Cancer, bacteria, cancer treatment by bacteria, Bacteriotherapy, Engineered Bacteria for Cancer Therapy, Bacterial Metabolites in Cancer Therapy, Bacterial DNA Vaccines, Genetic engineering, Artificial Immune Stimulation, immunotherapy, immune cells, recombinant vaccine, Toll-like receptors, exotoxins, antibiotics, bacteriocins, radiotherapy, chemotherapy,

1. By
Hasnat Tariq
and
Um-e-Ummara Shafi
Bacteria and
Cancer
Different sides of the same
coin

2. Contents
• Abstract
• Introduction
• Bacteriotherapy
• Engineered Bacteria for Cancer Therapy
• Bacterial Metabolites in Cancer Therapy
• Bacterial DNA Vaccines for Cancer Therapy
• Combination of bacterial therapy with conventional anticancer
therapies
• Concluding Remarks
• Drawbacks

3. Abstract 1
 Conventional cancer therapies have been unsuccessful for the proper eradication of
tumor cell so there is need of alternative approaches.
 Bacterial cancer therapy is an alternative anticancer approach that has promising
result on tumor cells in vivo.
 With the aid of genetic engineering, natural or genetically modified bacterial
strains can directly target hypoxic regions of tumor and secrete therapeutic
molecules.
 Additionally stimulation of immune cells by bacteria, bacterial cancer DNA
vaccines and antitumor bacterial metabolites are other therapeutic applications of
bacteria in cancer therapies.

4. Introduction
 What is Cancer ?
Cancer is a common cause of death worldwide
known as a group of diseases consists of abnormal
cell growth, which can invade and spread into
different organs of the body through metastasis.
 Statistical Analysis :
• According to WHO, 9.6million deaths in 2018.
• Globally, cancer causes about one in every six
deaths.
History of cancer treatment shows that conventional
cancer therapies like surgical therapy, chemotherapy,
radiation, immunotherapy, gene or hormonal therapy,
and bone marrow/stem cell transplantation have
serious drawbacks that can outweigh their merits

5. Why we need to find alternative approaches?
Multifactorial etiology
Development of resistance to traditional
therapies
Side effects and long term sequela of
traditional therapies
Chemotherapy causes formation of
multi-drug resistance in some cells

6. Alternative
Approaches
Gene therapy
Non-invasive
radiofrequency (RF)
cancer treatment
Insulin potentiation
therapy (IPT)
Hyperthermia
therapy
Photodynamic
therapy (PDT)
Telomerase therapy
Diet therapy Bacteriotherapy

7. Bacteriotherapy
Bacteriotherapy is the purposeful use of bacteria
or their products in treating an illness.
The promising approach to reduce the limitations of
conventional therapies is using therapeutic bacteria.
The use of bacterial cancer therapy has increased
considerably over the past few decades.

8. Why
Bacteriotherapy?
Bacteria
Regression of
tumor
Genetic
Manipulation
Migration, Colonization and
Secretion of Cytotoxic
Bacterial Products
Activation of
Immune System

10. History of Bacteriotherapy:
History of using bacteria in cancer therapy goes back to the 1890s
when William Coley injected two heat-inactivated bacteria,
Streptococcus pyogenes, and Serratia marcescence, which are
known as Coley's toxin to his cancer patients which caused tumor
regression.
Reliance of Bacteriotherapy:
The scientific concept of using bacteria relies on the pathogenic
substance produced by bacteria which is called ‘pathogen-associated
molecular patterns’ (PAMPs) that contributes to activation and
maturation of tumor antigen-loaded dendritic cells

11. Engineered bacteria
for Cancer Therapy
Gene therapy is used to engineer bacteria because of the following
reasons.
• Higher efficacy in targeting antitumor agents to the tumor cells.
• Provides attenuated bacteria with the increased therapeutic ability.
• Improvements in internalization, colonization, and multiplication
in tumor tissues.

12. Example of
Engineered Bacteria
1. Corynebacterium diphtheria M55 and Salmonella
typhimurium serovar VNP2000 are useful vectors
because of their selective tumor colonization.
2. Engineered bacteria expressing death inducers
like S. typhimurium secreting murine Fas ligand
(FasL) a pro-apoptotic cytokine.
3. S. typhimurium expressing Shiga toxin caused a
marked increase in necrosis

13. Immunotherapy of
bacteria in cancer therapy
 Artificial Immune Stimulation
• Why we Use it?
Escaping tumor cells from the immune system is the main
difficulty of efficient immune therapy.
 Advantages
• Probability of killing infected tumor cells increases when the
malignant cells are primarily recognized as infected cells
rather than cancer cell
• Increasing the antigenicity of poorly antigenic tumor cells can
be achieved through tumor-targeting bacterial infection, which
can activate and improve the function of the immune system,
such as tumor-associated macrophages (TAMs), CD4+ and
CD8+ T cells, regulatory T-cells (Tregs)

