Shaping cancerous landscapes with microbial communities Aditya Kulkarni, Ph.D. Department of ...

20 Pages·2016·0.75 MB·English

by  Partha Ramasastry

PostDoc Journal Journal of Postdoctoral Research
Vol. 4, No. 11, November 2016 www.postdocjournal.com
Shaping cancerous landscapes with microbial communities
Aditya Kulkarni, Ph.D.
Department of Biochemistry, University of Texas Southwestern Medical Center,
Dallas TX 75390
Email: [email protected]

Abstract
The dynamic interplay between resident microbiota, host immunity and anti-cancer therapy has
generated a captivating enigma underlying the assignment of cause-effect relationships among these
factors. The diverse effects of microbes on carcinogenesis, ranging from preventing or promoting cancer
to dictating therapeutic outcomes, complicates the understanding of the relationship between the
microbiota and the host. Understanding how host-microbe interactions are influenced by genes and
environment in carcinogenesis, and applying that knowledge for cancer detection and treatment are
gathering prime interest. This review scrutinizes the host-microbe relationship in the context of cancer
by discussing the latest findings involving the host-microbe-drug interaction axes.

Keywords: microbiome, anti-cancer therapy, immune system, bacteria mediated tumor therapy

I. Introduction
There are several microscopic communities or ability to respond to anti-cancer therapy
living on or inside our bodies. Together they include: 1) altering the balance of host cell
orchestrate a carefully balanced symbiotic proliferation and death through induction of
relationship. The human body provides a place pro- or anti-inflammatory programs, 2) guiding
for them to survive and thrive. Microorganisms immune system function, 3) influencing
maintain the body’s homeostasis by modulating metabolism (re-activation or detoxification) of
the epithelial tissue expression of genes host-produced factors, dietary components and
involved in nutrient uptake and metabolism, xenobiotics/ pharmaceuticals, 4) probiotic- or
mucosal barrier function, enteric nervous antibiotic-driven changes in abundance and/ or
system and motility, hormonal responses, localization of specific microbes, 5) inducing
angiogenesis, cytoskeleton and extracellular genotoxic stress in healthy or tumor cells and 6)
matrix, signal transduction, and general cellular loss of symbiosis-permissive mucosal barriers
functions [1]. It is an important relationship, between host and microbe. Carcinogenesis has
with any disequilibrium of these unique been hypothesized to be related to microbial
communities being linked to a number of dysbiosis under context-specific conditions, and
diseases, including cancer. has been relatively well documented in case of
colorectal cancer [2]. Loss of recognition of
Investigations in recent years delving into the microbial species by host [3], bacteria-induced
interplay between the commensal human mutations in host DNA [4], epigenetic
microbiota, cancer progression and alterations in host gene expression driven by
accompanying therapy are leading to the bacterial metabolites that act as cofactors,
definition of a new terminology called modifiers or allosteric regulators [5], infection-
‘oncomicrobiome’. This emerging concept in associated chronic inflammation [6, 7], indirect
cancer biology implicates the microbiota as a microbial effects on energy uptake and
powerful environmental factor modulating the
carcinogenic process. Some of the ways in
which the microbial community can enhance or
diminish a host’s risk of developing cancer and/
28 Journal of Postdoctoral Research November 2016: 27-46

metabolism [2] are few mechanisms that have Oxygen Species) and reduced cytotoxic effects.
been suggested to regulate host cell apoptosis, The production of ROS required for oxaliplatin
proliferation and migration directly or via genotoxicity in vivo were shown to be mostly
cytokines or hormones. In this review, I derived from tumor-associated inflammatory
specifically evaluate 1) how microbes may cells. Gene-expression analysis showed that
contribute to responsiveness to various kinds of induction of pro-inflammatory genes was
anti-cancer therapy, 2) how microbes may be decreased in the absence of microbiota after
genetically modified to improve anti-cancer oxaliplatin treatment, indicating that
therapeutic efficacy and limit toxicity, and 3) inflammation was essential to the anti-tumor
the opportunity for microbes to be developed effect of the drug [9]. If the microbiome is
as biomarkers for cancer diagnosis. altered in such a way that inflammation is
reduced, these therapeutic agents are less
II. Contribution of microbes to responsiveness effective. Upon depletion of myeloid cells, the
to anti-cancer therapy ability of oxaliplatin to induce tumor regression
Depending upon the type and stage of cancer, and to increase survival was impaired. This
various kinds of anti-cancer approaches have suggests that the reduced effect of oxaliplatin in
clinically been approved as standard of care antibiotic-treated or germ-free mice is partially
chemotherapy or are being included as due to reduced myeloid-cell ROS production.
