrophages to lyse The control of gene expression is crucial for sor of the recA promoter, induces gene tumour cells90. when expressed together, managing the timing and location of drug expression. This system is amplified by self- Il-12 and GM-cSF significantly reduce production. The incorporation of specific induction of RecA when lexA is cleaved. To tumour growth in mice, while limiting the promoter sequences upstream of genes reduce basal expression and increase radia- systemic toxicity that is associated with sys- that encode anticancer proteins enables the tion responsiveness, nuyts et al.100 showed temic cytokine injection90. control of transcription by external signals. that adding an extra cheo box into the recA The transfer of genes for two tumour Precise triggering of expression can be used promoter increased expression tenfold. antigens has been shown to be effective at to induce greater intratumoural effects To date, all environmental triggering reducing tumour growth in mouse models: while minimizing systemic toxicity23. Some strategies have been designed to sense α-fetoprotein (AFP) and vEGF recep- gene products require tighter control than hypoxia using the fumarate and nitrate tor 2 (vEGFR2; also known as FlK1)93–95. others; for example, cytotoxic molecules reduction (FnR) regulator19,101 (FIG. 3). FnR is an oxygen-responsive transcrip- table 3 | Published human trials using bacterial cancer therapies tion factor that is naturally present in Salmonella19,101,102. In the absence of oxygen, Strain Cancer type n responses refs iron-sulphide clusters induce the formation Clostridium squamous cell carcinoma, 5 Oncolysis (3 patients) 63 of FnR homodimers that bind to specific butyricum M-55 metastatic, malignant neuroma, dnA sequences and promote transcrip- leiomyosarcoma, melanoma and 19,101 sinus carcinoma tion . In the presence of oxygen, the clusters and FnR homodimers disassemble, C. butyricum M-55 vascular glioblastoma 49 Oncolysis 138 reducing transcription. Two artificial pro- Salmonella Metastatic melanoma and renal 25 Focal tumour 55 moters have been developed that contain typhimurium cell carcinoma colonization (3 patients) FnR-binding sites: FF+20* (REF. 19) and vNP20009 hypoxia inducible promoter 1 (HIP1)101 S. typhimurium Metastatic melanoma 4 tumour biopsy culture 54 (TABLE 2). These two promoters were created vNP20009 positive for vNP20009 by random19 and directed101 mutagenesis to (1 patient) amplify expression in hypoxia and reduce S. typhimurium squamous cell carcinoma and 3 Intratumoural bacterial 56 expression in normoxia19. To identify vNP20009 tAPet-cD adenocarcinoma colonization (2 patients) bacterial promoters that could be used

a b [18F]-2′-fluoro-2’-deoxy-1-β-d-arabino- L-arabinose Salicylate furanosyl-5-ethyl-uracil ([18F]-FEAU)36. + + + Both these methods have successfully been shown to identify bacteria accumulated in AraC nahR xylS2 mouse tumours34–36. + + + Conclusions and future perspectives AraC Gene xylS2 Gene Recently, many experiments have shown that bacterial therapies can successfully regress pC pBAD Psal Pm tumours and promote survival in mice. However, numerous challenges remain before cd γ-irradiation O2 bacteria can be used in the clinic, including limited drug production, intrinsic bacterial DNA damage toxicity, targeting efficiency, genetic instability and combination with other therapies. Tuning + + FNR LexA RecA drug production is necessary to synthesize + drugs at sufficient concentrations to induce RecA Gene Gene therapeutic effects but at concentrations not high enough to cause systemic toxicity (FIG. 2). controlling bacterial toxicity will be crucial to pRecA pRecA FNR-responsive promoter ensure safety and allow regulatory approval. Figure 3 | Gene triggering systems. a | the pBAD system, which responds to extracellularNature Revie lws-arabinose, | Cancer Both Clostridium and Salmonella have been contains two components: the arabinose-sensitive protein Arac and the pBAD promoter. the consti- shown to be non-pathogenic in multiple tutively expressed regulator Arac induces transcription by binding to the pBAD promoter. Arac is a animal species46,65 and in human trials54–56,63, positive and negative regulator of pBAD: it activates transcription in the presence of arabinose and represses transcription in its absence. b | the salicylate cascade system uses two salicylate-sensitive but any retained virulence could be prob- regulator proteins, nahr and xyls2, to maintain tight regulation. In the presence of salicylate, nahr lematic for immunocompromised late-stage activates transcription from the promoter Psal, leading to the expression of Xyls2. Xyls2, which is also cancer patients. variable targeting efficiency sensitive to salicylate, activates transcription from the promoter Pm. c | the recA system senses could lead to poor efficacy for large groups γ-irradiation, which causes DNA damage. this damage