14. 1. Myeloid Cells
2. Natural Killer Cells
3. CD4 and CD8 T cells
4. Gamma-Delta T-cells (γδ T cells)
5. Toll like Receptors
Targets Of Bacterial Cancer Immunotherapy

15. Myeloid Cells
treatment with mice carrying B16-F1 melanoma
cells
Attenuated strain
of S. typhimurium
• results in the maturation of intratumoral myeloid cells and reduction in their
immune suppressive function
The direct intratumoral (i.t.) injection to
Her2/Neu expressing CT26 tumors
Attenuated and
recombinant S.
typhimurium
vaccine
• resulted in a change in macrophage phenotypic characteristics from immature
immunosuppressive phenotype form to mature-type expressing TNF-α and
enhanced the antitumor action
immunotherapy on human or murine melanoma
cell line
L. monocytogenes
strain
• resulted in the transformation of malignant melanocytes into professional antigen-
presenting cells (APCs) that mediates regression and eradication of tumor with the
expression of phenotypic markers such as, MHC II, CD83, CD40 similar to mature
dendritic cells

16. Natural Killer Cells
Natural killer (NK) cells, as a part of innate immunity, are lymphoid cells
with the ability to destroy tumor cells without any previous antigen
exposure.
Salmonella strain
Express IL-12, IFN-γ, or
TNF-α through NK cell
activation
Causes induction
antimicrobial
response
Causes antitumor
responses

17. CD4+ & CD8+ T
Cells
CD4+ and CD8+ T cells function is related to their specific
subsets such as Th1 and Th17 cells
Th1
Directly kill cancer cells
by secretion of TNF-α or
IFN-γ.
Induce the activation of
CD8+ T

18. CD4+ & CD8+ T
Cells
Th17 Helps in inflammation and autoimmunity
Example.
Intraperitoneal treatment of B16-F1 melanoma model
with attenuated Salmonella strain resulted in more than
three-fold enhancement in the infiltration of CD4+ and
CD8+ T-cells into tumor cells

19. Gamma Delta T
Cells
 Gamma-Delta T-cells (γδ T cells) which characterized
by a surface antigen recognition complex type 2,
demonstrate the strong antitumor activity by releasing
significant amounts of IFN-α and TNF-α.
Example
Using Mycobacterium vaccae and M. obuense or BCG in
Mycobacterial immunotherapy induced the activation and
proliferation of gamma-delta T-cells, which causes Th1
cytokines production, such as IFN-γ and TNF-α leading to
regression of tumor cells reduction in tumor growth.

20. Toll-like Receptors
 Toll-like receptors and other receptors of innate
immune cells can recognize several conserved bacterial
ligands as an agonist and after binding to these
receptors, using the stimulation of intracellular
signaling cascades, the production of inflammatory
cytokines arises.
• Several exotoxins and other cytotoxic bacterial
components as anticancer agents can directly kill tumor
cells.
• Using Salmonella and Listeria in different types of
cancer immunotherapy, they can be utilized as delivery
vectors of immunogenic tumor antigens.

21. Table.1
Strain(s) Type of cancer Mechanism of action
Mycobacterium bovis
BCG
Bladder cancer
Stimulating the immune
system and increasing the
proinflammatory cytokines
activation of cancer cells
phagocytosis
Clostridium novyi Leiomyoma Capable of producing specific
enzymes and toxins that destroy
cancer cells or
Listeria monocytogenes
LADD strain
Cervical, Oropharyngeal,
Pancreatic, Lung and
Mesothelioma
Vaccines for cancer therapy
induction of antigen-specific T-
cell responses and more
targeted cancer cells
elimination

22. Bacterial metabolites
in cancer therapy
Numerous metabolites with different biologic activities are
expressed and secreted by bacteria and play a vital role in
bacterial growth and pathogenesis.
1. Antibiotics 3. Enzymes
2. Bacteriocins 4. Toxins
Bacterial
Metabolites

23. Table. 2
Bacterium Metabolite Molecular Weight Mode of Action
Antibiotics
Actinomyces antibioticus Actinomycin D 1.26kD Intercalation to DNA and
the stabilization of
cleavable complexes of
topoisomerases I and II
with DNA,
photodynamic activity
and free radical
formation
Bacteriocins
Lactococcus lactis Nisin A 3.5kDa Cell cycle arrest and
reduction in cell
proliferation cause
apoptosis by alteration in
calcium influx and cell
cycle arrestin G2 phase
as significantly higher
calcium influx

24. Enzymes
Escherichia coli l-Asparaginase 11.20kDa Reduction of
asparagine blood
concentration causing
a selective inhibition
of growth of sensitive
malignant cell
Toxins
Pseudomonas aeruginosa Exotoxin A 66kDa Targeting with tumor-
related antigens and
induction of cytotoxic
pathways

