Bacteria and genetically modified bacteria as cancer therapeutics: Current advances and challenges.

Cytokine xxx (2016) xxx–xxx

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Cytokine journal homepage: www.journals.elsevier.com/cytokine

Bacteria and genetically modified bacteria as cancer therapeutics: Current advances and challenges Shreeram C. Nallar a,⇑, De-Qi Xu b, Dhan V. Kalvakolanu a,⇑ a b

Department of Microbiology & Immunology, Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA Dalian Hissen Biopharm Co Ltd. E&T Development Zone, Dalian 116600, Peoples Republic of China

a r t i c l e

i n f o

Article history: Received 30 October 2015 Received in revised form 5 January 2016 Accepted 6 January 2016 Available online xxxx Keywords: Cancer Bio-therapy Bacterial vectors Tumor suppression Immune response

a b s t r a c t Bacteria act as pro- or anti- tumorigenic agents. Whole bacteria or cytotoxic or immunogenic peptides carried by them exert potent anti-tumor effects in the experimental models of cancer. The use of attenuated microorganism(s) e.g., BCG to treat human urinary bladder cancer was found to be superior compared to standard chemotherapy. Although the phase-I clinical trials with Salmonella enterica serovar Typhimurium, has shown limited benefits in human subjects, a recent pre-clinical trial in pet dogs with tumors reported some subjects benefited from this treatment strain. In addition to the attenuated host strains derived by conventional mutagenesis, recombinant DNA technology has been applied to a few microorganisms that have been evaluated in the context of tumor colonization and eradication using mouse models. There is an enormous surge in publications describing bacterial anti-cancer therapies in the past 15 years. Vectors for delivering shRNAs that target oncogenic products, express tumor suppressor genes and immunogenic proteins have been developed. These approaches have showed promising anti-tumor activity in mouse models against various tumors. These can be potential therapeutics for humans in the future. In this review, some conceptual and practical issues on how to improve these agents for human applications are discussed. Ó 2016 Published by Elsevier Ltd.

1. Introduction Chemotherapy, although widely used for treating many tumors, causes many off-target effects such as significant damage to normal tissues. In contrast biological therapeutics, such as antibodies, Abbreviations: BC, breast cancer; CC, cervical cancer; CMV, cytomegalovirus; CRC, colorectal cancer; HCC, hepatocellular cancer; Hly, hemolysin, an exotoxin produced by bacteria.; HSC, hematopoietic stem cells; LC, lung cancer; LPS, lipopolysaccharide; PC, prostate cancer; PSA, prostate-specific antigen; RCC, renal cell cancer; SCC, squamous cell cancer; SPI, Salmonella pathogenicity islands; DLT, dose-limiting toxicity – a single minimal dose of a therapeutic agent that results in unacceptable side effects or toxicity in a cohort; MTD, maximum tolerated dose – a single maximal dose of a therapeutic agent that produces the desired response(s) with acceptable side effects or toxicity in a general population. This value is less than DLT; Exotoxins, a family of bacterial proteins secreted or injected using type-I/ II secretory apparatus targeting specific host components. Toxins that target host cytosolic proteins include Cholera, Diphtheria, Pertussis and Shiga toxins while another group that creates pores on host cell membrane includes cholesteroldependent cytolysins and RTX toxins; Epitope spread, a phenomenon where multiple antigen-specific immune responses are triggered that are unrelated, in sequence or structure, to the primary immunogen. ⇑ Corresponding authors. E-mail addresses: [email protected] (S.C. Nallar), dkalvako@umaryland. edu (D.V. Kalvakolanu).

peptide-mimetics and a few cytokines, generally exert targetspecific effects and are relatively safer for human use. Although attenuated viruses and bacteria have been successfully used for generating immunity to potential pathogens, their general use for tumor therapy is still lagging. The isolation of Streptococcus pyogenes from sarcoma by Busch [1] and neck cancer by Coley [2] are historical landmarks that suggested the potential use of bacteria to treat human cancers. The use of microorganism(s) to treat human cancers provided a short term benefit, but eventually tumors recurred [3]. Recombinant viruses with tumoricidal activity have been developed over the years. Major obstacles with these viral vectors include lack of the specificity toward cancer cells, short half-life of the vector, limited tropism for antigenpresenting cells, pre-existing immunity, rapid development of neutralizing antibodies to the viral coat proteins, etc., which limits their utility as vehicles to elicit tumoricidal activities and/or gene therapy. A major step in the development of bacterial therapeutics is the identification of potential species and strains with minimal pathogenicity to the host. In the past century, many genera of bacteria have been isolated and/or identified in and around various tumors (see Cummins and Tangney [4] for additional details).

http://dx.doi.org/10.1016/j.cyto.2016.01.002 1043-4666/Ó 2016 Published by Elsevier Ltd.

Please cite this article in press as: S.C. Nallar et al., Bacteria and genetically modified bacteria as cancer therapeutics: Current advances and challenges, Cytokine (2016), http://dx.doi.org/10.1016/j.cyto.2016.01.002

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S.C. Nallar et al. / Cytokine xxx (2016) xxx–xxx

Extracellular and intracellular bacteria, both gram-positive and gram-negative, have been isolated. The ability of such bacteria to successfully colonize regions of implanted tumors in experimental animals has also been addressed and only a few had the potential to home into tumors [5] (see Table 1). Upon bacterial administration, each of these preferentially accumulated and grew in tumor vicinity in a time-dependent manner when compared to most other host tissues (Tables 2 and 3). Hence, it is possible that the tumor microenvironment may be more conducive for bacterial survival and/or growth as it may provide protection from the host immune system and/or nutrients (see Table 1). Even though the precise mechanism behind this selective homing is not understood, hemodynamics [6] and/or chemotaxis [7] appear to be essential for this process. Survival and growth of bacteria homed into tumors appears to be dependent on their oxygen requirement and other characteristics such as intra/extracellular mode of survival, and motility. So far, facultative and obligate anaerobic, intracellular and extracellular bacteria have shown tremendous success in colonizing tumors implanted in mouse. In this review we will focus more on Salmonella enterica subsp enterica serovar Typhimurium (hereafter S. Typhimurium), a rod-shaped flagellated gramnegative non-spore forming facultative anaerobic bacteria, closely related to Escherichia coli a commensal in the intestines of vertebrates, as a broad spectrum vaccine host and in cancer therapy/ immunity. The genus Salmonella, a gammaproteobacterial member, is represented by two species viz., bongori and enterica. There are Table 1 Bacteria home and replicate in tumor microenvironment. Bifidobacteria B. adolescentis B. animalis B. bifidum B. boum B. breve B. coryneforme B. dentium B. indicum B. infantis B. longum B. magnum B. pseudolongum Lactobacilli L. bifidus L. delbrueckii Clostridia C. absonum C. acetobutylicum C. bifermentans C. difficile C. histolyticum C. perfringens C. novyi – exhibited extensive spreading even in poorly-vascularized tumor areas C. sordellii – exhibited extensive spreading even in poorly-vascularized tumor areas Murine tumors B16 melanoma M27 lung carcinoma Spontaneous breast tumor

Tumor:Liver 12,000:1 10,000:1 700:1

Human tumor xenografts MDA-MB-231 breast carcinoma DU145 prostate HCT 116 colon carcinoma DLD1 colon carcinoma HTB177 lung carcinoma LOX melanoma A549 lung carcinoma SW-620 colon carcinoma