concurrent or combination adjuvant therapy. The commensal effect on Oxaliplatin’s
The impact of the diverse microbiota on antitumor cytotoxicity was proposed to be
response to such treatments is only recently related to microbial product sensing. Besides
becoming more evident, as discussed further in platinum complexes, drugs such as
this section. Reports from diverse model anthracyclines, alkylating agents,
systems, have displayed the direct involvement podophyllotoxins, and Camptothecin induce
of resident microbes in mediating an anti- ROS as part of their anticancer activity
neoplastic response. exhibiting a similar manner of regulation. The
gut microbiota can be said to prime myeloid
(i) Oxaliplatin cells for increased Reactive Oxygen Species
The platinum compound oxaliplatin has been (ROS) production in the tumor
used to treat various gastrointestinal (GI) microenvironment. The resultant intratumoral
malignancies, particularly advanced colorectal oxidative stress augments the Oxaliplatin-
cancer. Oxaliplatin initiates tumor cytotoxicity associated DNA damage. The microbiota and in
by forming platinum-DNA adducts and intra- turn the immune system cooperate to
strand cross-links eventually leading to potentiate the efficacy of Oxaliplatin.
irreparable DNA damage and apoptotic death of
the cancer cell [8]. Oxaliplatin chemotherapy (ii) Irinotecan
has been demonstrated to work in part by Irinotecan is a semisynthetic analogue of the
boosting inflammation. Oxaliplatin, that was natural alkaloid Camptothecin. Its main use is in
initially not linked to work via activation of the colon cancer, in particular, in combination with
body’s immune system, also surprisingly relied other chemotherapy agents. It has also been
on the gut microbiota for successful eradication used against lung and brain tumors as well as
of tumors in animal studies [9]. Antibiotic- refractory forms of leukemia and lymphoma.
treated and germ-free mice bearing tumors had Irinotecan, a prodrug, is hydrolyzed by
reduced tumor regression and survival carboxylesterases to its active metabolite, SN-
compared with control mice receiving platinum 38, an inhibitor of Topoisomerase I [10]. The
therapy, with the antibiotic-treated mice poisoning of the catalytic cycle of
exhibiting reduced production of ROS (Reactive Topoisomerase I by SN-38 eventually leads to
Kulkarni 29

inhibition of both DNA replication and potential β-glucuronidase blockers to be
transcription, especially in rapidly dividing cells, combined with Irinotecan therapy, along with
which would be ideal for targeting cancer cells. successful reduction in diarrhea, it is important
In clinical trials, however, it was observed that to note that the drugs should not alter the
the dose-limiting side effect of Campthothecin levels of active SN-38 in the bloodstream. This
and its derivatives including Irinotecan is severe strategy can thereby widen the therapeutic
diarrhea [11]. As part of routine hepatic window of Irinotecan. Oral administration of
biotransformation, active SN-38 is inactivated these inhibitors has been effective at alleviating
and detoxified to SN-38G by glucuronidation by the GI toxicity of Irinotecan in mouse models
uridine diphosphate glucuronosyltransferase [12], and this approach may allow the dose or
1A1 (UGT1A1). SN-38G is excreted via biliary duration of Irinotecan-based chemotherapy to
ducts into the GI tract. Once in the intestines, be ramped up in patients. One might expect
though, SN-38G serves as a substrate for these inhibitors to be effective in combination
bacterial β-glucuronidase enzymes of the with other chemotherapeutics that are
symbiotic commensal microbiota that scavenge glucuronidated in the liver and reactivated by
for and remove the glucuronide group as a bacterial β-glucuronidases in the gut.
carbon source, producing reactivated SN-38
[12]. This new toxic form of SN-38 destroys (iii) Cyclophosphamide
rapidly dividing intestinal epithelial cells and Anti-cancer chemotherapeutics often cause
causes severe GI distress including diarrhea. mucositis (a debilitating mucosal barrier injury
High SN-38 levels in the intestinal lumen associated with bacterial translocation) and
prevent dose intensification and efficacy neutropenia (an abnormally low concentration
achievement in up to 40% of treated patients of neutrophils), two complications that require
[12]. treatment with antibiotics, which in turn can
result in dysbiosis. Some anti-neoplastic agents
Employing antibiotics to reduce GI bacteria mediate part of their anti-cancer activity by
levels prior to Irinotecan treatment does not stimulating anti-cancer immune responses.
