CHAPTER ONE

INTRODUCTION

1.0 Background of Study

Cancer is a catastrophic disease with 18.1 million new cases and 9.6 million deaths per year. It

poses extensive disease burden to affected people, their families and health care systems (WHO,

2020). Development and advancement of therapeutic agents and preventive tactics to cure this

devastating disease critically relies on our understanding of cancer cells’ behaviors and relevant

mechanisms via which they emerge. Generally, carcinogenesis appears as a complex process

which involves multiple steps influenced by various factors and external stimuli. Up to a quarter

of human cancers are associated with microbes mostly caused by viruses, explaining the reason

for gaps in understanding bacterial association to oncogenesis (de Martel et al., 2020). Bacteria

as a transmissible cause of cancer was suspected as early as 16th century and since then the

bacterial association with cancer has been primarily considered opportunistic. More recently, the

involvement of bacteria in the development of several human cancers has been firmly

established. One strong evidence in this regard up to this date is Helicobacter pylori (H. pylori)

which are involved in the induction of gastric cancer (Goodman and Gardner, 2018). During

different phases of the infection cycle, bacteria can directly manipulate the host cell, further

supporting their involvement in altering host cells’ normal physiology and inducing

carcinogenicity. Furthermore, bacterial immune evasion and suppression mechanism also

strengthens their connection to oncogenesis (Damyanov et al., 2018).

Conversely, bacteria have shown great potential for cancer therapy. Bacteria of many species

demonstrate the surprising ability to invade and colonize solid tumors, which often results in

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neoplasm growth retardation, and in some instances, complete tumor clearance (Siegel et al.,

2017). Different strains of Clostridia, Bifidobacteria and Salmonella are capable of colonizing

the hypoxic area of the tumor and destroy the tumor cells. Therefore, they are potential strains

for selective tumor targeting therapy (Pucci et al., 2019). Bacteria create anti-tumor effects

through the depletion of nutrients required for cancer cell metabolism. The tumor tissues that are

deoxygenated nurture the accumulation of obligate anaerobic bacteria – which only survive in

the anoxic region (Janku et al., 2020). Observation has shown that the systemic administration of

Salmonella bacteria flushed into the solid tumor through severe haemorrhaging area, the area

which leads to necrotic regions in which bacteria proliferate, colonized the tumor and decreased

the proliferation of the tumor. The necrotic regions are formed because of the reduction of

oxygen and nutrient supply, which leads to the breaking down of blood vessels in the

hemorrhagic area. This causes the tumor cells in the center of the tumor to die from starvation

and suffocation. The tumor microenvironment may be conducive to bacterial survival and

growth, as it may provide protection from the host immune system and nutrients (WHO, 2020).

Bacteria mediated tumor therapy (BMTT) has been demonstrated for centuries. However, the

associated adverse side effects hinder its development. Adequate balance between the control of

infection and the therapeutic benefit of bacteria is an essential requirement for a successful

BMTT, but this can only be achieved via the heat-inactivation method (Alhunaidi and Zlotta,

2019). Recently, state-of-art genetic engineering has increased the ability to alter bacterial strains

(Han et al., 2020). Such alteration reduces bacteria side effects while increasing their therapeutic

benefits. For example, the wellestablished bacillus Calmette–Guerin (BCG) vaccine for the

treatment of human bladder cancer is arguably superior to intravesical chemotherapy for

superficial disease. The therapy is commonly used as the first-line adjuvant treatment (Hank et

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al., 2020). Similarly, tumordetecting bacteria provide a sensitive and minimally invasive method

to detect tumor recurrence, monitor treatment efficacy, and identify the onset of metastatic

disease. For example, probiotic Escherichia coli Nissle 1917 has been used to develop the orally

administered diagnosis that can noninvasively indicate the presence of liver metastasis by

producing easily detectable signals in urine. Such genetic engineering approaches have paved the

way for further development of promising bacteria-based cancer therapy (Deng et al., 2018).

1.1 Significance of Study

This review will answer the questions and explore the multidimensional association of bacteria

with cancer. This review will also establish associations between certain bacterial infections and

tumor development. This review will also discuss association between bacteria and cancer and

the limitations and advantages of bacterial therapies in cancer treatment.