activates recA, which induces autoproteolysis of patients and will affect which sites could of LexA. transcription is induced when LexA, a repressor of the pRecA promoter, releases from DNA. be effectively treated with bacteria. Targeting Feed-forward regulation increases the recA concentration when the system is active. d | the fumarate efficacy will also play a large part in the treat- and nitrate reduction (FNr) system turns on in hypoxic environments. the absence of oxygen promotes ment of metastatic disease because, to be the dimerization of FNr, which induces transcription. Multiple promoters bind FNr, including FF+20*, effective, bacteria will have to colonize a high HIP-1, pfle and ansB. proportion of distal sites. Genetic instability is a potential problem because mutations could in environmental-triggering strategies, have proved to be very efficient at identifying create ineffective or harmful phenotypes. The Arrach et al.103 developed a reporter system tumours in mice using whole-mouse rate of mutation will specify the upper time that they tested in tumour-bearing mice. imaging5,17,31,32,104–106. These light-based limit that bacterial colonies could be allowed The two most active promoters, pflE and mechanisms might have limited clinical to remain in tumours. Finally, determin- ansB, both contained FnR-binding sites applications, however, because of the poor ing the correct combination of bacteria and and are known to be oxygen dependent103. penetration of visible light through tissue. other cancer therapies14,18,107–110 (TABLES 1,2) These experiments did, however, identify Alternatively, magnetotactic bacteria will be crucial for creating strategies that can other promoters that were not oxygen could be injected and detected by MRI. For completely clear tumours and metastases. dependent and might rely on alternative example, Magnetospirillum magneticum Solving these challenges could overcome the environmental triggers. produces magnetite particles and has been limitations that have previously been seen shown to accumulate in tumours33. For in the clinic54–56 (TABLE 3): reduced toxicity Detection improved tumour targeting, the genes for will increase the maximum-tolerated dose; Being able to locate colonized bacteria is magnetite production could be transferred improved targeting will increase tumour colo- clinically important because it enables the into other bacterial strains33. Two differ- nization; and efficient drug production will detection of obscured tumours and metas- ent methods that have been used to detect promote tumour regression. tases. Four different strategies have been bacteria with PET are expression of an All these challenges can be addressed implemented to identify bacteria in tumours: exogenous viral tyrosine kinase34,35 and using synthetic biology techniques. Rates of bioluminescence, fluorescence, magnetic reliance on endogenous protein kinases36. protein drug production can be optimized resonance and positron emission (TABLE 2). when herpes simplex thymidine kinase by manipulating multiple factors111, includ- Bioluminescent bacteria are generated by (HSv1-TK) is expressed in Salmonella, ing gene copy number, promoter strength, transformation with plasmids containing the it selectively phosphorylates and traps optimized codons, bacterial metabolism, luxcdABE operon from Photobacterium the detectable marker 2′-fluoro-1-β-d- mRnA secondary structure112 and synthetic leiognathi 5,17,104–106, and fluorescent bacteria arabino-furanosyl-5-iodouracil (FIAU)35. ribosome-binding sites113. Both toxicity are generated by transformation with plas- Alternatively, the endogenous pro- and targeting are affected by the immune mids containing the gene for green fluores- tein kinases of E. coli nissle 1917 have response following injection and innate cent protein5,31,32. Both of these mechanisms been shown to phosphorylate and trap bacterial virulence. determining which

virulence factors are essential for targeting 1. Minchinton, A. I. & Tannock, I. F. Drug penetration in 25. Theys, J. et al. Stable Escherichia coli–Clostridium solid tumours. Nature Rev. Cancer 6, 583–592 acetobutylicum shuttle vector for secretion of murine and which introduce unnecessary toxic- (2006). tumor necrosis factor α. Appl. Environ. Microbiol. 65, ity can be achieved by screening knockout 2. St. Jean, A. T., Zhang, M. M. & Forbes, N. S. Bacterial 4295–4300 (1999). therapies: completing the cancer treatment toolbox. 26. Nuyts, S. et al. Increasing specificity of anti-tumor models of the pathogenicity genes that, Curr. Opin. Biotechnol. 19, 511–517 (2008). therapy: cytotoxic protein delivery by non-pathogenic for example, enable the evasion of the 3. Jain, R. K. The next frontier of molecular medicine: clostridia under regulation of radio-induced delivery of therapeutics. Nature Med. 4, 655–657 promoters. Anticancer Res. 21, 857–861 (2001). immune system, induce uptake into cells, (1998). 