34,000:1 24,000:1 17,000:1 15,000:1 4000:1 3000:1 300:1 275:1

Based on Dang et al. [5] and Sznol et al. [21] with minor modifications.

six subspecies viz., enterica, salamae, arizonae, diarizonae, houtenae and indica, of which subspecies enterica is a medical concern. The subspecies enterica is further classified into typhoidal serovars (represented by Typhi, Paratyphi (A, B, C), Sendai, etc.) and nontyphoidal serovars (NTS, represented by Choleraesuis, Dublin, Enteritidis, Infantis, Typhimurium, etc.) based on biochemical signatures on flagella, capsule, carbohydrates and LPS. More than 2500 serovars of S. enterica have been described. In a natural setting, the typhoidal serovars cause enteric or typhoid fever, a systemic disease mostly restricted to humans, while the NTS that cause a self-limiting enteritis and/or diarrhea is prevalent in many mammals. Currently, two commercially available vaccines (one administered orally and the other injectable) against serovar Typhi are used more commonly in poultry and swine industries. Although both of these vaccine formats are safe for human use, it requires regular boosters to maintain effective immunity. Thus, the collective natural features and the ease of genetic manipulation of S. Typhimurium have been exploited for targeting experimental tumors in mouse models. Such modified strain(s) i.e., rendered less toxic, when administered intravenously has the potential to target distant tumors. As the major cell wall component of gram-negative bacteria, LPS is a key determinant to the pathogen’s success in colonizing host tissues vis-à-vis infection. Biochemically LPS is a phosphoglycolipid; it can be separated into three modules viz., a central core oligosaccharide, a peripheral variable polysaccharide and a lipid portion. The central portion of core oligosaccharide is composed of 8-carbon sugars called KDO (3-deoxy-D-mannooctulosonic acid). One of the 8-carbon sugars is covalently linked to a 6-carbon sugar while the opposing 8-carbon sugar is covalently linked to a 7-carbon sugar. Core oligosaccharide is highly diverse among bacterial species or even within strains of a bacterial species [8]. The 6-carbon sugars are acylated to form the Lipid A component that is embedded in the bacterial outer membrane. The other side containing the 7-carbon sugars is glycosylated to form the O antigen that projects into the extracellular medium (Fig. 1). The O antigen is a highly variable polysaccharide component that forms the basis of ‘Rough’ and ‘Smooth’ strain classification. The core oligosaccharide and 6-carbon sugars attached to the Lipid A bear phosphate residues that are essential for virulence. The common feature among the experimental strains is that they are rendered less toxic i.e., well tolerated, to host cells as they are administered in high doses directly into systemic circulation. This has been accomplished either by direct inactivation of genes coding for the enzymatic components responsible for the linkages in the LPS or by indirect means by targeting gene products that contribute to survival in specific niches (Table 2 and Fig. 1). Since these two components are interdependent virulence factors, strong immune reactions from the host are dampened. In these strategies, bacteria are selected for those that retain the ability to colonize host tissues i.e., tumors and incapable of causing disease e.g., S. Typhimurium strain VNP20009 cannot synthesize wild-type LPS, so it is less immunogenic to humans while strain LH430 is deficient in pho regulators; a two-component system essential for environmental sensing, adaptation and survival [9]. Also some strains are engineered with an additional mutation(s) in other locus/loci to prevent unanticipated reversion to wild-type genetic background (Table 2) e.g. strain VNP20009 is also a purine auxotroph. However, reversion is common in strains mutant for aromatic and/or hydrophobic amino acid metabolism; though the mechanism remains unknown. Hence, such vectors are not suitable for targeting tumors growing in vivo. The development of an attenuated serovar Typhimurium VNP20009 that retained the ability colonize experimental tumors in mice without eliciting strong immune and/or toxic side effects paved way for phase-I clinical trials in humans [10–12] and


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S.C. Nallar et al. / Cytokine xxx (2016) xxx–xxx Table 2 Bacteria tested in controlling experimental tumors. Host

Tumor

Site

Route

Outcome(s)a

Effector(s)

Mice (Nude)

Melanoma, breast, lung, colorectal cancer cell lines – Allo/Xenografts

s.c.

i.v.

Tumor growth reduced [20,21]

Independent of T or B cells

BALB/c nude BALB/c

s.c. s.c.

i.v. p.o.

C57BL/6

SiHa (CC – Xenograft) CMS5 (Sarcoma – Syngeneic) expressing human NY-ESO-1 H22 (HCC – Syngeneic)

Orthotopic

p.o.

Tumor growth reduced [60] Only NY-ESO-1-positive tumors are eliminated [36] Tumor growth reduced [57]

C57BL/6 C57BL/6

RM1 (PC – Syngeneic) RM1 (PC – Syngeneic)

Orthotopic s.c.

i.v. i.v.

Tumor growth reduced [56] Tumor growth reduced [59]

Apoptosis CD8+ cell mediated CD8+ cell mediated Apoptosis Apoptosis

BALB/c DBA/2

CT-26 (CRC – Syngeneic) P815 (Mastocytoma – Syngeneic)

s.c. s.c.

i.v. p.o.


Nude mice C57BL/6 BALB/c

MARY-X (BC – Xenograft) B16G3.26 (Melanoma – Syngeneic) CT-26 (CRC – Syngeneic)

Orthotopic s.c. i.v.

i.v. p.o. p.o.

Tumor growth reduced [96] Tumor growth and metastases reduced [38]

BALB/c nude C57BL/6 BALB/c BALB/c NZW

HCT116 (CRC – Xenograft) B16 (Melanoma – Syngeneic) CT-26 (CRC – Syngeneic) RENCA (CRC – Syngeneic) VX2 (rabbit HCC – Syngeneic)

s.c. s.c. s.c. s.c. s.c.

i.v. i.v. i.v. i.v. i.v.

Prolonged survival with reduced tumor burden [97] Tumor-specific immunity with variable penetrance [37]

Not reported

E. coli K-12

BALB/c BALB/c

CT-26 (CRC – Syngeneic) 4T1 (BC – Syngeneic)

s.c. s.c.

i.v. i.v.

Tumor growth reduced [98] Metastases reduced [99]

Not reported Fibrotic reaction

E. coli Nissle 1917f

BALB/c C57BL/6

4T1 B16

Orthotopic s.c.

i.v. i.v.