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 CHAPTER TWO

Literature Review

2.0 Mechanisms behind Bacteria Driven Carcinogenesis

Bacteria may induce carcinogenesis via several mechanisms, such as inducing chronic infection

or immune evasion and immune suppression. Chronic infections can alter the cell growth by

disturbing the cell cycle resulting in the DNA damage similar to that caused by oncogenes. To

name a few mechanisms, several bacteria may influence the oncogenesis through effects on the

transformation of cells, production of toxins, deleterious alterations in the physiological host

process, and induction of hormones which increases the epithelial cell proliferation, and antigen-

lymphoproliferation (Sohn et al., 2017). Bacteria may use actively as well as passive

mechanisms non-specifically to target the tumors. Host and its resident microbiota constitute a

complex “superorganism” with a symbiotic relationship that benefits the host in several key life

components. Once this symbiotic relationship is disturbed by defects in the host’s regulatory

circuits that regulate bacterial sensing, or changes in the microbiome, via environmental changes

(chronic infection, diet, or lifestyle), it may lead to the stimulation of the diseases (Fenner, 2018).

Bacterial chronic infections are of great importance because they stimulate the immune response

and increase the oxidative stress in the infected cells, resulting in the release of reactive oxygen

species (ROS) and leading to the cell membrane and DNA leakage. In other reports, some gut

bacteria were able to produce beta-glucuronidase, an enzyme responsible for producing

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compounds that can bind with DNA and cause mutations (Raskov et al., 2021). Beta-

glucuronidase leads to the deconjugation of conjugated toxins and bile acids and potentially

stimulate the carcinogenicity (Davar et al., 2021). A study reported that in the lungs of smokers,

Chlamydia pneumonia (C. pneumonia) invades the lung and produces nitric oxide (NO) and

other oxygen radicals which result in damage to the lung tissue and DNA driving the lung cancer

(Hamada et al., 2019). Several microorganisms such as Fusobacterium nucleatum (F. nucleatum)

promote colorectal cancers by chronic infections, interaction with cell surface molecules,

immune evasion and immune suppression via chronic inflammation and biosynthesis of

genotoxins (Gholizadeh et al., 2017). Virulence factors of the microorganisms, for example,

lipopolysaccharide (LPS) and cell wall extracts may act as effector molecules driving the

transformation of normal epithelial cells into cancerous cells. In one report, the progression of

colorectal cancer with driver and passenger bacteria was studied. The driver bacteria initiates

tumorigenesis by altering the intestinal environment, which leads to the overgrowth of passenger

bacteria and finally the development of colorectal cancer (Huang and Shi, 2019).

2.1 Bacteria as Causative Agents of Cancers

2.1.1 Bacteria Associated Oral Cancer

Oral cancer remains a major health issue because of its advanced stage detection. In patients with

periodontal diseases, bacteria-induced chronic inflammation has been suggested to increase the

risk of oral cancer in addition to the other etiologies such as alcohol consumption, smoking,

Human papillomavirus, genetic factors and nutrition deficiencies (Zhao et al., 2017). The oral

cavity is inhabited by many bacterial species that are considered harmless until the homeostasis

of the oral cavity is maintained. However, an alteration in the homeostasis can contribute to an

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“ecological shift” or “dysbiosis” which in turn can lead to the development of oral diseases,

including oral cancer (Bray et al., 2018).

2.1.2. Bacteria Associated Esophageal Carcinoma

Esophageal cancer is one of the most aggressive malignancies and the sixth leading cause of

cancer-related deaths (Bray et al., 2018). It is the eighth most common cancer type worldwide

with a 5-year survival rate of less than 25 % (Sung et al., 2021). The primary risk factors for

esophageal cancer included smoking, drinking, obesity, ingestion of hot food, gastroesophageal

reflux disease, and barrett’s esophagus (Then et al., 2020). Gastroesophageal reflux disease and

barrett’s esophagus are considered major bacterial risk factor of esophageal adenocarcinoma (A

subtype esophageal cancer highly prevalent in the West). Researchers have demonstrated that

decreased microbial richness in the upper digestive tract is associated with esophageal squamous

dysplasia which is regarded as a precursor lesion of esophageal squamous cell carcinoma

(Another subtype of esophageal cancer highly prevalent in the East) (Corning et al., 2018).