27. Nuyts, S. et al. Radio-responsive recA promoter promote intracellular replication and stimu- 4. Pawelek, J. M., Low, K. B. & Bermudes, D. Tumor- significantly increases TNFα production in targeted Salmonella as a novel anticancer vector. recombinant clostridia after 2 Gy irradiation. Gene 114 late cytokine synthesis . Other targeting Cancer Res. 57, 4537–4544 (1997). Ther. 8, 1197–1201 (2001). mechanisms can be enhanced by the genetic 5. Yu, Y. A. et al. Visualization of tumors and metastases 28. Loessner, H. et al. Remote control of tumour-targeted in live animals with bacteria and vaccinia virus Salmonella enterica serovar Typhimurium by the use manipulation of endogenous chemorecep- encoding light-emitting proteins. Nature Biotech. 22, of L-arabinose as inducer of bacterial gene expression tors16, selective control of bacterial prolif- 313–320 (2004). in vivo. Cell. Microbiol. 9, 1529–1537 (2007). 6. Parker, R. C., Plummer, H. C., Siebenmann, C. O. & 29. Stritzker, J. et al. Tumor-specific colonization, tissue eration in tumours and strategies to avoid Chapman, M. G. Effect of histolyticus infection and distribution, and gene induction by probiotic sequestration by neutrophils. Similarly, toxin on transplantable mouse tumors. Proc. Soc. Exp. Escherichia coli Nissle 1917 in live mice. Int. J. Med. Biol. Med. 66, 461–467 (1947). Microbiol. 297, 151–162 (2007). genetic stability could be enhanced by incor- 7. Malmgren, R. A. & Flanigan, C. C. Localization of the 30. Nuyts, S. et al. The use of radiation-induced bacterial porating engineered genes on the bacterial vegetative form of Clostridium tetani in mouse tumor promoters in anaerobic conditions: a means to control following intravenous spore administration. Cancer gene expression in clostridium-mediated therapy for chromosome and by limiting homologous Res. 15, 473–478 (1955). cancer. Radiat. Res. 155, 716–723 (2001). recombination and horizontal gene transfer. 8. Kohwi, Y., Imai, K., Tamura, Z. & Hashimoto, Y. 31. Zhao, M. et al. Tumor-targeting bacterial therapy with Antitumor effect of Bifidobacterium infantis in mice. amino acid auxotrophs of GFP-expressing Salmonella This moment in history is a turning point Gann 69, 613–618 (1978). typhimurium. Proc. Natl Acad. Sci. USA 102, for bacterial therapies. The preliminary 9. Bhatnagar, P. K., Awasthi, A., Nomellini, J. F., Smit, J. 755–760 (2005). & Suresh, M. R. Anti-tumor effects of the bacterium 32. Hoffman, R. M. & Zhao, M. Whole-body imaging of proof-of-concept experiments have dem- caulobacter crescentus in murine tumor models. bacterial infection and antibiotic response. Nature onstrated the vast capacity of bacteria for Cancer Biol. Ther. 5, 485–491 (2006). Protoc. 1, 2988–2994 (2006). 10. Pan, Z. K., Weiskirch, L. M. & Paterson, Y. Regression 33. Benoit, M. R. et al. Visualizing implanted tumors in mice treating cancer and have illustrated the large of established B16F10 melanoma with a recombinant with magnetic resonance imaging using magnetotactic number of effective tools that these robot Listeria monocytogenes vaccine. Cancer Res. 59, bacteria. Clin. Cancer Res. 15, 5170–5177 (2009). 5264–5269 (1999). 34. Tjuvajev, J. et al. Salmonella-based tumor-targeted factories possess. The ultimate bacterial 11. Kim, S. H., Castro, F., Paterson, Y. & Gravekamp, C. cancer therapy: tumor amplified protein expression therapy will consist of a collection of strains High efficacy of a Listeria-based vaccine against therapy (TAPETTM) for diagnostic imaging. J. Control. metastatic breast cancer reveals a dual mode of Release 74, 313–315 (2001). designed for specialized purposes rather action. Cancer Res. 69, 5860–5866 (2009). 35. Soghomonyan, S. A. et al. Positron emission than a single perfect strain. Successful treat- 12. Arakawa, M., Sugiura, K., Reilly, H. C. & Stock, C. C. tomography (PET) imaging of tumor-localized Oncolytic effect of Proteus mirabilis upon tumor- Salmonella expressing HSV1-TK. Cancer Gene Ther. ment could use these strains cooperatively bearing animals. II. Effect on transplantable mouse 12, 101–108 (2005). and in combination with molecular chemo- and rat tumors. Gann 59, 117–122 (1968). 36. Brader, P. et al. Escherichia coli Nissle 1917 facilitates 13. Maletzki, C., Linnebacher, M., Kreikemeyer, B. & tumor detection by positron emission tomography and therapy: a detectable facultative anaerobe Emmrich, J. Pancreatic cancer regression by optical imaging. Clin. Cancer Res. 14, 2295–2302 could be used for diagnosis; an engineered intratumoural injection of live Streptococcus pyogenes (2008). in a syngeneic mouse model. Gut 57, 483–491 37. Nagakura, C. et al. Efficacy of a genetically-modified immunogenic stain could be used to sen- (2008). Salmonella typhimurium in an orthotopic human sitize the immune system; an obligate 14. Dang, L. H., Bettegowda, C., Huso, D. L., Kinzler, K. W. pancreatic cancer in nude mice. Anticancer Res. 29, & Vogelstein, B. Combination bacteriolytic therapy for 1873–1878 (2009). anaerobe could be used to treat inoperable the treatment of experimental tumors. Proc. Natl 38. Lambin, P. et al. Colonisation of Clostridium in the primary tumours; and a motile Salmonella Acad. Sci. USA 98, 15155–15160 (2001). body is restricted to hypoxic and necrotic areas of 15. Kasinskas, R. W. & Forbes, N. S. Salmonella tumours. Anaerobe 4, 183–188 (1998). strain that controllably produces a cytotoxic typhimurium specifically chemotax and proliferate in 39. Minton, N. P. Clostridia in cancer therapy. Nature Rev. agent could be used to treat diffuse tumours heterogeneous tumor tissue in vitro. Biotechnol. Microbiol. 