Not reported Not reported

Species (Strain) S. Typhimurium (VNP20009)

b

S. Typhimurium (LH430)c

S. Typhimurium (amino acid(s) auxotroph(s))d

C. novyi (NT)e

a b c d e f

Not reported CD8+ cell mediated Not reported CD8+ cell mediated

CD8+ cell mediated

With respect to control(s). A purine auxotroph (Dpur) lacking the secondary myristyl chain (DmsbB) in LPS. A two-component system mutant (DphoP DphoQ). Amino acid auxotrophs (Daro, Dhis, Darg, Dleu etc) – single or double mutants. A gram-positive soil-dwelling spore-forming obligate anaerobic opportunistic pathogen lacking phage-encoded major systemic toxin (Da toxin). A pro-biotic strain used to ease gastrointestinal discomfort in humans.

pre-clinical trials in dogs [13]. Since the outcomes of these trials were far below expectation, subsequent strategies aimed at eradication or reduction in growth/spread of xeno/allografted tumor(s) in mouse by S. Typhimurium has utilized various paradigms to target experimental tumors either in orthotopic or heterotopic settings. These include native toxicity of the bacterium, delivery of immune effectors, expression of cytotoxins, host antigens, growth-controlling agents, gene-silencing agents, activation of prodrugs, etc. Bacterial Type-I Secretion System (T1SS), Type-3 Secretion System (T3SS), intracellular release of mammalian expression cassettes, etc. have been utilized in such scenarios (Table 3). The following sections highlight the outcome(s) of such initiatives. Since it is not possible to cover every published article on this topic, due to space limits, we will cite select studies that offer significant improvements or rationale or a newer perspective. 2. Mechanisms of tumor suppression Bacteria naturally secrete exotoxins via their T1SS, to favor their survival. T1SS is a chaperone-dependent machinery utilizing proteins from hly and tol gene clusters. The secreted cargo can be ions, small molecules, proteins and polysaccharides. Clinically important T1SS cargo is the exotoxin e.g. a-hemolysin (HlyA) is a virulence factor for uropathogenic E. coli. This group of exotoxin creates pores on host cells and thus has the ability to lyse blood cells as well as tumor cells. The T1SS machinery was employed to deliver chimeric human PSA that invoked CD8+ cell-mediated responses against a PSA-expressing syngeneic mouse mastocytoma [14]; a mast cell tumor not known to express Klk1b22, the mouse version of PSA. Using the T1SS mechanism, later studies demonstrated reduction in tumor growth when HlyE was secreted by recombinant S. Typhimurium using arabinose-inducible [15] and hypoxia-inducible bacterial promoter [16] in syngeneic hosts

although the nature of anti-tumor response appeared not primarily dependent on CD8+ cells. An acidic pH-responsive promoter was developed to secrete Shiga toxin (Stx2) in the tumor microenvironment by recombinant S. Typhimurium [17] that induced tumor necrosis. When E. coli expressed a heterologous bacterial toxin viz., Listeriolysin-O (LLO), it could evoke CD8+ cell mediated antitumor response [18]. LLO, an exotoxin produced by the bacterium Listeria monocytogenes, is very similar to E. coli hemolysin. Similarly, when S. Typhimurium secreted a chimeric heterologous antigen, CD8+ cell mediated anti-tumor responses could be evoked [19]. So there are at least two ways of initiating anti-tumor responses viz., with the help of the immune system and even without it; earlier experiments performed before these reports had used nude mice to control experimental tumors with success [20,21]. With additional controllable gene component(s) present in the bacterium, it appears that anti-tumor response(s) can be increased much beyond what is achievable using native bacterial toxicity. Cell death-inducing agents: The most prevalent clinical approach to reduce tumor cells is to kill using radio/chemotherapy. The latter agents do not discriminate tumors from normal cells and so they affect non-target tissues. An alternative would be to use endogenous cell death-inducing molecules that can be delivered in a localized and controlled manner i.e., spatio-temporal control. As mentioned previously, the natural tumor tropism of S. Typhimurium to concentrate around tumors comes in handy to achieve this goal. TNF-related apoptosis-inducing ligand (TRAIL) is a secreted cytokine that induces cell death of tumor cells [22]. As a ligand for death receptors 4 and 5 (DR4 and DR5), it first activates initiator caspase-8 to stimulate downstream effector caspases (-3, -6 and -7) leading to apoptosis. Recombinant S. Typhimurium expressing murine Trail under the control of radiation-inducible recA promoter reduced syngeneic breast tumor growth in BALB/c


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Table 3 Bacteria engineered to express or deliver anti-tumor agents in mouse models. Agent(s) – adjuvant

Host

Tumor(s)

Outcome(s)

Effector(s)

Bacterial toxin S. Typhimurium secreting HlyE S. Typhimurium secreting Stx2 E. coli expressing LLO

BALB/c Nude mice C57BL/6

CT-26, 4T1 B16, HCT116, HeLa MBL2 (Leukemia – Syngeneic) TRAMP-C (PC – Syngeneic)

Reduction in tumor mass [15,16] Reduction in tumor mass [17] Reduction in tumor mass [18]

Not reported Necrosis CD8+ cell mediated

BALB/c

WEHI-164 (Fibrosarcoma) cells expressing Lm-p60

Antigen-specific tumor inhibition [19]

CD8+ cell mediated

BALB/c

CT-26 D2F2 (BC – Syngeneic) 4T1

Reduction in tumor mass [26]

Neutrophils

Tumor growth reduced [23]

Apoptosis

Bacterial antigen S. Typhimurium secreting L. monocytogenes Iap217–225 (Lm-p60) Death inducer S. Typhimurium secreting murine Fasl S. Typhimurium secreting murine Trail Diablo/Trail

Cytokine Ccl21

IL2

BALB/c BALB/c C57BL/6

4T1 LL/2 (LC – Syngeneic) B16F10 (Melanoma)

Tumor growth inhibition with prolonged survival [44]

Apoptosis

C57BL/6

D121 (LC – Syngeneic)

CD8+ cell mediated

BALB/C

D2F2, CT-26

Suppression of angiogenesis and growth of pulmonary metastasized tumors [101] Tumor-limited inflammatory reaction with a substantial reduction in tumor burden [34]

C57BL/6

MCA-38 (Adenocarcinoma – Syngeneic) K7M2 (Osteosarcoma – Syngeneic)

Hepatic metastases reduced [27]

NK cells

Pulmonary metastases reduced compared to saline control [102]

NK cells

BALB/C

CD4+ and CD8+ cell mediated

IL4

C57BL/6

B16F1A (Melanoma)

Increased survival time [31]

Not reported

IL18

BALB/C

D2F2, CT-26

Reduced tumor growth and pulmonary metastases [32] Increased survival time [31]

Granulocyte, NK, CD4+, CD8+ cell mediated Not reported

C57BL/6

B16F1A (Melanoma)

Tnfsf14 (LIGHT)

BALB/C

D2F2, CT-26

Primary and metastatic tumor growth inhibited [33]

NK, CD4+, CD8+ cell mediated

Target antigen KLK3 (PSA)

DBA/2

P815 cells expressing human PSA

CD8+ cell mediated

CTAG1B (NY-ESO-1)

BALB/c

Birc5 (Survivin)

C57BL/6

CMS5 cells expressing human NY-ESO-1 D121

Direct i.m. DNA vaccination was better than serovar Typhimurium-delivered immunogen [14] NY-ESO-1-positive tumors are eliminated [36]

CD8+ cell mediated

Vegfr2 (Kdr or Flk1) full-length protein

BALB/c C57BL/6

CT-26 B16G3.26 (Melanoma) D121 MC-38 (CRC – Syngeneic)

Suppression of angiogenesis and pulmonary metastasized tumors [101] Micro vessel destruction retarded tumor growth and metastases. Healing of skin wounds slightly delayed. Immunological memory persisted at 120 days post immunization [38]

Growth inhibitor(s) Bcl2 shRNA

C57BL/6

B16F10

Apoptosis

Nude mice


Apoptosis

Nude mice

Hep-2 (Laryngeal cancer) DU145 (PC – Xenograft) MDA-MB-231 (BC – Xenograft) SiHa

Survival time of tumor-bearing mice prolonged Complete tumor regression not observed [58] Tumor growth reduced [62,63]