Interestingly, unlike other digestive organs, the esophagus is either sterile or contains only a few

transient microbes swallowed from the oropharynx or ejected from the stomach by

gastroesophageal reflux. Although there are a few studies which indicated the effect of

esophageal microbiota on esophageal carcinoma, further data is needed to reveal the precise

underlying association (Zhao and Lim, 2020).

2.1.3. Bacteria Associated Stomach Cancer

Stomach Cancer or gastric cancer is an extremely aggressive malignancy, and the 4th most

common cause of cancer-related deaths worldwide, with approximately 1 million new cases

diagnosed every year (Ternes et al., 2020). H. pylori associated chronic gastric inflammation is

the single strongest risk factor for gastric cancer. H. pylori selectively colonizes the gastric

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epithelium and is overall associated with over two-fold increased risk of gastric cancer (Yang et

al., 2019). International agency for cancer research has also listed this bacteria as a class I

carcinogen. Though a bunch of other bacteria such as Streptococcus pseudopneumoniae (S.

pseudopneumoniae), Streptococcus parasanguini (S. parasanguini), Streptococcus oralis (S.

oralis), have also been reported recently to be associated with gastric cancer, their link with

gastric carcinogenicity is not as strong as H. pylori (Yang et al., 2019).

2.1.4. Bacteria Associated Colorectal Cancer/Colon Cancer

Colorectal cancer is the 3rd most prevalent malignancy and the 2nd most common cause of

cancer-associated deaths, causing over 600,000 cancer deaths each year, and accounting for over

1 million new cancer diagnoses each year (Sung et al., 2021). Approximately 70 % of the human

microbiome is found in the colon, and therefore bacterial association to colorectal cancer is very

crucial (Yu and Schwabe, 2017). The large intestine is significantly enriched (10 11 cells per ml)

with the microbial density than the small intestine (10 3 -104 cells per ml), and the same trend

follows for the risk of acquiring cancer (Yu and Schwabe, 2017). Imbalance of gut microbiome

(dysbiosis) is presented as a pathological signature in colorectal cancer patients. Therefore, this

complex interplay between colorectal cancer and GIT (Gastrointestinal Tract) microbiome has

become a highly topical area in current colorectal cancer research (Yu and Schwabe, 2017).

2.1.5. Bacteria Associated Liver Cancer

The commonest type of primary liver cancer is hepatocellular carcinoma, which is the 3rd most

common cause of cancer-related deaths worldwide (Sung et al., 2021). Bacterial connection to

liver cancer is relatively less defined as the liver is generally considered sterile. The direct

interaction of liver with GIT (which harbor the highest number of microbes) via the hepatic

portal and bile secretion systems makes it vulnerable to bacteria. This could be why increased

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translocation of GIT associated bacteria is considered the hallmark of chronic liver infections,

which can further contribute to liver carcinogenicity development (Molina-Romero et al., 2020).

The main bacterial species which are thought to contribute to liver carcinogenicity are,

Helicobacter hepaticus (H. hepaticus), Helicobacter pylori (H. pylori), Atopobium cluste (A.

cluste) and, Escherichia coli (E. Coli). Recently other bacterial genera are also found

predominantly in liver cancer compared to healthy control suggesting the possible association of

bacteria with this cancer. However, recent reports tried to explicate the potential mechanisms

associated GIT microbiome and liver cancer (Yang et al., 2019). Yet, precise mechanisms that

can govern liver cancer’s bacterial-induced carcinogenicity are still required to be explored. In

this regard, a comprehensive and in-depth GIT microbiome research in connection to liver cancer

can provide great support to uncover a bacterial link to liver cancer (Yang et al., 2019).