1, 237–242 (2003). Bioeng. 94, 710–721 (2006). 40. Forbes, N. S., Munn, L. L., Fukumura, D. & Jain, R. K. and metastatic disease. All bacterial thera- 16. Kasinskas, R. W. & Forbes, N. S. Salmonella Sparse initial entrapment of systemically injected pies will be in used in combination with typhimurium lacking ribose chemoreceptors localize in Salmonella typhimurium leads to heterogeneous 14,18,107–110 tumor quiescence and induce apoptosis. Cancer Res. accumulation within tumors. Cancer Res. 63, other therapeutics (TABLES 1,2), 67, 3201–3209 (2007). 5188–5193 (2003). which will have a synergistic effect: small 17. Nguyen, V. H. et al. Genetically engineered Salmonella 41. Leschner, S. et al. Tumor invasion of Salmonella typhimurium as an imageable therapeutic probe for enterica serovar Typhimurium is accompanied by molecules would kill cancer cells close to cancer. Cancer Res. 70, 18–23 (2010). strong hemorrhage promoted by TNF-α. PLoS ONE 4, blood vessels and bacteria would kill cells far 18. Jiang, S. N. et al. Inhibition of tumor growth and e6692 (2009). metastasis by a combination of Escherichia coli- 42. Sznol, M., Lin, S. L., Bermudes, D., Zheng, L. M. & from vessels (FIG. 2). The greatest strength of mediated cytolytic therapy and radiotherapy. Mol. King, I. Use of preferentially replicating bacteria for bacterial therapies is their genetic flexibility, Ther. 18, 635–642 (2010). the treatment of cancer. J. Clin. Invest. 105, 19. Ryan, R. M. et al. Bacterial delivery of a novel cytolysin 1027–1030 (2000). which enables tuning for individualized to hypoxic areas of solid tumors. Gene Ther. 16, 43. Clairmont, C. et al. Biodistribution and genetic therapy, targeting to multiple tumour sites 329–339 (2009). stability of the novel antitumor agent VNP20009, a 20. Loeffler, M., Le’Negrate, G., Krajewska, M. & genetically modified strain of Salmonella typhimurium. and the precise control of cytotoxicity. Once Reed, J. C. Attenuated Salmonella engineered to J. Infect. Dis. 181, 1996–2002 (2000). perfected, anticancer bacteria are expected produce human cytokine LIGHT inhibit tumor 44. Lee, C. H., Wu, C. L. & Shiau, A. L. Endostatin gene growth. Proc. Natl Acad. Sci. USA 104, therapy delivered by Salmonella choleraesuis in murine to be an essential clinical tool, able to carry 12879–12883 (2007). tumor models. J. Gene Med. 6, 1382–1393 (2004). out functions that are unachievable by other 21. Loeffler, M., Le’Negrate, G., Krajewska, M. & 45. Zheng, L. M. et al. Tumor amplified protein expression Reed, J. C. Salmonella typhimurium engineered to therapy: Salmonella as a tumor-selective protein therapies, and which can detect, prevent, and produce CCL21 inhibit tumor growth. Cancer delivery vector. Oncol. Res. 12, 127–135 (2000). treat tumours and metastases. Immunol. Immunother. 58, 769–775 (2009). 46. Low, K. B. et al. Lipid A mutant Salmonella with 22. Gentschev, I. et al. Use of a recombinant Salmonella suppressed virulence and TNFα induction retain tumor- Neil S. Forbes is at the enterica serovar Typhimurium strain expressing C-Raf targeting in vivo. Nature Biotech. 17, 37–41 (1999). for protection against C-Raf induced lung adenoma in 47. Theys, J. et al. Repeated cycles of Clostridium-directed University of Massachusetts, Amherst, mice. BMC Cancer 5, 15 (2005). enzyme prodrug therapy result in sustained Department of Chemical Engineering, 23. Ganai, S., Arenas, R. B. & Forbes, N. S. Tumour- antitumour effects in vivo. Br. J. Cancer 95, 159 Goessmann Laboratory, targeted delivery of TRAIL using Salmonella 1212–1219 (2006). Amherst, Massachusetts 01003–9303, USA. typhimurium enhances breast cancer survival in mice. 48. Zhao, M. et al. Targeted therapy with a Salmonella Br. J. Cancer 101, 1683–1691 (2009). typhimurium leucine–arginine auxotroph cures e-mail: [email protected] 24. Loeffler, M., Le’Negrate, G., Krajewska, M. & Reed, orthotopic human breast tumors in nude mice. Cancer J. C. Inhibition of tumor growth using Salmonella Res. 66, 7647–7652 (2006). doi:10.1038/nrc2934 expressing Fas ligand. J. Natl Cancer Inst. 100, 49. Streilein, J. W. Unraveling immune privilege. Science Published online 14 October 2010 1113–1116 (2008). 270, 1158–1159 (1995).