Apoptosis

Nude mice

PC3


Apoptosis

C57BL/6

H22


BIRC5 shRNA NDUFA13 (GRIM-19) BIRC5 shRNA TNFSF15 (VEGI) HPV E6 shRNA TP53 MDM2 shRNA TP53 – Cisplatin Stat3 shRNA

Stat3 shRNA Col18A1Endo

Nude mice

CD8+ cell mediated

CD8+ cell mediated

Apoptosis

C57BL/6

RM1


Apoptosis and CD8+ cell mediated Apoptosis

C57BL/6

RM1


Apoptosis



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Fig. 1. Structures of gram-negative bacterial LPS grown in vitro. LPS is a conjugate of 3-hydroxy fatty acids with a glucosamine disaccharide bearing diametrically-placed phosphate residues. R1 to R4 represent the 11-carbon portion of myristoyl acid (C14:0). In Salmonella, R5, R6 and R7 represent the 11-, 13- and 15-carbon portion of lauric (C12:0), myristic (C14:0) and palmitic (C16:0) acids, respectively. The hexa-acylated LPS containing 12–14 carbons in each chain is a potent activator of human TLR4. Deviations from this structure is not recognized by the human immune system. In S. Typhimurium (DmsbB, VNP20009), LPS lacks the acyl chain (R6). In serovar Minnesota, PagP (PhoP–PhoQ two-component responsive gene) catalyzes the addition of palmitic acid. This PagP activity is not observed in S. Typhimurium grown in vitro. In Yersinia pestis grown in vitro at 26 °C, R5 and R6 represent the 15- and 11-carbon portion of palmitoleic (C16:1D9) and lauric (C12:0) acids, respectively. When grown at 37 °C, LPS lacks these two acyl chains. Tetra-acylated LPS is a weaker agonist of mouse Tlr4.

mice [23]. FASL, another cytokine from the TNF family, can be membrane bound or secreted [24]. Binding of FASL to its receptor, FAS, is known to induce apoptosis, regulate immune system and cancer progression (reviewed in [25]). Recombinant S. Typhimurium secreting murine Fasl under a weak constitutive ompC promoter reduced syngeneic breast and colorectal tumor growth in BALB/c mice [26]. The ompC promoter is induced by envZ-ompR two-component signaling system responsive to osmolar changes. Immune effectors: The host immune system is believed to control transformed or malignant cells from getting a foot hold. A proper functioning of the immune system is dependent on a well-orchestrated network of cytokine signaling events. As majority of the cytokines have recognized systemic toxicity, they are less preferred for anti-cancer therapy. However, tumor growth can be reduced significantly upon localized synthesis and delivery of a few cytokines by S. Typhimurium, as seen by the activation of immune response in juxta-tumoral regions (Table 3). Interleukin2 (IL2), IL4, IL18, CC chemokine-21 (CCL21), TNFSF14 (commonly known as LIGHT), etc., have been demonstrated to cause significant tumor regression. In these situations, expression of one cytokine drives the synthesis of other cytokines in the tumor milieu, hence, affects immune cell infiltration and/or functions. IL2 primarily activates cytotoxic T cells; it is involved in tolerance, effector immunity, lymphocyte proliferation and immunological memory decisions. It is an FDA-approved biological therapeutic for RCC and metastatic melanoma. Synthesis of human IL2 by recombinant S. Typhimurium led to NK cell activation and reduction of metastatic liver tumors [27] and was recently reported to reduce pulmonary metastatic burden in a murine osteosarcoma model [28].

IL4 helps in the differentiation of naïve T helper (Th0) to humoral T helper (Th2) cells, stimulation and differentiation of activated B cells to antibody-secreting plasma cells [29], etc. IL18, similar to IL2, promotes T and NK cell proliferation and enhances their cytokine synthesis [30]. Expression of IL4 and/or IL18 in tumors resulted in increased levels of IFN-c in the serum [31]. IL18 suppressed tumor neo-vascularization [32] by inhibiting growth of stromal fibroblasts, a major source of angiogenic factors. CCL21– CCR7 signaling is important for the migration of immune cells and organization of secondary lymphoid structures. TNFSF14 is a cytokine belonging to the TNF family that stimulates T cell proliferation, induces dendritic cell growth and triggers apoptosis of cancer cells. Recombinant S. Typhimurium expressing CCL21 and TNFSF14 were shown to attract leukocytes and neutrophils to the tumor resulting in cancer cell growth inhibition [33,34]. Tumor immunization: The immune system restricts and/or inhibits tumor growth. Recent studies have suggested the importance of adequate innate immune system stimulation to maintain efficient acquired immune responses. As tumors evolve in vivo they have been suggested to go undergo ‘immunoediting’ [35]. This process involves three stages viz., elimination, equilibrium and escape. Elimination involves the active killing of immunogenic tumor, which restricts the size significantly. After elimination, the residual tumor cells and the tumor-specific immune effectors exist in a state of equilibrium. Here the tumor neither grows aggressively nor does the immune system attack it. This could be a result of clonal evolution which allows the outgrowth of minor population of immune-attack resistant tumor cells (that may not express tumor antigens). This may exist for several years, and the individual does


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not manifest progressive disease. Overtime tumors escape because of age-dependent weakening of immune system. Alternatively, reformatting of tumor microenvironment, by recruiting immune suppressor cells to keep a check on killer cells and recruitment cum proliferation of angiogenic cell types into it favors cancer cell survival. This last phase occurs when a tumor mutates sufficiently to evade elimination by the immune system and grows out. Thus, a resupply of antigenic proteins might provide an upper hand to the immune system during equilibrium phase to eliminate most of the tumor. Since S. Typhimurium has been used as a carrier to deliver heterologous antigens e.g., influenza, lymphocytic choriomeningitis virus, etc., T3SS could be used to elicit potent immune responses against tumors. T3SS is composed of approximately 30 different protein components and the assembled apparatus resembles a syringe and needle. Unlike the broad variety and size range of T1SS cargoes, T3SS cargoes are invariably proteins. Pathogenic bacteria inject virulence factors using T3SS e.g., Shiga-like toxin by enteropathogenic E. coli and sop/sip gene products in Salmonella. In Salmonella slightly variant T3SS, due to SPI-derived gene products, are known to deliver different effector proteins. Cell-mediated immunity (CD8+) towards a melanoma-expressed antigen (NYESO-1) could be enhanced when S. Typhimurium was engineered to deliver sopE-NY-ESO-1 fusion protein via T3SS [36]. Conversely, when a cancer cell line expressed a part of the protein that was a T3SS cargo of recombinant S. Typhimurium also resulted in a robust development of CD8+ cell responses [19]. These studies suggested that tumor-specific CD8+ cells were present but not active in a tumor milieu. Upon immune system activation, achieved through antigen-expressing S. Typhimurium, tumor growth was controlled by rekindling of CD8+ and CD4+ T cells. A very strong support for this mechanism comes from experiments performed earlier using Clostridium novyi. Here BALB/c mice that completely rejected syngeneic CT26 tumors, due to C. novyi-NT spore-induced oncolysis, were completely resistant to a subsequent challenge of the same tumor while a different syngeneic RCC (RENCA) went on to form tumor mass [37]. Similarly mice immunized against vascular endothelial growth factor receptor 2 (Vegfr2, also known as Kdr or Flk-1), using S. Typhimurium, were resistant to subsequent syngeneic tumor challenges even after 10 months post vaccination [38]. Adoptive transfer experiments further demonstrated that this response was mediated by CD8+ cells and occurred independently of CD4+ cells. These studies also demonstrated the importance of ‘Epitope spread’ [39] in stimulating immune responses against other tumor antigen(s). ‘Epitope spread’ is a process wherein epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an ongoing immune response. It extends immune response to additional epitopes or to other proteins that are part of the target tissue. Therefore, it was suggested by Nishikawa et al. [36] that pre-existing CD8+ T cells against commonly occurring viral antigens in human populations, from prior vaccination or convalescent immunity, could be used in such a scenario. Importantly, cancer immunotherapy would benefit from S. Typhimurium T3SS antigen delivery platform as it does not require a prior knowledge of the tumor antigen composition. These studies suggested that tumor eradication is dependent on the immune system. The importance of B cells in controlling S. Typhimurium was recently reported [40]. Here mice immunized with heat-killed S. Typhimurium, were refractory to the oncolytic activity of S. Typhimurium and the immune serum effectively blocked S. Typhimurium from infecting cells in culture suggesting to the presence of neutralizing antibodies [40]. Presence of such serum IgG against LPS appeared to confer resistance to complement-mediated lysis in humans. Bacterial gene delivery systems for tumor growth control: In cultured non-phagocytic cells, S. Typhimurium has the ability to