2.1.6. Bacteria Associated Lung Cancer

Lung cancer has been continuously ranked as the leading cause of cancer-related deaths

worldwide (Sung et al., 2021). Although, smoking is the major risk factor, yet latest research

indicates that several additional factors, including systemic inflammatory responses and

microbial infections with certain microorganisms may contribute to the development of lung

cancer (Trabert et al., 2019). A strong correlation between bacteria and specific lung diseases

leading to carcinogenesis has put forth the complex interactions between the lung microbiome

and lung health. For instance, Mycobacterium tuberculosis (M. tuberculosis) and lung cancer

were mechanically interpreted by chronic inflammation-associated carcinogenesis. It is very well

established that strong and chronic inflammatory responses promote cancer development and

progression via different mechanisms. Some hypothesis also link lung epithelium DNA damage

with both free-radical-induced DNA damage and the maintenance of a pro-inflammatory and

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immunosuppressive cytokine network (Molina-Romero et al., 2020). Pulmonary fibrosis and

over production of tumor necrosis factor (TNF) caused by M. tuberculosis are considered factors

initiating lung carcinogenesis. Furthermore, the presence of Acidovorax, Klebsiella, Rhodoferax,

Comamonas, and Polarmonas species in small-cell carcinoma of lung strongly supports an

important role of bacteria in lung cancer (Vranic et al., 2018).

2.1.7. Bacteria Associated Ovarian Cancer

Globally, ovarian carcinoma is a commonly occurring cancer type of the genital tract in women

and one of the most lethal cancers (Momenimovahed et al., 2019). Several microbes associated

with sexually transmitted diseases, including Chlamydia trachomatis (C. trachomatis) and

Mycoplasma genitalium (M. genitalium) have been reported to be associated with an increased

risk for ovarian cancer (Trabert et al., 2019). These microbes are associated with female

reproductive disorders such as tubal infertility and pelvic inflammatory disease, suggesting that

they can localize from lower to upper genital tract resulting in infection and persistent

inflammation of fallopian tubes and ovaries. They also induce chronic and asymptomatic

infection and increase the risk of neoplastic transformation of the ovarian surface epithelium,

resulting in highly aggressive ovarian carcinomas. Although many bacteria are isolated and

found to be involved in causing ovarian cancer, more studies are needed to explore the precise

mechanisms of microbial associated ovarian carcinogenicity (Trabert et al., 2019).

2.1.8. Bacteria Associated Cervical Cancer

Cervical cancer is the fourth most commonly occurring female malignancy worldwide (Sung et

al., 2021). Human papillomavirus infection with high-risk Human papillomavirus (16 and 18) is

one of the common causes of cervical cancer. Some other pathogens such as Chlamydia

trachomatis trachomatis and Epstein-Barr virus are found to be involved in cervical carcinoma

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acting either independently or as co-factors in predisposing cervical epithelial cells to

malignancy (de Lima et al., 2019). Chlamydia trachomatis infection has been found to be a

major risk factor for cervical cancer where it is reported to hinder the process of apoptosis in

infected cells by blocking the pathways required for initiating the apoptosis. Furthermore it

promotes the entry of infected cells into the S phase of cell cycle resulting in chronic

inflammation and ultimately tumorigenesis. Studies also demonstrated the involvement of

Chlamydia trachomatis as co-factors in predisposing cervical epithelial cells to malignancy by

increasing the risk of Human papillomavirus infection (Ohno, 2019).

2.1.9. Bacteria Associated Urothelial Carcinoma

Urothelial carcinoma accounts for about 90–95 % of all bladder cancers and 5–10 % of all

urothelial malignancies (Ohno, 2019). Several researchers have reported the bacterial link to this

cancer and suggest that bacteria may induce urothelial cancer. The precise mechanism of the

interaction between bacteria and host that augments the development of urothelial carcinoma is

not well understood. Clinically, the development of infection, urinary stones and indwelling

catheters are some of the factors linked with urothelial cancer. Other studies suggested that the

mechanism may be linked to the production of carcinogenic compounds (nitrosamines) which

can be detected during urinary tract infections (Miyazaki and Nishiyama, 2017). A study has

reported that rats with chronic urinary tract infections produce increasing urinary levels of N1 N-

dimethylnitrosamine over a 24-week period. The production is correlated with hyperplasia and

early neoplasia of the bladder epithelium (Jones et al., 2020). Another study from the same

research group suggested that strain-dependent virulence factors may support the development of

urothelial hyperplasia and neoplasia (Eyerich et al., 2018). Researchers suggested that virulence

factors may stimulate the macrophages to produce nitrosamine in infected tissues, thereby

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contributing to the urothelial neoplasia. Urinary tract infections have been reported to be directly

associated with the risk of bladder cancer in retrospective observational studies. Although all

these findings support the hypothesis that Urinary tract infections might act as inducers of

carcinogenesis yet the precise mechanisms behind this association call for further investigations

(Sung et al., 2021).

