The document discusses using bacteria, specifically Salmonella, to target and destroy tumors in three ways: 1) Delivery of anti-cancer compounds to the tumor site using genetically engineered Salmonella. 2) Sensitizing the immune system to the presence of tumors by using Salmonella to deliver immune-sensitizing compounds or antigens. 3) Directly activating caspase-3, a key enzyme in apoptosis, by engineering Salmonella to up-regulate caspase-3 while also introducing anticancer drugs. Salmonella is a promising approach for cancer therapy due to its ability to preferentially target hypoxic tumor environments, deliver therapeutic payloads, and exploit apoptotic pathways.

1. Nimisha Tiwari
Under supervision of Dr. A.
Pal
In-vivo testing facility
Molecular Bio-prospection
CSIR-CIMAP
Targeting Tumors with Bacteria

2. Introduction
 Regarding the industrialized countries of the
western world, cancer accounts for about
one fifth of all deaths. One person out of
three will be treated for a severe cancer in
their lifetime.
 Conventional cancer treatments, such as
surgery, chemotherapy and radiotherapy,
often fail to achieve a complete cancer
remission.

3.  In 1890, William B. Coley, a surgeon in the Memorial
Hospital in New York, described for the first time
bacteria as anticancer agents. He observed that
several patients showed tumor regression after being
infected with pathogenic bacteria.
 Directly targeting cancer cells, killing these cells
through innate bacterial toxicity, competition for
nutrients or delivery of anti-cancer agents.
 Salmonella, Clostridium, Escherichia, Shigella ,
Bifodobacterium, Caulobacter and Listeria species
have all been tested for their potential use.

4.  S.Typhimurium is motile, easily genetically
manipulated and grows as a facultative anaerobe
in the presence or absence of oxygen.
 Salmonella also has some attractive properties
well suited for the design of a chemotherapeutic
agent.
 Salmonella migrate towards the tumor by their
flagella, attracted by the high concentrations of
nutrients available within the tumor
microenvironment.

5.  Metabolic genes have been removed from S.
Typhimurium, (purI in the mutant strain VNP20009),
rendering the mutant bacterium auxotrophic for
certain compounds that are found in very high
concentrations at tumor sites (Purines).
 Two notable receptors : TAR receptor, which detects
aspartate secreted by cancerous tissues and the TRG
receptor, which aids in migration towards ribose found
in necrotic tissues.
 The aspartate receptor controls migration towards
tumors, the serine receptor initiates penetration, and
the ribose/galactose receptor directs Salmonella into
necrotic regions.

6. Presently, S.Typhimurium is being used to target
and destroy tumors in three specific ways:
i) delivery of anti cancer compounds
ii) sensitizing the immune system to the presence
of tumors
iii) using bacterial toxins to directly activate
caspase-3, a key enzyme of the apoptotic
pathway

7. i) Delivery of anti cancer
compounds
 “Tiny programmable robot factories”
 Numerous compounds that can be delivered via
bacteria to a tumor site (cytotoxic agents, green
fluorescent protein, DNA and small interfering RNAs
(siRNA) ).
 One of the key challenges with conventional
chemotherapy is delivery of potentially toxic
agents to the appropriate areas of the tumor while
preventing damage to healthy tissue.

8.  Temporal control over bacterial delivery of therapeutic agents is a key
consideration as delivery of these compounds during transit to the
tumor site will distribute products systemically.
 Detection of small molecules by the bacteria, or irradiation at the point
where expression is required, have both been used successfully in
vivo along with the use of pro-drugs introduced at the tumor site for
conversion into an anticancer agent by the bacteria upon contact.
 recA, the radiation inducible promoter, was linked to tumor necrosis
factor (TNF)-related apoptosis inducing ligand (TRAIL) to control its
expression by Salmonella during infection of tumors. S. Typhimurium
at the tumor site was then induced to express TRAIL by doses of
radiation, which simultaneously treated the tumor.
 A second approach for controlling therapeutic delivery is the
construction of bacteria that produce enzymes converting harmless
pro-drugs into active agents inside the tumor. Toward this goal
Salmonella has been engineered to express cytosine deaminase
(CDase) that cleaves the pro-drug 5-fluorocytosine to the active
chemotherapeutic 5-fluorouricil.