50. Westphal, K., Leschner, S., Jablonska, J., Loessner, H. 75. Sorenson, B. S., Banton, K. L., Frykman, N. L., 97. Yang, N., Zhu, X., Chen, L., Li, S. & Ren, D. Oral & Weiss, S. Containment of tumor-colonizing bacteria Leonard, A. S. & Saltzman, D. A. Attenuated Salmonella administration of attenuated S. typhimurium carrying by host neutrophils. Cancer Res. 68, 2952–2960 typhimurium with interleukin 2 gene prevents the shRNA-expressing vectors as a cancer therapeutic. (2008). establishment of pulmonary metastases in a model of Cancer Biol. Ther. 7, 145–151 (2008). 51. Lee, C. H., Wu, C. L. & Shiau, A. L. Systemic osteosarcoma. J. Pediatr. Surg. 43, 1153–1158 (2008). 98. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. administration of attenuated Salmonella choleraesuis 76. Sorenson, B. S., Banton, K. L., Frykman, N. L., Tight regulation, modulation, and high-level expression carrying thrombospondin-1 gene leads to tumor- Leonard, A. S. & Saltzman, D. A. Attenuated by vectors containing the arabinose PBAD promoter. specific transgene expression, delayed tumor growth Salmonella typhimurium with IL-2 gene reduces J. Bacteriol. 177, 4121–4130 (1995). and prolonged survival in the murine melanoma pulmonary metastases in murine osteosarcoma. Clin. 99. Royo, J. L. et al. In vivo gene regulation in Salmonella model. Cancer Gene Ther. 12, 175–184 (2005). Orthop. Relat. Res. 466, 1285–1291 (2008). spp. by a salicylate-dependent control circuit. Nature 52. Lee, C. H., Wu, C. L., Tai, Y. S. & Shiau, A. L. Systemic 77. Al-Ramadi, B. K. et al. Potent anti-tumor activity of Methods 4, 937–942 (2007). administration of attenuated Salmonella choleraesuis systemically-administered IL-2-expressing Salmonella 100. Nuyts, S. et al. Insertion or deletion of the Cheo box in combination with cisplatin for cancer therapy. Mol. correlates with decreased angiogenesis and enhanced modifies radiation inducibility of Clostridium promoters. Ther. 11, 707–716 (2005). tumor apoptosis. Clin. Immunol. 130, 89–97 (2009). Appl. Environ. Microbiol. 67, 4464–4470 (2001). 53. Vaupel, P., Kallinowski, F. & Okunieff, P. Blood flow, 78. Barnett, S. J. et al. Attenuated Salmonella 101. Mengesha, A. et al. Development of a flexible and oxygen and nutrient supply, and metabolic typhimurium invades and decreases tumor burden in potent hypoxia-inducible promoter for tumor-targeted microenvironment of human tumors: a review. Cancer neuroblastoma. J. Pediatr. Surg. 40, 993–997 (2005). gene expression in attenuated Salmonella. Cancer Res. 49, 6449–6465 (1989). 79. Feltis, B. A. et al. Liver and circulating NK1.1+CD3- Biol. Ther. 5, 1120–1128 (2006). 54. Heimann, D. M. & Rosenberg, S. A. Continuous cells are increased in infection with attenuated 102. Strauch, K. L., Lenk, J. B., Gamble, B. L. & Miller, C. G. intravenous administration of live genetically modified Salmonella typhimurium and are associated with Oxygen regulation in Salmonella typhimurium. Salmonella typhimurium in patients with metastatic reduced tumor in murine liver cancer. J. Surg. Res. J. Bacteriol. 161, 673–680 (1985). melanoma. J. Immunother. 26, 179–180 (2003). 107, 101–107 (2002). 103. Arrach, N., Zhao, M., Porwollik, S., Hoffman, R. M. & 55. Toso, J. F. et al. Phase I study of the intravenous 80. Saltzman, D. A. et al. Antitumor mechanisms of McClelland, M. Salmonella promoters preferentially administration of attenuated Salmonella typhimurium attenuated Salmonella typhimurium containing the activated inside tumors. Cancer Res. 68, 4827–4832 to patients with metastatic melanoma. J. Clin. Oncol. gene for human interleukin-2: a novel antitumor (2008). 20, 142–152 (2002). agent? J. Pediatr. Surg. 32, 301–306 (1997). 104. Min., J. J. et al. Noninvasive real-time imaging of tumors 56. Nemunaitis, J. et al. Pilot trial of genetically modified, 81. Lee, S. R. et al. Multi-immunogenic outer membrane and metastases using tumor-targeting light-emitting attenuated Salmonella expressing the E. coli cytosine vesicles derived from a MsbB-deficient Salmonella Escherichia coli. Mol. Imaging Biol. 10, 54–61 (2008). deaminase gene in refractory cancer patients. Cancer enterica serovar typhimurium mutant. J. Microbiol. 105. Min., J. J., Nguyen, V. H., Kim, H. J., Hong, Y. J. & Gene Ther. 10, 737–744 (2003). Biotechnol. 