release plasmids into the host cytosol [41]. Even though the mechanism behind this delivery is currently unclear, its benefit has been evaluated in mouse models. Similar to DNA transfection, the plasmid finds its way into the nucleus to express the cloned recombinant gene product. In fact expression of the cloned gene, from vertebrate promoter(s), is seen when no bacteria can be cultured from such cells. To deliver recombinant DNA into tumor mass, the natural tropism of attenuated S. Typhimurium is exploited. Since most tumors need blood supply to grow and spread to other tissues, preventing neo-vascularization was the first to be tested. Here, mice were orally immunized with S. Typhimurium bearing a mammalian expression plasmid containing full-length Vegfr2 (Flk-1) cassette [38]. Vegfr2 is a receptor tyrosine kinase that is activated upon Vegf binding. For endothelial cells to proliferate and form new blood capillaries, Vegfr2 upregulation is needed. By targeting this receptor, tumor growth and spread to other tissues can be controlled. Since, Vegfr2 is expressed by the noncancerous cells in the tumor; virtually all tumors can be controlled independently of cancer cell protein expression. Presence of Vegfr2-specific spleen-derived CTLs indicated a break in peripheral tolerance against a self-antigen. A later publication by the same group demonstrated even mini-gene versions of Vegfr2 resulted in T cell-mediated suppression of angiogenesis with tumor protective immunity [42]. Alternatively an immune-independent route of tumor growth control was also reported. Here S. Typhimuriumdelivered expression cassettes coding for endogenous angiogenesis inhibitors viz., endostatin (proteolytically processed c-terminal fragment of type-XVIII collagen) and THBS1 (Thrombospondin-1) reduced growth by limiting new blood formation in already established tumors. Both these proteins are present in the extra-cellular matrix supporting epithelial and endothelial cells. Endostatin competes with basic fibroblast growth factor and Vegf to block neovascularization and/or cause endothelial cell apoptosis. THBS1 induces Fasl expression to induce endothelial cell apoptosis. However, small tumors cannot be targeted using this approach as they do not have a good blood supply and S. Typhimurium accumulates poorly in such tumors [6]. Thus, this is a major barrier to target poorly-vascularized tumors. The rationale to use a promoter that is active in all tumor types and stages began with the telomerase promoter-driven expression of pro-apoptotic proteins viz., TNFSF10 (TRAIL) and DIABLO (SMAC) that activate CASP8 and CASP9, respectively. DIABLO, also known as SMAC is a mitochondrial protein that promotes caspase activation along with cytochrome-c. SMAC antagonizes and downregulates caspase inhibition by inhibitor of apoptosis proteins (IAP) [43]. A synergistic tumor growth inhibition was reported upon TRAIL and DIABLO co-expression from a human telomerase reverse transcriptase (hTERT) promoter compared to individual agents [44]. So by having two apoptosis-inducing proteins, far better tumor growth inhibition can be achieved. This approach has certain limitations as CASP8-driven apoptosis is cell-type specific and CASP9-driven apoptosis needs healthy mitochondria that are invariably rare in advanced tumors [45]. In cancer cells activation of multiple genetic events converts benign cells into malignant and metastatic variants. Oncogenes are central players in these processes. Certain oncoproteins themselves are transcription factors and in other cases oncogenic signals activate other transcription factors to enhance the expression of proliferation, malignancy and metastasis-associated genes. Thus, a down regulation of oncogenic transcription factors leads to the suppression of pro-growth gene expression to restrain tumor growth. A reverse strategy would be depriving tumor cells of molecules that are crucial for its survival using RNAi principle, a complementarity-driven dsRNA degradation and/or translational inhibition process. Most cells have the protein components of this machinery and just by providing appropriate RNA; it has been



possible to deplete proteins of choice. Briefly, certain structural and spatial aspects of an RNA molecule need to be fulfilled for it to function as an inhibitor [46]. After this computation, the dsRNA molecule can be synthesized in vitro or cloned as dsDNA sequence into an expression plasmid. Additional processing steps take place in the cytoplasm of the cell before the correct RNA strand (guide strand, 21 to 23 bases) is loaded into the RISC complex. Upon finding an appropriate complementarity between the guide strand and an mRNA, Argonaute proteins cleave the mRNA and it cannot be translated by the ribosome [47]. Transcription factor STAT3 has been identified a major oncoprotein in recent years. STAT3 is a latent cytoplasmic transcription factor that is crucial to signaling through cytokine receptors. Its activity is controlled through a regulated addition and removal of a phosphate moiety on a critical tyrosyl residue (Y705) by JAKs and feedback inhibitors, respectively. In cytokine-stimulated pathways e.g. IL6, JAK-induced tyrosyl phosphorylation of STAT3 results in nuclear translocation and transcriptional induction of several target genes including that of an inhibitor, Suppressor of Cytokine Signaling 3 (SOCS3) (see [48] for a review). During the same time, STAT3 is dephosphorylated and exported from the nucleus [49]. SOCS proteins bind to JAKs and prevent STAT from getting restimulated. However, in malignant cells persistent activation of STAT3 appears to occur independently of JAKs. A number of activated tyrosine kinase oncoproteins e.g. Src family members [50], p56Lck, Etk, Eyk, Ros, etc. and deregulated receptor tyrosine kinases [51] e.g. growth factor receptors, promote STAT3 activation persistently. Such STAT3 causes cell growth-associated gene expression, immune suppression and metastases. Consequently, a reduction in the physical levels of STAT3 will contain tumor growth better. A very similar pattern of oncogenic activity was seen with STAT5, another oncogenic member of the STAT family of transcription factors, in certain cancers [52]. A feature observed in many tumors is the over expression of STAT3, anti-apoptotic BCL2 family members and BIRC5 (Survivin, an IAP family member). Bcl2 proteins regulate outer mitochondrial membrane integrity. At least 25 Bcl2-like proteins are known to date. Some members are anti-apoptotic e.g. Bcl-XL and Mcl1 while some are pro-apoptotic e.g. Bcl-XS, Bax, Bim, etc. [53]. IAP family members are structurally and functionally related endogenous protein inhibitors of caspases that function in different contexts [54]. At least 6 known members containing the baculoviral IAP repeat (BIR) are known to date. BIRC5 (Survivin), as the name suggests, is important for cancer cell survival as is involved in numerous cellular processes. Inhibition of Bax/Fasl-induced apoptosis, regulation of cytokinesis, acquired resistance to chemotherapeutics, etc. has been attributed to over expressed Survivin in cancer cells (see [55] for a review). Genes responsive to STAT3 are involved in stimulation of cell growth e.g. cyclin D1, or inhibition of apoptosis e.g. Bcl-XL. Growth of syngeneic prostate cancer cell line (RM1) reduced when Stat3 was depleted by S. Typhimurium-delivered RNAi in an orthotopic setting [56]. Transcriptional targets of Stat3, such as Bcl2, Ccnd1 and Vegf levels were down regulated in the tumors, and prevented new blood vessel formation. In addition, in vivo grown RM1 cells were vulnerable to apoptosis due to reduced levels of Myc protein along with depleted Stat3 [56]. In addition to these gene products, Hif-1a and Mmp2 were also down regulated in orthotopicallygrown syngeneic liver cancer cell line (H22) upon Stat3 depletion [57]. Reduced numbers of regulatory T cells (CD25+ Foxp3+) along with increased numbers of NK and CD8+ T cells were observed in the spleen when Stat3 was depleted in the tumor [57]. A direct depletion of BCL2 was reported to reduce tumor size along with prolonged survival in mice [58]. Similar to expression of dual apoptosis-inducing proteins mentioned earlier, S. Typhimurium-delivered dual expression cassettes encoding a shRNA targeting an oncogenic product along with a