9. ii) Sensitizing the immune system to the
presence of tumors
 The ignorance of the immune system to the presence of
tumors within the body is a significant consideration in
attempting to treat cancer.
 S. Typhimurium infection of cancer cells was shown to
upregulate the cellular protein connexin43, resulting in
gap junction formation not only between tumor cells but
also between cancer cells and antigen presenting cells
(APCs).
 APCs access to pre-processed tumor antigens, which
can be presented to T-cells, sensitizing the immune
system to the presence of tumors and activating an anti-

10.  Other approaches employ S. Typhimurium to
deliver cytokines to the tumor site in an
attempt to activate immune cells or elicit an
immune response against the tumor.
 Antigens have also been linked to bacterial
toxins that are highly immunogenic to
sensitize the immune system against cancer
cell antigens.
 Approach harnesses the ability of S.
typhimurium:
a) delivery of immune sensitizing compounds
at the tumor site to alert circulating immune
cells

12. iii) Using bacterial toxins to directly activate
caspase-3, a key enzyme of the apoptotic
pathway
 Recent evidence indicates that S. Typhimurium
may activate specific apoptotic enzymes .
 Activation of caspase-3 by a single effector
protein of S. Typhimurium increases the
infectivity of this pathogen, as caspase-3
directed the processing of S. Typhimurium
secreted effectors into their functional subunits
upon their delivery into the host cell.
 An unprecedented means of promoting infection
indicates that the interaction of bacteria with
apoptotic pathways may be more intimate than
previously recognized, and in fact, may be quite
common amongst pathogens.

13. Can we use bacteria to exploit apoptotic pathways,
and in particular activation of caspase-3, opening
a new front in the fight against cancer?
 Direct activation of caspase-3 in the treatment of cancer
through using procaspase-3 activating compounds has
been attempted previously using pan activating caspase-1
(PAC-1).
 Rather than deliver large quantities of non native proteins,
it may be possible to channel the built-in toxicity of S.
Typhimurium to destroy tumors.
 S. Typhimurium also harbors several promoters that are
induced under hypoxic conditions, and using these
promoters to control expression of specific effectors.
 May perhaps be engineered to up-regulate caspase-3
through effector expression while at the same time
introducing potent anticancer drugs can’t be successfully
expelled by the tumor.

14. Tumor-targeting bacterial therapy
 Low cost of production at large scale and more
importantly broad diversity of their effects on the immune
system.
 Almost all tumors have the same microenvironment of low
oxygen tension or hypoxia, an environment obligate
anaerobes prefer.
 Bacteria may be easily manipulated, engineered to
overcome the limitations that hamper current cancer
therapies.
 Bacterial therapy achieves adequate tissue penetration,
which other treatments, including chemotherapy and
radiation, do not.
 Bacteria exhibit intrinsic genetic instability. Although
advanced recombinant DNA technology has rendered

15. RBM5 inhibits tumor growth
in vivo. Tumor-bearing mice
were treated with attenuated
Salmonella carrying
pcDNA3.1 or
pcDNA3.1-RBM5 by
injection two times (on day
28 and 35).
(A) Comparison of tumor
sizes in two groups on day
42 after
implantation.
(B) Tumor growth curve (day
7 to 42 after implantation.
(C) Tumor wet weights were
measured when the mice
were sacrificed on day 4
after implantation.

16. References
 Bernardes, N., Seruca, R., Chakrabarty, A., & Fialho,
A. 2010. Microbial-based therapy of cancer: Current
progress and future prospects. Bioengineered Bugs,
1(3): 178-190.
 Wall, D., Srikanth, C., & McCormick, B. 2010.
Targeting Tumors with Salmonella Typhimurium-
Potential for Therapy. Oncotarget, 1(8): 721-728.
 Shao, C., Yang, B., Zhao, L., Wang, S., Zhang, J., &
Wang, K. 2013. Tumor suppressor gene RBM5
delivered by attenuated Salmonella inhibits lung
adenocarcinoma through diverse apoptotic signaling
pathways. World J Surg Onc, 11(1): 123.
 Shao, C., Yang, B., Zhao, L., Wang, S., Zhang, J., &
Wang, K. 2013. Tumor suppressor gene RBM5
delivered by attenuated Salmonella inhibits lung
adenocarcinoma through diverse apoptotic signaling
pathways. World J Surg Onc, 11(1): 123.