19, 1271–1279 (2009). Choy, H. E. Quantitative bioluminescence imaging of 57. Hall, S. S. A Commotion in the Blood: Life, Death, and 82. Nishikawa, H. et al. In vivo antigen delivery by a tumor-targeting bacteria in living animals. Nature the Immune System (Henry Holt, New York, 1997). Salmonella typhimurium type III secretion system for Protoc. 3, 629–636 (2008). 58. Coley, W. B. Contribution to the knowledge of therapeutic cancer vaccines. J. Clin. Invest. 116, 106. Cheng, C. M. et al. Tumor-targeting prodrug-activating sarcoma. Ann. Surg. 14, 199–220 (1891). 1946–1954 (2006). bacteria for cancer therapy. Cancer Gene Ther. 15, 59. Nauts, H. C., Swift, W. E. & Coley, B. L. The treatment 83. Groot, A. J. et al. Functional antibodies produced by 393–401 (2008). of malignant tumors by bacterial toxins as developed oncolytic clostridia. Biochem. Biophys. Res. Commun. 107. Gericke, D. & Engelbart, K. Oncolysis by Clostridia.II. by the late William B. Coley, MD, reviewed in the light 364, 985–989 (2007). Experiments on tumor spectrum with variety of of modern research. Cancer Res. 6, 205–216 (1946). 84. Sizemore, D. R., Branstrom, A. A. & Sadoff, J. C. Clostridia in combination with heavy metal. Cancer 60. Fensterle, J. et al. Cancer immunotherapy based on Attenuated Shigella as a DNA delivery vehicle for DNA- Res. 24, 217–221 (1964). recombinant Salmonella enterica serovar Typhimurium mediated immunization. Science 270, 299–302 (1995). 108. Dang, L. H. et al. Targeting vascular and avascular aroA strains secreting prostate-specific antigen and 85. Darji, A. et al. Oral somatic transgene vaccination compartments of tumors with C. novyi-NT and anti- cholera toxin subunit B. Cancer Gene Ther. 15, 85–93 using attenuated S. typhimurium. Cell 91, 765–775 microtubule agents. Cancer Biol. Ther. 3, 326–337 (2008). (1997). (2004). 61. Mottram, J. C. Factors of importance in 86. Weiss, S. & Chakraborty, T. Transfer of eukaryotic 109. Bettegowda, C. et al. Overcoming the hypoxic barrier radiosensitivity of tumors. Br. J. Radiol. 9, 606–614 expression plasmids to mammalian host cells by to radiation therapy with anaerobic bacteria. Proc. (1936). bacterial carriers. Curr. Opin. Biotechnol. 12, Natl Acad. Sci. USA 100, 15083–15088 (2003). 62. Möse, J. R. & Möse, G. Oncogenesis by clostridia. I. 467–472 (2001). 110. Cheong, I. et al. A bacterial protein enhances the Activity of Clostridium butyricum (M-55) and other 87. Palffy, R. et al. Bacteria in gene therapy: bactofection release and efficacy of liposomal cancer drugs. Science nonpathogenic clostridia against the Ehrlich versus alternative gene therapy. Gene Ther. 13, 314, 1308–1311 (2006). carcinoma. Cancer Res. 24, 212–216 (1964). 101–105 (2006). 111. Voigt, C. A. Genetic parts to program bacteria. Curr. 63. Carey, R. W., Holland, J. F., Whang, H. Y., Neter, E. & 88. Fu, W., Chu, L., Han, X. W., Liu, X. Y. & Ren, D. M. Opin. Biotechnol. 17, 548–557 (2006). Bryant, B. Clostridial oncolysis in man. Eur. J. Cancer Synergistic antitumoral effects of human telomerase 112. Pfleger, B. F., Pitera, D. J., Smolke, C. D. & Keasling, 3, 37–46 (1967). reverse transcriptase-mediated dual-apoptosis-related J. D. Combinatorial engineering of intergenic regions 64. Lee, C. H., Wu, C. L. & Shiau, A. L. Salmonella gene vector delivered by orally attenuated Salmonella in operons tunes expression of multiple genes. Nature choleraesuis as an anticancer agent in a syngeneic enterica serovar Typhimurium in murine tumor Biotech. 24, 1027–1032 (2006). model of orthotopic hepatocellular carcinoma. Int. models. J. Gene Med. 10, 690–701 (2008). 113. Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated J. Cancer 122, 930–935 (2008). 89. Li, Y. H. et al. Prophylaxis of tumor through oral design of synthetic ribosome binding sites to control 65. Thamm, D. H. et al. Systemic administration of an administration of IL-12 GM-CSF gene carried by live protein expression. Nature Biotech. 27, 946–950 attenuated, tumor-targeting Salmonella typhimurium attenuated Salmonella. Chin. Sci. Bull. 46, (2009). to dogs with spontaneous neoplasia: Phase I 1107–1112 (2001). 114. Ohl, M. E. & Miller, S. I. Salmonella: a model for evaluation. Clin. Cancer Res. 11, 4827–4834 (2005). 