represent a preferred treatment option because Cyclophosphamide is a prominent DNA
the indiscriminate killing of bacteria can be alkylating chemotherapy agent used in
deleterious. Intestinal microbiota plays hematologic malignancies and solid tumors. It
essential roles in carbohydrate metabolism, has been known to disrupt the intestinal
vitamin production, and the processing of bile epithelial barrier, affecting mucosal integrity
acids, sterols, and xenobiotics [13]. Thus, the and causing the gut to leak certain bacteria.
removal of GI bacteria is not recommended for Cyclophosphamide treatment induces mucosa-
patients already challenged by neoplastic associated microbial dysbiosis and provokes
growths and chemotherapy. In addition, selective translocation of distinct Gram-positive
elimination of symbiotic GI flora increases the bacterial species (Lactobacillus johnsonii,
chances of infections by pathogenic bacteria Lactobacillus murinus and Enterococcus hirae)
including enterohemorrhagic Escherichia coli in secondary lymphoid organs such as the
and Clostridium difficile. Through structural and lymph nodes and the spleen [14]. Bacteria now
chemical biology advances, potent and selective accumulated in lymphoid tissue outside the gut
pharmacological inhibitors of β-glucronidases stimulate the generation of memory T helper 1
from various bacterial species, but not the (Th1) cells and a specific subset of “pathogenic”
mammalian version, are being developed to T helper 17 (pTh17) effector cells that
eliminate the GI toxicity of Irinotecan without subsequently migrate to the tumor and kill it.
killing the bacterial symbiotes required for Tumor-bearing mice that were germ-free or
intestinal health [12]. During the screening of that had been treated with antibiotics like
30 Journal of Postdoctoral Research November 2016: 27-46

Vancomycin to kill Gram-positive bacteria checkpoint blockade immunotherapy in
showed a reduction in pTh17-mediated immune preclinical tumor models [16, 17].
response and their tumors were resistant to
Cyclophosphamide. Adoptive transfer of pTh17 Antibodies that inhibit either CTLA-4 or PD-L1
cells partially restored the anti-tumor efficacy of have shown particular promise by triggering a
Cyclophosphamide [14]. These results suggest checkpoint blockade that unleashes a robust
that the composition of the gut microbiota immune response against cancer cells. Although
helps shape the anticancer immune response. killer T cell infiltration of solid tumors has been
associated with favorable patient outcomes, the
(iv) Immune checkpoint blockade therapy mechanisms responsible for variable immune
Multiple mechanisms underlying inherent responses between individuals are not
prevention of an effective anti-cancer immune completely understood. Interestingly, the
response have been described, including efficacy of anti-CTLA4 and anti-PD-L1
signaling via immunosuppressive and anti- treatments was found to be dependent on the
inflammatory factors, such as nitric oxide, composition of the patient gut microbiota and
arginase, Transforming Growth Factor-β (TGF-β) its ability to induce the maturation of dendritic
and IL-10 that are produced by both classically cells. This involves a vigorous mobilization of
and alternatively activated macrophages, other tumor infiltrating cytotoxic T lymphocytes.
myeloid cell subsets, and regulatory T cells. In Distinct bacterial species were associated with
addition to malignant tumor cells, stromal cells augmented dendritic cell function leading to
and hematopoietic cells also express ligands enhanced and microbe-specific killer T cell
such as the B7 family molecules and PD-L1/2 priming and accumulation in the tumor
that trigger the immune checkpoint T cell microenvironment. Presence of Bifidobacterium
receptors, CTLA-4 and PD-1 respectively. They species promoted anti-PD-L1 efficacy [16]
cause blunting of T cell-mediated antitumor whereas Bacteroides promoted anti-CTLA4
activity [15]. Thus, inflammation and immunity efficacy [17]. Fecal microbial transplantation
should be considered inherent characteristics of from humans to mice confirmed that treatment
cancer, and “local chronic inflammation” and of melanoma patients with antibodies against
“evasion of the immune system” are now CTLA-4 favored the outgrowth of Bacteroides
included among the hallmarks of cancer. fragilis with anti-cancer properties [16].