2.1.10. Bacteria Associated Skin Cancer

Carcinoma and cutaneous squamous cell carcinoma, tops the list of global incidences of any

form of cancer (Jones et al., 2020). A large number of the microbiome inhabits human skin, and

therefore, the association of bacteria with skin cancer is coming into focus recently. Skin is the

body’s largest organ and acts as a natural physical barrier between invading pathogens and the

body. Under homeostasis, microbes, keratinized skin cells and immune cells work in association

to maintain and protect physical and immune barriers of skin (Eyerich et al., 2018).

Keratinocytes and melanocytes, are the primary skin cell types involved in melanoma and non-

melanoma skin cancers and express toll like receptors (TLRs) which induce inflammatory

responses against invading pathogens (Sherwani et al., 2018). Uncontrolled activation of TLRs

can lead to chronic inflammation that gives rise to skin cancer. Exact correlation between skin

microbiome and cancer is yet to be established and the research focusing on this association is

still in early stages. Psoriasis and acne, chronic inflammatory diseases involving Th17 immune

stimulation, are some of the inflammatory skin conditions where microbiome is speculated to be

associated with skin cancer via inflammation. It can be hypothesized that unchecked

inflammation along with microbiome dysbiosis, may elevate the risk of skin cancer. Also, the

disruption of normal skin microflora may add to the inflammatory mechanisms that might induce

carcinogenesis (Mrazek et al., 2019).

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2.2 Bacteria for Cancer Prevention and Treatment

2.2.1. Bacteria as Therapeutic Agents of Cancer and Associated Mechanisms

In the middle of 18th-century bacteria were used as anticancer agents by German physicians,

Busch and Fehleisen. Still, the research did not progress any further due to the high mortality by

bacterial infections and other associated complexities. The first successful utilization of

microscopic bacterial organisms was performed by Morales, Eidinger, and Bruce. The group

treated bladder cancer with Bacillus Calmette–Guerin (Lu et al., 2021). The recent development

in understanding tumor microenvironment and biotechnological advancements has made bacteria

well-suited to be ‘perfect’ anticancer agents (Sehrawat et al., 2021). Many bacterial

strains/species have been reported to possess underlying oncolytic potentials to invade and

colonize solid tumors and are in clinical trials. One of the major advantages of using bacteria for

cancer therapy is their ability to specifically colonize and target the tumors. The bacterial

accumulation in the tumors depends on oxygen tolerance (Sehrawat et al., 2021). Since tumor

microenvironment is mostly hypoxic, different Clostridia strains, Bifidobacteria and Salmonella

are capable of colonizing the hypoxic area of the tumor and destroy the tumor cells without

posing any risk to the surrounding normal cells. Most microbes exert anti-tumor effects by

depleting the tumor microenvironment of the nutrients needed for the cancer cell metabolism.

The tumor tissues are the perfect breeding ground for the anaerobic bacteria, which only survive

in the anoxic region (Naller et al., 2017). One report showed that when the Salmonella bacteria

was flushed into the solid tumor, it colonized the tumor and reduced the tumour cells’

proliferation (Yaghoubi et al., 2019). Furthermore, the tumor microenvironment is conducive to

bacterial survival and growth, protecting the host immune system and nutrients (Yaghoubi et al.,

2020). When interacting with the host as a pathogen, Bacteria may trigger the immune response

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by activating the inflammasome pathways and thereby damaging the tumor cells. Some

anaerobic bacteria have also been reported to engulf the solid tumors and indirectly stimulate

host defense mechanism initiation. Other mechanisms that are able to kill cancer cells are

bacterial based peptides and toxins. Bacterial toxins are also called bacteriocins such as bovicin

HC5, colicins, laterosporulin 10, nisin A, and phenazine 1,6-di-carboxylic acid have been shown

to be efficacious against several tumors (Weerakkody and Witharana, 2019).