25. Bacterial DNA vaccines
for cancer therapy
DNA vaccines are based on bacterial plasmids that
encode antigens with the ability to encode the
immunostimulatory molecules like interleukin-2 (IL-2)
and GM-CSF.
Routes of
Delivery
• Mucosal
• Intramuscular (IM)
• Intradermal (ID)
• Subcutaneous (SC)

26. Delivering System of DNA Vaccines
Physical Strategies which include
Transport the DNA vaccine to the nucleus of host
cells
Leads to the expression and presentation of major
histocompatibility molecules (MHC) which leads
to activation of T-cell
Electroporation , Sonoporation , DNA tattooing and
Gene gun

27. Advantages of DNA
Vaccines
• Activating both CD4 and CD8 cells occurs as a
consequence of encoded antigen presentation by MHC
class I and class II, and eventually, the humoral
immunity is activated.
• When cytosolic sensors recognize the double-stranded
DNA, innate immune response would be activated.
• DNA vaccine can stimulate immunological memory.

28. Strategies to Improve
Vaccine-Induced Immune
Response
The first strategy is the selection and insertion of best
antigens into the plasmid DNA in favor of elevated
immune system activation.
Two Main Tumor Antigens
Mutational antigens Tumor-associated antigens
Derived mutated self-proteins Differentiation and universal tumor
antigens
Products of a mutated oncogene
(TSA) and neoantigens
Overexpressed by tumor cells and
are mostly unmutated

29. The second strategy combined anticancer
therapy, which comprised of exploiting
complementary therapies in association with
DNA vaccines that leads to improvement in the
activity and the number of different immune cells
in the immunosuppressive moieties of growing
tumor cells.

30. Combination of bacterial
therapy with conventional
anticancer therapies
Combination of Radiotherapy and Bacteriotherapy
Why?
Presence of hypoxic region make tumor cell resistant to
radiotherapy so we utilize facultative or obligate anaerobes for
reaching the poorly vascularized hypoxic intratumoral sites.
• Lowered doses of radiotherapy would be administered, and it can
significantly reduce the damage of the healthy tissues.
Example
1. Genetically engineered Salmonella can be used as an antitumor
vector.
2. Tumor-targeting bacteria can replicate inside the tumors and
secrete effector proteins like the herpes simplex thymidine
kinase and can cause tumor growth suppression.

31. Combination of Chemotherapy and Bacteriotherapy
Why?
Chemotherapy, as a prominent anticancer treatment, has several
shortcomings like
systemic toxicity, drug resistance, and insufficient drug penetration in
tumor sites.
• Bacterial exotoxins can kill tumor cells, and bacterial endotoxins
can be utilized to fight against cancer in combination with other
efficient chemotherapeutic agent.
• Probiotic tumor-targeting bacteria can reduce the severe side
effects of gastrointestinal toxicity induced by chemotherapy.

32. Example
Administration of Lactobacillus to the patients
who have been during chemotherapy showed
gastrointestinal problems less than others, which
leads to a significant reduction in the doses of the
chemotherapeutic drugs and shortened the period
of chemotherapy.

33. Express and secrete toxins that disturb regulation of cell
growth by damaging signaling pathway
Toxic substances and Enzymes e.g. several
oral bacteria produce peptidyl arginine
deaminase (PAD), which is correlated with
pancreatic adenocarcinoma
Inflammation that is noticeable factor of tumor.
Quorum-sensing peptides such as stimulating competency
(CSP) and extracellular death factors (EDF), from S.mitis
and E.coli respectively are effective tumour invasion
promoters and help in the angiogenesis of the collagen I
extracellular matrix, which, in turn, promotes tumour
metastasis.

34. Examples of
Notorious Bacteria
associates with hepatobiliary cancerSalmonella typhi
associates with lung cancerMycobacterium
tuberculosis
associates with colorectal cancerCitrobacter
rodentium

35. Role of bacteria in cancer. Some bacteria are notorious as carcinogenic bacteria, which cause
tumorigenesis, while some bacteria have the potential of cancer therapy, targeted to hypoxic
tumor region and leading to the eradication of tumor cells by releasing of therapeutic
molecules.

36. Concluding Remarks
• Hypoxic condition within tumor is leading cause of
failure and inefficiency of chemical treatment but
bacterial agents can selectively target and proliferate in
hypoxic region of tumor.
• Bacteriotherapy have no side effects as compare to
conventional therapies
• Bacterial anticancer agents have cytotoxic activity
against MDR cancer cells.
• Bacteriotherapy has ability of post-administration
control, direct and selective killing of cancer cells that
have attracted the attention of cancer researchers

37. Drawbacks
• Lack of well-designed clinical trials
• Innate bacterial toxicity
• Short half-life
• DNA instability

38. THANK YOU