Additionally, therapeutic feeding of these
In the past few years there has been very particular immunostimulatory bacteria
promising progress in the therapy of melanoma, improved the efficacy of immunotherapy in
kidney and lung cancers with respect to mouse models lacking those bacteria. Dendritic
boosting the patient's immune response against cells from microbe-fed mice in these studies
the tumor using immune checkpoint inhibitors showed elevated expression of genes
such as antibodies blocking the CTLA-4 or PD-1 associated with antitumor immunity and
receptors or PD-L1 ligand. Many recent studies heightened capability for T cell activation [16,
reporting the role of the commensal microbiota 17].
in modulating the response to cancer
immunotherapy, immunogenic chemotherapy, All the pieces of evidence described above
and adoptive T cell transfer have raised the demonstrate an unsuspected role for
possibility that the microbiota may also commensal microbiota in regulating various
modulate the clinical efficacy of this new class anti-cancer therapeutic strategies either
of anticancer drugs. Two path breaking studies positively through microbial shifts enhancing
have addressed this question by identifying anti-tumor immunity or negatively through
specific gut-resident bacteria as drivers of influencing drug metabolism.
Kulkarni 31

III. Manipulating microbes for anti-cancer expression of prominent Microbial Associated
therapy Molecular Patterns (MAMPs) such as
Any immunotherapy essentially works by Lipopolysaccharides (LPS, the immunogenic
hacking your immune system. The therapeutic element in bacterial cell membranes) and
moiety teaches the immune system how to flagella (regardless of their functionality in
recognize and attack the previously hidden motility or chemotaxis) [19]. Interference with
cancer cells that it would otherwise ignore. these virulence factors or restricting the in vivo
Origins of one of the most promising areas of survival of bacteria by metabolic auxotrophies
cancer immunotherapy can be traced back to can serve as amenable strategies for
about a century ago with the rather modification. However, such modifications can
serendipitous introduction of Coley’s toxins: easily lead to over-attenuation of strains that
controlled bacteria that might be the most might increase their safety but at the same time
powerful tool yet to turn the immune system compromise their ability to mount an adequate
into a cancer-fighting machinery [18]. anti-tumor response.
Appreciating the relevance of microbes as key
modulators of benefits and adversities One of the frequently distinguishing
associated with anti-cancer therapy, scientists characteristics of solid tumors is the formation
have been attempting to engineer microbes and of a necrotic, hypoxic zone with low partial
their derivatives in different ways that can pressure of oxygen in the interior of the tumor.
improve the balance between therapy-linked Obligate anaerobic bacteria like Clostridia were
efficacy and toxicity. This is leading to a wave of preferentially chosen for anti-cancer therapy
Bacteria-Mediated Tumor Therapy (BMTT) since their spores germinate only in the absence
trials. BMTT differs from conventional of oxygen. Although germination of Clostridia
immunotherapy in the sense that a bacterial was confined to hypoxic regions, the related
infection itself can exert toxic effects on toxicity led to high mortality rates using this
individual cancer cells, rather than recruiting type of bacteria. To increase the safety of
the immune system. The battle in this approach Clostridia, virulence factors like the lethal α-
is to fine-tune the bacterium’s ability to reach a toxin were deleted from potential therapeutic
solid tumor, thrive in the local environment and strains. Besides experimental studies in mice,
execute its engineered function or manifest its Clostridium novyi-NT (non-toxic) has already
virulence in a manner that ultimately kills most been tested in preclinical and clinical trials using
of the tumor cells while preventing damage to dogs as well as human patients [20]. A human
healthy tissues. patient with advanced leiomyosarcoma was
chosen for treatment with an intratumoral
(i) Optimizing microbial strain design injection of C. novyi-NT spores. This treatment
Exploiting the uniqueness of the tumor biology resulted in regression of the tumor within and
and understanding the appropriate microbial surrounding the bone [20]. C. novyi-NT was
features for optimizing strain design will be thereafter suggested to precisely eradicate
critical factors governing the success of the neoplastic tissues, warranting further clinical
BMTT strategy. Salmonella, Clostridium and trials of this agent in selected patients. C. novyi-
Listeria species have been among the few NT is unique because it thrives in a low-oxygen
microorganisms that have exhibited potential in environment where it begins to divide and