2.2.2. Engineered Bacteria for Cancer Therapy

As a result of the ease of manipulation, bacteria can be engineered to overcome the limitations

surrounding the previously existing cancer therapies (mentioned in detail in the latter part of the

review). Utilizing the top-down engineering approach, an ideal cancer therapy can be envisioned:

to give readers some idea of this approach, these engineered bacteria can be tiny programmable

‘robot factories’ which will specifically target the tumors by being selectively cytotoxic to only

cancer cells, can be self-propelled, and responsive to external signals and can sense the local

environment and will be externally detectable (Weerakkody and Witharana, 2019). The

functional advantage of each of above-mentioned features of the engineered bacteria is as

follows. The specific targeting by these robot factories will allow the use of more toxic

molecules without worrying about the systemic effects. Self-propulsion feature of these

engineered bacteria would enable penetration into otherwise inaccessible tumor regions.

Responsiveness to external signals would enact the absolute control of the location and timing of

cytotoxicity. Sensing the local environment would allow for the ‘smart’, responsive therapies

which can decide on its own about where and when drugs should be administered. Finally, the

dexterity to be detected externally would help retrieve the critical information about the state of

the tumor, the success of localization and the efficacy of treatment (Weerakkody and Witharana,

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2019). Attenuated bacteria created using conventional strategies have been used as vaccines all

over the globe. The same attenuation strategies have also been applied in using live bacteria as

therapeutic agents. One such example is the generation of auxotrophic mutants, which can

colonize and thrive only in a specific environment. These auxotrophic mutants cannot colonize

very well in an environment lacking one or more specific nutrient(s) required by the mutant

strain. Thus, incorporating a selective advantage to the engineered bacteria to replicate only in

the targeted sites thereby reducing the cytotoxicity to the unwanted tissues (Rahimi et al., 2019).

Different bacterial strains from multiple bacterial genera are now successfully used against

various cancers and some of them are even in phase II and III clinical trials. For instances

Salmonella typhimurium (S. typhimurium) A1-R mutant was developed to target the tumors.

This mutant strain of S. typhimurium has demonstrated selective tumor colonization and potent

antitumor activity in various cell lines from multiple cancer types including melanoma, sarcoma,

glioma, breast, pancreatic, colon, cervical, prostate, and ovarian cancers (Rahimi et al., 2019).

This genetically modified bacterial strain is also shown to overcome nab-paclitaxel resistance in

cervical cancer. When used in combination with nab-paclitaxel, S. typhimurium A1-R

significantly reduced tumor growth in comparison to nab-paclitaxel only (Yaghoubi et al., 2020).

Similarly, other bacterial strain such as Clostridium novyi (C. novyi), Listeria monocytogenes (L.

monocytogenes) also showed strong therapeutic potential against numerous cancers. For

instance, engineered Clostridium novyi-NT (nontoxic; lacking the alpha toxin) showed high

selectivity towards hypoxic and necrotic areas of tumors, induced lysis of tumor masses and

initiated strong immune response in a number of animal models and human subjects (Murakami

et al., 2019). Another in vitro study using Attenuated Listeria monocytogenes (Lmat-LLO)

showed that Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety

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of melanoma cells (Vitiello et al., 2019). Furthermore a recombinant strain of Lactococcus lactis

producing Interleukin-17A showed significant decrease in tumor size compared to Lactococcus

lactis WT (Teran-Navarro et al., 2019). A study which used Diphtheria Toxin-Based gene

therapy showed a significant inhibition of Hepatocellular Carcinoma cells (Kamimura et al.,

2020).

2.2.3. Bacteria in Delivery of Anticancer Drug

Salmonella typhimurium Ty21a carrying gold nanoparticles has been shown to be efficient

delivery vehicle for the gold nanoparticles to the tumours’ hypoxic regions (Chen et al., 2018).