7

growth-suppressive protein produced a synergistic tumor growth-inhibitory effect compared to either individual gene products (Table 3). Suppressing Stat3 protein levels alone or in conjunction with expressed endostatin was reported to be beneficial in controlling syngeneic prostate cancer cell (RM1) tumor [59]. Tumor suppressor TP53 is frequently mutated in many human cancers. In human cervical carcinomas caused by high-risk HPV infection, p53 protein levels are downregulated without accumulation of mutations in the TP53 gene. The high-risk HPV encoded E6 oncoprotein targets p53 to ubiquitin–proteasome mediated degradation. Expression of wild-type p53 in conjunction with a shRNA against high-risk HPV-E6 [60] or MDM2 [61] was reported to retard growth of xenografted cervical or prostate cancer, respectively, in mice. Similarly, depletion of BIRC5 (Survivin) along with expression of GRIM-19 was shown to slow growth of xenografted prostate [62] and laryngeal [63] cancer cells while expression of vascular endothelial growth inhibitor (TNFSF15, VEGI) was shown to reduce breast cancer cell growth [64] in nude mice. These studies suggest that tumor growth can be controlled without the help from the immune system. In spite of the tremendous success in eradicating experimental tumors in mouse transplant models, pre-clinical and phase-I trials of using bacteria alone to control tumors have been disappointing (Table 4). A literature survey for clinical trials that used bacterial therapy to control cancer revealed none of them have posted the results of the study and some have been terminated. However, a few publications are available from these studies. Even a direct intra-tumoral injection of S. Typhimurium, expressing E. coli cytosine deaminase, resulted in short-term tumor colonization with no tumor shrinkage [11] (Table 4). These barriers need to be investigated to exploit the full potential of S. Typhimurium as a tumor control agent. Lastly, the potential of recombinant gene delivery systems (shRNAs and tumor suppressors) have not been fully exploited in clinical studies. Results from pre-clinical models are encouraging and more effort is needed toward human-directed applications of these technologies.

3. Impediments to bacterial therapy and issues to be addressed in future studies A vaccination strategy was envisioned to help break peripheral tolerance towards self-antigens that are often poorly immunogenic on their own. Here immunized mice, a self-antigen delivered or expressed by S. Typhimurium and processed by macrophages/ antigen-presenting cells, are challenged by an antigen-expressing tumor cells. Tumor growth was controlled due to the presence of antigen-specific circulating CD8+ T cells in the host. Host antigens that are expressed in particular situations, like tumor neovascularization, can also be targeted using this approach as demonstrated by mice immunized against the extra-cellular domain of Vegfr2 (Flk-1) suppressed tumor growth and prevented metastasis of Lewis lung carcinoma [65]. In some cases expression of immune system effectors or chemotherapeutics synergized with the immunogen i.e., DNA vaccine [14], to boost host responses against the tumor (Table 3). More interesting is that antigens from a different species (microbial [19] or mammalian [14]) when expressed by syngeneic tumor cells is not recognized as foreign by the immune system in an unstimulated state. However, upon immunization, they are immediately recognized as foreign and destroyed. Although the immunogenic properties of such antigens are not the focus of this review, it is worth visiting in the future. Thus, these studies suggest that tumor growth is controlled mainly with the help from the immune system. In a natural setting, Salmonella invade the host through the cells of the lower gastrointestinal tract; in mouse the preferred entry


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Table 4 Bacterial therapy for controlling tumor growth – a summary of pre-clinical and clinical trials. Species (strain)

Identifier

Tumor (sample size)

Outcome(s)

Effector(s)

C. sporogenes (M55)

– –

5 types (1 each) Vascular glioblastoma (49)

Oncolysis, limited utility to treat tumors Oncolysis, tumor recurrence unaffected [3]

Not reported Not reported

C. novyi (NT)

NCT00358397 NCT01118819 NCT01924689 Pre-clinical Dog

Solid tumor (20) Solid tumor (5) Solid tumor (18) 6 types (6) Soft-tissue sarcoma (12)

Terminated – due to a design problem Terminated – reason(s) not disclosed In progress DLT observed. Responders: 5 (stable) [103] Responders: 2 (complete), 3 (partial), 5 (stable) [104]

– – Awaited – Not reported

C. histolyticum

NCT01613313 NCT02249052

Lipoma (14) Lipoma (20)

Not available and/or published In progress

Not reported Awaited

S. Typhimurium (VNP20009)

NCT00004216

Metastatic melanoma (24)

Not applicable

NCT00004988

Metastatic melanoma (4)

NCT00006254

Squamous cell carcinoma (3)

Pre-clinical Dog

7 types (35)

Focal tumor colonization in 3 patients No tumor shrinkage [12] Tumor biopsy culture positive in 1 patient No tumor shrinkage [10] Tumor colonization in 2 patients No tumor shrinkage [11] Responders: 4 (complete), 2 (partial), 2 (stable) Overall survival was better in complete responders [13]

S. Typhimurium (v4550)b S. Typhi (VXM01)c

NCT01099631 NCT01486329

Hepatocellular carcinoma (22) Pancreatic cancer (72)

Mixed Bacterial Vaccine

NCT00623831

Malignant tumors positive for CTAG1B (NY-ESO-1) (17)

a

a b c

Terminated – reason(s) not disclosed Reduction in tumor perfusion after vaccination. Fate of tumors not disclosed [95] Not available and/or published