entering, colonizing and destroying cancer cells. grow, and in the process kills cancer cells. The
They have been modified in various ways to bacteria then stop growing at the tumor
make them more suitable as BMTT agents. The boundary, where there is more oxygen,
intrinsic anti-tumor response generated by preventing them from intruding any further into
bacteria is most likely connected to their healthy cells. Furthermore, orthotopic
32 Journal of Postdoctoral Research November 2016: 27-46

glioblastomas were successfully targeted with structure of LPS or downregulate the expression
C. novyi spores upon intravenous infection in a of flagella. To counteract such mechanisms, a
rat model, in turn indicating that the spores are promising recombinant strategy would be to
able to pass the blood brain barrier under reinstate the immunogenicity of Salmonella via
certain conditions [21]. The mechanism of the modification of immunogenic targets or
antitumor effect by Clostridia is poorly MAMPs. For example, a hexa-acylated Lipid A
understood. Although these bacteria are able to structure was shown to be highly efficient at
successfully target neoplastic tissue without immune stimulation, whereas tetra-acylated
seriously harming the host, their application is Lipid A acted antagonistically [24]. In addition, it
limited to large solid tumors with hypoxic was shown that Salmonella variants bearing
centers. both flagella proteins FliC and FljB trigger an
increased host immune response upon oral
To overcome the limitation of confinement to administration [25]. These examples
hypoxic regions and to address the problem demonstrate that the immunogenicity of
that tumors grow out from viable oxygenated attenuated bacteria can be enhanced when the
tissue, facultative anaerobic bacteria like MAMPs are modified in such a way that host
Salmonella typhimurium became the focus of pattern recognition receptors are more
initial BMTT experiments. However, as efficiently stimulated.
Salmonella can grow under aerobic conditions,
they are not restricted to merely colonizing Modifying the expression or activity of certain
tumors but are also able to disseminate to MAMPs could have pleiotropic effects, some
healthy organs like spleen and liver. Therefore, detrimental, that may affect the gene
to ensure safe application, Salmonella needs to regulatory circuits of bacteria in a more general
be adapted. The prominent Salmonella strains way. Therefore, a wild-type like phenotype of
VNP20009 (in vitro) and A1-R (in vitro and in bacteria that is only conditionally modified may
vivo) were created by passaging bacteria from be the next step in strain design. Currently, two
tumor to tumor either in cell culture or in mice, concepts are being evaluated using this
so as to develop a tumor-adapted phenotype rationale, namely, delayed attenuation and
concomitantly exhibiting high tumor specificity delayed lysis. Genetically modified mutants
[22]. Auxotrophy for purines or Arginine and generated in these two approaches exhibit a
Leucine, respectively, rendered these wild-type like phenotype upon in vivo
Salmonella variants metabolically deficient and administration whereas manifestation of the
highly tumor-specific [23]. However, the intended ultimate phenotype usually driven by
isolation of spontaneously appearing mutant loss of an inducer kicks in later. For instance,
bacterial clones through selective pressure auxotrophic or attenuated bacteria may
represents a challenge to appropriately tailor maintain viability through gene
bacterial strains. Due to the uncertainty complementation by expressing a gene product
associated with such a non-specific method of under an inducible promoter like P or P in
BAD tet
attenuation, targeted gene editing would be a the presence of arabinose or
better choice for customizing bacteria for anti- anhydrotetracycline, respectively [26]. Such
cancer therapy. bacteria can be stably induced and
complemented in culture. In vivo, the inducers
The immune recognition of Salmonella and are diluted out and are no longer available. As a
induction of an immune response are factors consequence, the bacteria will lose their wild-
that directly correlate with the presence of type phenotype and become attenuated after a
various MAMPs. To survive in a hostile few rounds of replication. This delayed
environment Salmonella may either modify the attenuation system was recently deployed for
Kulkarni 33

Salmonella to modify the LPS structure under this promising idea is beset with its own set of
the control of P . The effect was evaluated in challenges. At least two components including
BAD
a murine tumor model [26]. Compared to the (i) a tumor-specific microbe-based platform and
bacteria harboring a complete gene deletion (ii) a cytotoxic compound or payload that can be
and affecting LPS expression immediately upon synthesized and actively secreted or delivered
inoculation into the mice, the initial wild-type by the microbes, are required.