90. Li, Y. H. et al. Oral cytokine gene therapy against bacterial pathogenesis. Annu. Rev. Med. 52, 66. Jia, L. J. et al. Oral delivery of tumor-targeting murine tumor using attenuated Salmonella 259–274 (2001). Salmonella for cancer therapy in murine tumor typhimurium. Int. J. Cancer 94, 438–443 (2001). 115. Engelbart, K. & Gericke, D. Oncolysis by Clostridia.V. models. Cancer Sci. 98, 1107–1112 (2007). 91. Qi, H., Li, Y. H. & Zheng, S. B. Oral gene therapy via Transplanted tumors of the hamster. Cancer Res. 24, 67. Chen, G. et al. Oral delivery of tumor-targeting live attenuated Salmonella leads to tumor regression 239–243 (1964). Salmonella exhibits promising therapeutic efficacy and and survival prolongation in mice. Nan Fang Yi Ke Da 116. Thiele, E. H., Boxer, G. E. & Arison, R. N. Oncolysis by low toxicity. Cancer Sci. 100, 2437–2443 (2009). Xue Xue Bao 26, 1738–1741 (2006). Clostridia.III. Effects of Clostridia and chemotherapeutic 68. Bermudes, D., Low, B. & Pawelek, J. Tumor-targeted 92. Yoon, W. S., Choi, W. C., Sin, J. I. & Park, Y. K. agents on rodent tumors. Cancer Res. 24, 222–233 Salmonella. Highly selective delivery vectors. Adv. Exp. Antitumor therapeutic effects of Salmonella (1964). Med. Biol. 465, 57–63 (2000). typhimurium containing Flt3 ligand expression 117. Mohr, U., Boldingh, W. H., Behagel, H. A. & 69. Hedley, D., Ogilvie, L. & Springer, C. plasmids in melanoma-bearing mouse. Biotechnol. Emminger, A. Oncolysis by a new strain of Clostridium. Carboxypeptidase-G2-based gene-directed enzyme- Lett. 29, 511–516 (2007). Cancer Res. 32, 1122–1128 (1972). prodrug therapy: a new weapon in the GDEPT 93. Zuo, S. G. et al. Orally administered DNA vaccine 118. Weibel, S., Stritzker, J., Eck, M., Goebel, W. & Szalay, armoury. Nature Rev. Cancer 7, 870–879 (2007). delivery by attenuated Salmonella typhimurium A. A. Colonization of experimental murine breast 70. Brown, J. M. & Wilson, W. R. Exploiting tumour targeting fetal liver kinase 1 inhibits murine Lewis lung tumours by Escherichia coli K-12 significantly alters hypoxia in cancer treatment. Nature Rev. Cancer 4, carcinoma growth and metastasis. Biol. Pharm. Bull. the tumour microenvironment. Cell. Microbiol. 10, 437–447 (2004). 33, 174–182 (2010). 1235–1248 (2008). 71. Walczak, H. et al. Tumoricidal activity of tumor 94. Feng, K. et al. Anti-angiogenesis effect on glioma of 119. Luo, X. et al. Antitumor effect of VNP20009, an necrosis factor-related apoptosis-inducing ligand attenuated Salmonella typhimurium vaccine strain attenuated Salmonella, in murine tumor models. in vivo. Nature Med. 5, 157–163 (1999). with flk-1 gene. J. Huazhong Univ. Sci. Technol. Med. Oncol. Res. 12, 501–508 (2001). 72. Barbe, S. et al. Secretory production of biologically Sci. 24, 389–391 (2004). 120. Rosenberg, S. A., Spiess, P. J. & Kleiner, D. E. active rat interleukin-2 by Clostridium acetobutylicum 95. Chou, C. K., Hung., J. Y., Liu, J. C., Chen, C. T. & Hung., Antitumor effects in mice of the intravenous injection DSM792 as a tool for anti-tumor treatment. FEMS M. C. An attenuated Salmonella oral DNA vaccine of attenuated Salmonella typhimurium. Microbiol. Lett. 246, 67–73 (2005). prevents the growth of hepatocellular carcinoma and J. Immunother. 25, 218–225 (2002). 73. Saltzman, D. A. et al. Attenuated Salmonella colon cancer that express α-fetoprotein. Cancer Gene 121. Zhao, M. et al. Monotherapy with a tumor-targeting typhimurium containing interleukin-2 decreases Ther. 13, 746–752 (2006). mutant of Salmonella typhimurium cures orthotopic MC-38 hepatic metastases: a novel anti-tumor agent. 96. Zhang, L. et al. Intratumoral delivery and suppression metastatic mouse models of human prostate cancer. Cancer Biother. Radiopharm. 11, 145–153 (1996). of prostate tumor growth by attenuated Salmonella Proc. Natl Acad. Sci. USA 104, 10170–10174 (2007). 74. Loeffler, M., Le’Negrate, G., Krajewska, M. & Reed, enterica serovar typhimurium carrying plasmid-based 122. Kimura, H. et al. Targeted therapy of spinal cord J. C. IL-18-producing Salmonella inhibit tumor growth. small interfering RNAs. Cancer Res. 67, 5859–5864 glioma with a genetically modified Salmonella Cancer Gene Ther. 15, 787–794 (2008). (2007). typhimurium. Cell Prolif. 43, 41–48 (2010).