Not applicable Not applicable Not reported – Teffector cells? Not reported

A gram-positive soil-dwelling spore-forming obligate anaerobic non-pathogenic bacterium. A triple mutant (asd, crp, cya) secreting human IL2. A Ty21a host carrying mammalian expression plasmid for full-length human VEGFR2. See [105] for details about Ty21a.

point are the microfold cells of Peyer’s patches. Since, the gastrointestinal tract is a niche abundant with numerous microflora, the host needs to discriminate bacteria using specific pattern recognition to deploy appropriate counter measures. Mucosal immunity provided by IgA is the first line of defense to control enteric pathogens. In humans, innate immunity offered by the resident cells in the mucosa viz., dendritic cells and macrophages, normally control Salmonella using the lysosomal machinery; neutrophils are also recruited for controlling bacterial loads in certain instances (see [66] for a review). However, typhoidal serovars are known to evade this and gain access to deeper tissues by minimizing mucosal immune stimulation (see [67] for a review). Even though T3SSmediated delivery of virulence factors encoded by SPI has been extensively studied, clinical-grade disease is influenced by numerous host factors. T3SS effectors imparting virulence in a host species-specific manner are known in literature (see [68] for a review). In rare cases, S. Typhimurium persists, in the gut, for a long time period that is often asymptomatic. Contribution from the normal gut microflora is also believed to control S. Typhimurium colonization and/or persistence. By rearing experimental mice devoid of their natural gut microflora, have we introduced an unintended bias in experiments that skewed immune system maturation? Recently the human restricted S. Typhi was found to infect Tlr11null mice resembling pathogenesis as occurring in humans [69]. This could pave way for the identification and development of pan Salmonella vaccine targets even though WHO places Salmonella in its ‘Moderate risk’ category. As mentioned earlier S. Typhimurium causes typhoid fever-like disease in mouse as it enters into systemic circulation. In mouse, when S. Typhimurium colonizes non-conventional sites (such as implanted tumors) via systemic circulation, neutrophils and lymphocytes are additionally recruited to control further dissemination of such bacteria [70]. The role of cytokines in this process has revealed TNF to be potent inducer of vascular destruction [71] in the peri-tumoral regions that changes into a blood clot thus blocking the necessary blood supply that keeps them alive. This converts the tumor into a necrotic mass where the bacteria thrive

for some time before the arrival of immune cells which clear the debris. During this process, a small portion of tumor cells in the rim do survive and go on to reform the tumor mass at a later time when bacteria are absent [37]. Since most of the studies in mouse are short term in nature, one must excise caution in interpreting the initial reduction in tumor size; although rechallenge experiments have been successfully demonstrated only in BALB/c strain using syngeneic CT26 colorectal cancer cells [37]. Therefore in future studies, complete responders need to be re-challenged, at different time points, with the same tumor to confirm the nature of response(s) and characterize any specific cell type if involved. They also need to be tested for ‘Epitope Spread’ using different syngeneic tumors and whether they confer protection in nonresponders and/or partial responders. These will be informative for targeting new tumor antigens. Even though S. Typhimurium has an upper hand when infecting (cancer) cell lines in vitro, reports of culturable isolate(s) from intracellular sources are extremely rare while it is more prevalent in necrotic areas [72] and/or intercellular spaces [57,70]. To our knowledge, the presence of live intracellular S. Typhimurium in human tumor cells has not been demonstrated in vivo although macrophages are known to harbor them in some cases or certain situations. Most likely this may have contributed to recovery of the inoculated bacterium from a tumor mass, as it is a heterogeneous mixture of host cells. That said, the major shortcoming in the phase-I clinical study was the absence of attenuated S. Typhimurium VNP20009 in blood after 6 h. This mutant synthesizes a penta-acylated LPS [73] (Fig. 1) that is a weak TLR4 agonist in humans so it can be safely administered with minimal septic shock. It is well known in literature that human TLR4-MD2-CD14 complex recognizes a hexa-acylated LPS while mouse Tlr4-Md2Cd14 does not discriminate LPS based on its acyl chains [74]. Unfortunately, VNP20009 LPS reduces fitness when subjected to stress such as increased concentration of CO2 and/or osmolarity in laboratory growth conditions [75]. However, VNP20009 strain when delivered via oral route in mouse does not encounter such problems to induce immunity [76] while it stayed in mouse blood



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Fig. 2. A proposed model for bacterial accumulation in tumors. Physiological, tissue repair and tumor-associated vasculogenesis share many molecular features. Protein factors secreted by non-cancerous cells, cell–cell and cell–stromal interactions in the tumor microenvironment govern the nature of disease progression. Slow-growing tumors need very little of a direct blood supply, hence S. Typhimurium cannot reach such places. Tumors with periodic and frequent neovascularization need a direct blood supply and such vessels show abnormal features. Tumors with a very active stroma have chaotic and unstable capillaries. S. Typhimurium can home to such tumors but cannot persist due to direct contact with tumor-resident immune cells. When S. Typhimurium enters non-immune cells in the tumor, it can replicate within them and then induces host-cell lysis. When newly-forming endothelial cells and/or pericytes come in contact with S. Typhimurium, they can release TNF-a and other cytokines to activate platelets that leads to a micro-blood clot. This physiological in situ method of restraining a pathogen alerts and attracts immune cells to the site. The resident and circulating immune cells either control or eliminate S. Typhimurium.

when delivered intravenously contrary to the in vitro observation [33]. So it is possible that blood CO2 concentration does not affect VNP20009 survival when it is not growing. Hence, it is likely that human immune system and/or blood constituents are working somehow against attenuated S. Typhimurium VNP20009 (via rapid clearance) which needs to be determined in future studies. During an infection, neutrophils are the first cells of the innate immune system to respond and control pathogens via phagocytosis, oxidative burst or trapping microbes in extracellular structures called Neutrophil Extracellular Traps (NETs) [77,78]. Chronic granulomatous disease is a condition where catalase-positive microbes cause recurrent infections due to defects in oxidative burst capacity of granulocytic phagocytes; catalase-negative microbes are easily controlled. Even though S. Typhimurium is catalase positive, survival in the host appears to be independent of it. Hence, NETs likely appear to control systemically administered S. Typhimurium numbers in humans similar to peri-tumoral neutrophils controlling S. Typhimurium in experimental tumors in mouse [70]. In humans, it is known that S. Typhimurium can enter the blood stream of immuno-compromised individuals especially who have HIV or active malarial infection [79]. Even though adoptive transfer experiments in mice have demonstrated the effective control of S. Typhimurium was dependent on CD8+ cells, interference with CD4+ cell function during the early phase of infection resulted in frequent reinfection i.e., specific immunity did not develop for long-term protection [80]. So why neutrophils do not control S. Typhimurium needs to be determined in HIV-positive individuals or those down with malaria. Could there be a potential cross talk between CD4+ cells and neutrophils? Apart from this, in vivo and/or specific growth characteristics of other S. Typhimurium strains are also lacking to draw meaningful conclusions. Even though the blood volume to body mass is comparable between humans and mice, the effective bacterial