like phenotype of the delayed attenuation
strain induced a stronger immune response that In this context, a few concepts are currently
significantly enhanced its anti-tumor activity under investigation. The first one employs
[26]. None of the mice succumbed to the prodrug converting enzymes produced by
infection and the health status of the mice was bacteria. This strategy relies on enzymes that
only transiently affected after bacterial are capable of converting a systemically
administration. administered inactive prodrug into an active
cytotoxic drug. As the enzyme would be present
Similarly, in a delayed lysis Salmonella system, primarily in vicinity of the bacteria and facilitate
cell wall synthesis is abrogated in the absence local conversion at only this site, this method
of arabinose in vivo [27]. The bacteria are thus provides good tumor specificity. The
not able to establish a systemic infection. therapeutic benefit of enzymes like cytosine
However, the sudden microbial death in vivo deaminase and nitroreductase expressed by
might cause complications like septic shock in either Clostridia or Listeria has been tested [31,
the host due to release of large amounts of 32]. However, while they showed promising
bacterial endotoxins. Nevertheless, the system activity in vitro, no significant improvement of
was successfully tested to vaccinate mice therapeutic effects was observed in vivo. This
against influenza viruses, by a targeted release could most likely be attributed to low enzyme
of intracellular virus-specific antigens by the expression levels, low enzyme secretion
bacteria [28]. This system may show efficiency or low prodrug conversion inside the
comparable utility in a cancer model and should cancerous environment and needs to be
thus be explored. These studies demonstrate optimized for further attempts. However,
that modern strategies are more widely attenuated Salmonella strains as carriers of
effective when both attenuation and enzymes like cytosine deaminase [33] (which
optimization are accommodated in the same was effective in treating subcutaneously
therapeutic strain. implanted colon tumors in mice by converting
5-Fluorocytosine to 5-Fluorouracil), thymidine
(ii) Microbes as delivery vehicles for kinase [34] (which caused dose-dependent
therapeutic molecules suppression of tumor growth and prolonged
Bacteria could further be exploited as survival in melanoma-bearing mice, in addition
opportunistic infectious agents designed to to that seen with the bacteria alone, by
shuttle therapeutic agents directly into phosphorylation of Ganciclovir to its active
cancerous tissue. This should maximize their form) or carboxypeptidase G2 [35] (which
intended effect while reducing systemic side showed tumor growth reduction in cases of
effects. Various preclinical trials have shown the mouse melanoma, and human breast and colon
ability of different bacterial strains to migrate to carcinomas by converting a range of mustard
tumor sites, locally produce therapeutic agents, prodrugs to active DNA cross-linking agents)
and mediate highly effective and specific have been successfully used in preclinical and
therapeutic responses [29, 30]. Exploiting clinical setups. Nevertheless, the patient cohort
bacteria as live vector systems could represent needs to be enlarged to significantly validate
the next generation of strain design. However, any promising results.
34 Journal of Postdoctoral Research November 2016: 27-46

The second approach concerns production and process: (i) genetic alteration to display GB1, an
subsequent secretion of therapeutically active immunoglobulin G (IgG)-binding domain of
compounds by the bacteria themselves during protein G on the biosilica surface, enabling
tumor colonization. Therapeutic molecules attachment of the tumor cell-targeting antibody
include bacterial toxins like α-Hemolysin or specific for the p75 neurotrophin receptor
Azurin [36, 37], recombinant effector proteins (p75NTR) which specifically and readily
such as TNF-α and IL-2 [38], or small hairpin attached to neuroblastoma cells but not to
RNAs [39] (shRNAs) targeting, for instance, fibroblasts, and (ii) incorporation of the
STAT3 (in case of Hepatocellular Carcinoma) chemotherapeutic agent Camptothecin or SN-
[40] or IDO (in case of melanoma) [41]. The 38 into the silica-binding carriers using an
transport of these molecules across bacterial established method to encapsulate
membranes to the extracellular environment hydrophobic drug molecules into cationic
around or within the tumor is critical. The basic micelles and liposomes, in turn minimizing the
idea is to fuse therapeutic agents to signaling off-target toxicity. The algae-based drug-
molecules that determine release via a delivery vehicle is biodegradable and
particular bacterial secretory pathway, ensuring completely harmless to healthy cells. Such
continuous secretion. However, the fusion to a chemotherapy delivered by microalgae led to
signaling peptide could be a limiting factor in appreciable tumor regression in the mouse
this strategy. The agent may lose its activity due neuroblastoma model employed in this study.