123. Jia, L. J. et al. Enhanced therapeutic effect by 133. Fu, W., Lan, H. K., Liang, S. H., Gao, T. & Ren, D. M. combination of tumor-targeting Salmonella and Suicide gene/prodrug therapy using Salmonella- endostatin in murine melanoma model. Cancer Biol. mediated delivery of Escherichia coli purine nucleoside Ther. 4, 840–845 (2005). phosphorylase gene and 6-methoxypurine 124. Platt, J. et al. Antitumour effects of genetically 2′-deoxyriboside in murine mammary carcinoma 4T1 engineered Salmonella in combination with radiation. model. Cancer Sci. 99, 1172–1179 (2008). Eur. J. Cancer 36, 2397–2402 (2000). 134. Mei, S., Theys, J., Landuyt, W., Anne, J. & Lambin, P. 125. Shilling, D. A. et al. Salmonella typhimurium Optimization of tumor-targeted gene delivery by stimulation combined with tumour-derived heat shock engineered attenuated Salmonella typhimurium. proteins induces potent dendritic cell anti-tumour Anticancer Res. 22, 3261–3266 (2002). responses in a murine model. Clin. Exp. Immunol. 135. Hayashi, K. et al. Cancer metastasis directly eradicated 149, 109–116 (2007). by targeted therapy with a modified Salmonella 126. Al-Ramadi, B. K. et al. Attenuated bacteria as effectors typhimurium. J. Cell. Biochem. 106, 992–998 (2009). in cancer immunotherapy. Ann. N. Y. Acad. Sci. 1138, 136. Hayashi, K. et al. Systemic targeting of primary bone 351–357 (2008). tumor and lung metastasis of high-grade 127. Liu, S. C., Minton, N. P., Giaccia, A. J. & Brown, J. M. osteosarcoma in nude mice with a tumor-selective Anticancer efficacy of systemically delivered anaerobic strain of Salmonella typhimurium. Cell Cycle 8, bacteria as gene therapy vectors targeting tumor 870–875 (2009). hypoxia/necrosis. Gene Ther. 9, 291–296 (2002). 137. Dresselaers, T. et al. Non-invasive 19F MR 128. Liu, S. C. et al. Optimized Clostridium-directed enzyme spectroscopy of 5-fluorocytosine to 5-fluorouracil prodrug therapy improves the antitumor activity of the conversion by recombinant Salmonella in tumours. Br. novel DNA cross-linking agent PR-104. Cancer Res. J. Cancer 89, 1796–1801 (2003). 68, 7995–8003 (2008). 138. Heppner, F. & Mose, J. R. The liquefaction (oncolysis) 129. Dubois, L. et al. Efficacy of gene therapy-delivered of malignant gliomas by a non pathogenic Clostridium. cytosine deaminase is determined by enzymatic activity Acta Neurochir. (Wien) 42, 123–125 (1978). but not expression. Br. J. Cancer 96, 758–761 (2007). 130. Jazowiecka-Rakus, J. & Szala, S. Antitumour activity of Acknowledgements Salmonella typhimurium VNP20047 in B16(F10) This work was partly supported by the US National Institutes murine melanoma model. Acta Biochim. Pol. 51, of Health, National Cancer Institute grant CA120825. 851–856 (2004). 131. Friedlos, F. et al. Attenuated Salmonella targets Competing interests statement prodrug activating enzyme carboxypeptidase G2 to The author declares no competing financial interests. mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy. Clin. Cancer Res. 14, 4259–4266 (2008). FURTHER INFORMATION 132. Fu, W. et al. Synergistic antitumor efficacy of suicide/ Neil s. Forbes’s homepage: ePNP gene and 6-methylpurine 2′-deoxyriboside via http://www.ecs.umass.edu/~forbes/ Salmonella against murine tumors. Cancer Gene Ther. all linkS are aCTive in The online pdf 15, 474–484 (2008).