concentration is dependent on blood volume for a given dose of S. Typhimurium. Thus, circulating bacteria in good numbers is a pre-requisite for effective tumor colonization. Apart from this, there are species-specific differences between mouse and human immune cells viz., human blood is neutrophil-rich while mouse blood is lymphocyte-rich, nitric oxide production is inducible in mouse macrophages while human immune cells cannot produce nitric oxide upon stimulation, etc. If one considers the effective bacteria to neutrophil numbers, in mouse blood it would give bacteria the advantage while in human blood bacteria will have a disadvantage as they can get stuck in NET. Mice are known to be tolerant to high levels of cytokines in blood. A similar high level of cytokines in humans is known to cause serious systemic toxic effects and/or acute adverse reactions making the administration of higher titers of the bacterium impractical. So the balance between effective tumor targeting and MTD/DLT i.e., therapeutic window, ultimately decides the success of this technology that meets or exceeds the set clinical criteria for an objective response. The ideal system to evaluate this balance will be humans (see [81] for a perspective). At least available S. Typhimurium vaccine strains can be safely administered intravenously in healthy subjects to address bacterial dynamics in human blood. Alternatively, a mouse model that has engrafted human HSC may provide some clues to enhance S. Typhimurium persistence in mouse vessels when they encounter human blood cells (see [82] for a review). Although this model is significantly expensive to use and requires operational expertise, its feasibility and/or benefit needs to be addressed in upcoming experimental tumor targeting studies. The use of fluorescent or luminescent protein-expressing S. Typhimurium will allow for easy real-time visualization of bacterial dynamics in these chimeric mice. If this system yields valuable insights, one can move forward to develop and test more robust tumor targeting approaches as has been demonstrated by S. Typhimurium


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Fig. 3. Delivering therapeutic bacteria to hypoxic tumor area(s). Green circular arrows depict bacteria that have exited the blood capillaries and housed/growing near cells of the tumor mass. Due to hypoxia, not all cells in the tumor get to encounter bacteria. Recombinant bacteria expressing enzymes to convert pro-drugs to active drugs in tumor microenvironment (see Ref. [11]]). Adapted from Jordan and Sonveaux (Front Pharmacol, 2012). Targeting tumor perfusion and oxygenation to improve the outcome of anticancer therapy. Figure obtained from http://openi.nlm.nih.gov under open access. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

VNP20009 expressing single chain antibody fragment against human carcinoembryonic antigen in experimental tumors [83]. The host immune system is believed to potently discriminate foreign from self-antigens. In some niches, the immune system is activated against specific microbial antigens in spite of the plethora of foreign antigens, as seen in the gut mucosa. The immune system, however, is activated against these microbes and/or antigens when presented outside this niche. Even in a niche tolerant to microbial communities, immune system gets activated to protect the host as seen in food-borne illnesses and would suggest that tumor-vicinal bacteria will also be controlled by the immune system. If this is to be true, it raises an important question as to how bacteria could be isolated from solid tumors in the first place [84]. When bacteria were isolated form lung cancer patients, many of them had lung-associated inflammation [85] while patients with gastrointestinal neoplasms had a higher incidence of gut-associated inflammation [86]. Fungal, to a lesser extent bacteria, infections are common in patients with hematological malignancies undergoing a chemotherapeutic regimen [87]. We have not been able to find studies addressing progression/disease-free and/or overall survival in such scenarios without additional antimicrobial treatments [88]. There are reports of bacteria in the lymph nodes or nearby tissue(s) of patients who underwent surgical excision of tumors [89]. Survival benefit(s) have not been addressed in such studies. The only historically well-documented case for a microbe known to positively impact patient survival is HPV in SCC (head and neck, oral and cervical). Even though high-risk HPV increases the chances of developing SCC, it is a favorable prognostic factor for treatment regimen(s) and overall survival compared to HPVnegative SCCs. Incidentally HPV-positive tumors have wild-type tumor suppressors (p53, p16 and GRIM-19) as well as near intact mitochondrial protein composition when compared to HPVnegative SCCs that harbor genetic aberrations in p53 and p16. In non-immune cells, GRIM-19 protein was upregulated by Salmonella, but not E. coli, that contributed to innate immune responses by activating NF-jB via NOD2 signaling axis [90]. In vitro

cultured human monocytes/macrophages upregulated GRIM-19 protein level when challenged by purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant [91] while only a live Porphyromonas gingivalis could upregulate GRIM-19 protein levels, but not its LPS or its fimbrial proteins, in peripheral blood-derived monocytes [92]. Recently, Grim-19+/- male mice was reported to be prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus, and had compromised immune response [93]. Interestingly, this innate immune response is conserved even in insects as a differential response and enrichment of GRIM-19 expressed sequence tags were observed in hemocytes from mosquitoes (Armigeres subalbatus and Aedes aegypti) when challenged with E. coli and Micrococcus luteus [94]. This innate response to induce GRIM-19, may serve as a brake on endogenous STAT3-dependent gene expression that promotes tumor growth. Therefore, it is possible that a microbial presence in certain types of tumors can provide some treatment and/or survival benefit(s). It is likely that the genetic makeup of tumors dictates how favorable the response will be towards bacterial-based therapy. Future studies should first address whether tumors arising in genetically-engineered mouse models and/or naturally tumorprone mouse strains can be targeted by attenuated bacteria as anti-tumor agents. 4. Conclusions With the emergence of microbiome as a major player in many human diseases, bacteria as therapeutics are gaining significant interest. While bacteria alone may not offer full therapeutic benefits, modifying them with anti-tumor agents, anti-oncogenes or immunogenic antigens, either alone or in combination, will prove to be beneficial. Such therapeutics may become a future alternative or adjunct regimen along with conventional chemotherapy and radiotherapy. A recent phase-I trial report of S. Typhi to evoke immune responses against the neovasculature [95] is encouraging even though S. Typhimurium or Clostridium sp. have lagged on this front. The success of bacteria, or the product(s) they deliver, to



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Microbiome therapeutics - Advances and challenges.

Genetically modified T cells in cancer therapy: opportunities and challenges.

Current Review of Genetically Modified Lactic Acid Bacteria for the Prevention and Treatment of Colitis Using Murine Models.

Oral bacteria and cancer.

Gut bacteria and cancer.

Genetically modified bacteria as a tool to detect microscopic solid tumor masses with triggered release of a recombinant biomarker.

Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.

Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

Mitoriboscins: Mitochondrial-based therapeutics targeting cancer stem cells (CSCs), bacteria and pathogenic yeast.

Oxidative Stress and Cancer: Advances and Challenges.

Targeting Head and Neck Cancer Stem Cells: Current Advances and Future Challenges.

Challenges to production of antibodies in bacteria and yeast.

Current advances and remaining challenges in human transplant immunology.

Management of severe sepsis: advances, challenges, and current status.

Artificial Mitochondria Transfer: Current Challenges, Advances, and Future Applications.

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

Editorial: Current advances and challenges in understanding plant desiccation tolerance.

Bacteria as insect pathogens.

Modified lactic acid bacteria detect and inhibit multiresistant enterococci.

Distribution of folates and modified folates in extremely thermophilic bacteria.

Drugs in development for toxoplasmosis: advances, challenges, and current status.

Detection of genetically engineered traits among bacteria in the environment.

Isolation of Genetically Tractable Most-Wanted Bacteria by Metaparental Mating.

Diagnostic methods for platelet bacteria screening: current status and developments.