to, for example, conformational alteration or This system served as a good foundation for
non-native refolding of the fusion complex subsequent studies demonstrating how
upon secretion. However, proof of principle has engineered microbial sources may be used as
been successfully demonstrated by Singer et al., versatile vehicles for the targeted delivery of
where recombinant neuroactive peptides were anticancer drugs to tumor sites. For instance, in
delivered via the flagellar type 3 secretion a recent report by Felfoul et al. [45]
system (fT3SS) [42]. Furthermore, the efficacy immunodeficient mice were injected with
of fT3SS for delivery was assessed in a cancer Magnetococcus marinus, the microorganism
vaccine, where the codon-optimized human employed to transport nanoliposomes
tumor-associated antigen Survivin, an encapsulating SN-38 into tumor hypoxic
oncoprotein overexpressed in most human regions, coupled with navigation via an external
cancers, was genetically fused to the Salmonella energy source. The authors suggested that
secreted effector protein SseJ, and delivered in harnessing swarms of bacteria exhibiting
the cytosol of antigen-presenting cells. As a magneto-aerotactic migration behavior and
result, complete tumor regression in very low immunogenicity can dramatically
lymphoma-bearing mice was observed [43]. improve the therapeutic index of drug-loaded
Thus, delivery via the fT3SS of Salmonella may nanocarriers, while reducing systemic toxicity
represent a promising foundation for active and ensuring safety.
delivery. Complex constructs with multiple
domains for direct or indirect recognition of, For the most part, BMTT has incorporated
binding to and killing of cancer cells could in laboratory-made strains, and while results in
principle be engineered in various microbial murine models have been impressive,
strains. outcomes in patients have been inconsistent,
with the inherent pathogenicity and
In another path breaking effort, scientists have immunogenicity of the bacteria employed
created a nanoporous biosilica created from the outweighing therapeutic responses in patients
diatom microalga Thalassiosira pseudonana [13]. Still, the development of microbial vectors
[44]. This diatom was engineered in a two-step as delivery vehicles for therapeutic agents is an
Kulkarni 35
exciting area of research that is gaining addressed before optimized bacteria can be
acceptance by clinicians and regulatory implemented in the clinic for anti-cancer
authorities for its potential to deliver positive therapy.
clinical outcomes.
Immunosuppression in cancer patients can
Use of Clostridial species for targeted tumor occur via neutropenia, disruption in the barriers
killing and attenuated Salmonella or Listeria to infection and shifts in the microbial flora,
vectors for oral vaccination or tumor gene caused by the malignancy itself, treatment
delivery, represent the most widely applied procedures (surgery, radiation, chemotherapy)
bacterial vectors at the clinical trial level [46, 47, or reduced utilization of nutrition. These
48, 49, 50]. In a clinical trial performed on constitute some of the risk factors generally
metastatic pancreatic ductal adenocarcinoma making a cancer patient more prone to primary,
(PDA) patients, administration of CRS-207, a secondary or nosocomial infections. Due to low
live-attenuated Listeria monocytogenes white blood cell count, patients may not have
expressing the cancer antigen mesothelin, along the usual signs and symptoms when developing
with low dose Cyclophosphamide and GVAX an infection such as redness, swelling, pus
(another vaccine evaluated in PDA) significantly formation, cough, nasal drainage etc. Making
extended overall survival with minimal toxicity the distinction between patients at low and
[51]. high risk depending upon their bone marrow
function is critical in determining clinical
Similarly, a number of live attenuated strains of success. Applying BMTT in such situations raises
Listeria have been developed expressing a even higher safety concerns regarding the
broad range of tumor antigens, such as Her- pathogenicity and immunogenicity of microbial
2/neu [52, 53] (an oncoprotein associated with species supposed to mediate therapy, as they
a wide variety of cancers), Melanoma are known to cause life-threatening infections
Associated Antigen (MAGE) [54] and prostate in clinical practice. Through BMTT related
specific antigen (PSA) [55, 56], and HPV16 E7 bacterial translocation, endogenous
[57]. The cytoplasmic location of L. microorganisms can move into the
monocytogenes is crucial as this potentiates bloodstream, resulting in bacteremia [58].
entry of the antigen into the Class I MHC Although the mortality attributed to such
antigen-processing pathway leading to priming infections has decreased over the years, due to
of specific CD8+ T-cell responses. Intravenously the development of beta-lactam antibiotics and
administered attenuated L. monocytogenes fluoroquinolones, the types of infections have
expressing HPV16 E7 was recently used in phase changed as new resistant and opportunistic
I clinical trial on patients with metastatic microorganisms emerge [58]. Efforts to refine
cervical cancer [57]. Apart from some flu-like the process have involved prophylactic
symptoms and fever-related hypertension in antibiotic regimens [59], developing more
some patients, the vector was well tolerated. In specific antibiotics targeting the desired
addition, 30% tumor reduction was noted with microbial class [59], and genetic engineering of
an increase in overall survival, indicating the strains to enable encoding of additional
safety and efficacy of listerial vectors in patients heterologous genes [60, 61, 62] Both
and paving the way for clinical development of Clostridium and Salmonella have been shown to
this vector strategy. be non-pathogenic in multiple animal species
Persistent infection after chemotherapy, [63, 64] and in human trials [50, 65, 66], but any
genetic instability of engineered strains and retained virulence could be problematic for
determining the correct combination therapies immunocompromised late-stage cancer
are additional challenges that remain to be patients.
36 Journal of Postdoctoral Research November 2016: 27-46

Genetic instability of attenuated strains is a to engineer microbes that are well tolerated by
potential problem because mutations could patients and seamlessly integrate with existing
create ineffective (loss of function) or harmful clinical methods. The host strain Escherichia coli
(gain of function) phenotypes, leading to failure Nissle 1917 (EcN) has an established safety
of therapy and/ or exaggerated infection. The record in clinical trials for oral delivery to GI
rate of mutation will need to be estimated for disorders and has therefore been used to
each strain to be able to specify the maximum develop an orally administered diagnostic
permissible time limit that bacterial colonies agent. This agent functions non-invasively and
could remain in tumors before being eliminated indicates the presence of primary or metastatic
using specific antibiotics. Genetic stability could liver cancer by producing visibly detectable
be enhanced by creating clean deletions in signals in urine. Motivated by the need for an
virulence factors, identifying and modifying accessible, highly sensitive and specific tool for
multiple virulence factors to reduce the detection of micrometastases that are beyond
probability of reversion, or by incorporating the reach of existing diagnostic tools, Danino et
engineered genes on the bacterial chromosome al. [68] engineered EcN, with a series of
thereby limiting homologous recombination expression cassettes. The corresponding
and horizontal gene transfer [67]. diagnostic platform called PROP-Z
(programmable probiotics with lacZ) is made up
The ultimate BMTT can be thought to consist of of probiotic EcN bacteria transformed with a
a collection of strains designed for specialized dual-stabilized, high-expression lacZ vector as
purposes rather than a single perfect strain. well as a genomically integrated luxCDABE
Successful treatment could utilize these strains cassette that allows for luminescent
cooperatively and in combination with visualization without providing exogenous
chemotherapy by means of a detectable luciferin. Upon oral delivery, these probiotics
facultative anaerobe for diagnosis; an rapidly (within 24 hours) translocate across the
engineered immunogenic stain to sensitize the GI tract and selectively expand within tumor
immune system; an obligate anaerobe to treat cells present in the liver. The natural
inoperable primary tumors; and a motile strain reticuloendothelial filtration of gut-derived
controllably producing a cytotoxic agent to treat venous outflow to the liver maximizes liver
diffuse tumors and metastatic disease [67]. The exposure of gut bacteria, and thus, orally
genetic flexibility of bacterial strains can be delivered microbes selectively colonize liver
exploited to tune them for individualized tumors. EcN robustly colonized tumor tissue in
therapy, targeting to multiple tumor sites and rodent models of liver metastasis after oral
precise control of cytotoxicity. Once perfected, delivery but did not colonize healthy organs or
anti-cancer bacteria are expected to be an fibrotic liver tissue. No deleterious health
essential clinical tool, able to perform functions effects were observed on the mice for more
unachievable by other therapies, such as detect, than 12 months after oral delivery [68]. PROP-Z
prevent, and treat tumors and metastases. expresses high levels of the enzyme β-
galactosidase which can cleave systematically
IV. Microbes as biomarkers for cancer injected, cleavable substrates. Cleavage
diagnosis products of the substrates filter through the
Rapid advances in the engineering of genetic renal system to generate a high-contrast urine
circuitry in living cells has positioned synthetic signal for detection. Probiotics can thus be
biology as a remarkable tool to address programmed to safely and selectively deliver
numerous biomedical problems, including synthetic gene circuits to diseased tissue
disease diagnosis. One challenge in exploiting microenvironments in vivo.
synthetic biology for translational applications is