Furthermore another attenuated strain of Salmonella named S. typhimurium YS1646 efficiently

delivered and maintained the therapeutic efficacy of low- temperature sensitive liposome

encapsulated with doxorubicin inside colon cancer cells (Ektate et al., 2018). Escherichia coli

(E.coli Nissle 1917) when used as a targeted transport vehicle to deliver p53 and Tum-5 protein

to tumor hypoxic regions, inhibited the liver cancer growth (Gurbatri et al., 2020). The same

strain of E. Coli has been successfully engineered for the controlled production and intratumoral

release of nanobodies targeting programmed cell death–ligand 1 and cytotoxic T lymphocyte–

associated protein-4 (Gurbatri et al., 2020). Results from this study showed enhanced therapeutic

response compared to analogous clinically relevant antibodies and also displayed significant

tumor regression. An attenuated non-toxic and non-pathogenic bacterial strain (Listeria

monocytogenes) from genus Listeria was found to selectively deliver 32-Phosphorus in

pancreatic tumors and metastases regions (Chandr et al., 2017). This study showed that Listeria

monocytogenes is potentially able to penetrate the transgenic KPC (conditionally express

endogenous Kras-G12D and p53-R172H mutant alleles) pancreatic tumors and metastases.

These results were very significant since KPC exhibit a stromal barrier, which prevents most

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drugs from penetrating the pancreatic tumors. Similarly another strain from genus

Bifidobacterium (Bifidobacterium infantis) was successfully used for the construction of

Bifidobacterium infantis-mediated thymidine kinase suicide gene therapy system and showed

significant inhibition of bladder tumor growth (Janku et al., 2021). Altogether abovementioned

studies have suggested that tumors microenvironment can offer perfect hypoxic conditions

required for the anaerobic bacterial growth, which further demonstrates a potential opportunity

for precise local tumor targeting. However, both dose-dependent side effects arising from

bacteria, and low therapeutic efficacies that the bacteria may cause have been observed. Since

then, enormous work on reducing systemic toxicity by chemically and genetically modifying the

bacteria has been carried out as discussed earlier. More recently, various bacterial therapeutics

have been successfully implemented in humans as anticancer agents, and some of them have

moved into advanced clinical trials (Janku et al., 2021).

2.2.4 Bacteria as Biomarkers in Cancer

So far, a wide variety of molecules such as transcription factors, mRNA, DNA, proteins,

enzymes and receptors have been exploited as the cancer biomarkers. From the reports discussed

earlier in this review, it has become extremely perceptible that several bacteria may directly be

involved in the onset and progression of various human cancers. These experimental evidences,

not only provides more insight into bacterial connection to oncogeneses but also suggest the

possibility of use of these bacteria as biomarkers in early stage detection of carcinogenicity.

Bacterial dysbiosis, which is well documented in many cancers, particularly in GIT cancers, also

indicates the potential of relevant bacterial species in detecting relevant malignancies. For

instance, many studies have profiled the microbiome as a biomarker in lung cancer patients by

metagenomic sequencing (Shirazi et al., 2019). The results revealed that Streptococcus viridans

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(S. viridans) was significantly more prevalent, followed by Granulicatella adiacens (G.

adiacens), E. coli, Acinetobacter junii (A. junii), and Streptococcus species in lung cancer

patients. Moreover, in all lung cancer patients G. adiacens, Streptococcus intermedius (S.

intermedius), and M. tuberculosis were found. Another study that scrutinized and validated

potential bacterial biomarkers for lung cancer revealed Capnocytophaga and Veillonella as

potential bacterial markers for detecting lung cancer (Cameron et al., 2017). Some bacterial

species have also been implicated in colorectal cancer (Cameron et al., 2017). However, various

studies in different age groups, conditions, and technical and biological variations have

documented different predominant bacterial species. Cumulatively, all microbial studies

suggested that F. nucleatum is a potential bacterial biomarker for colorectal cancer. Another

recent study has characterized the urinary microbial profile of bladder cancer by 16S rRNA gene

sequencing and found that Acinetobacter is associated with bladder cancer and suggested that it

can be used as a potential microbial marker of bladder cancer (Bi et al., 2019). Several potential

bacterial biomarkers were also suggested by several studies in oral cancer and pancreatic cancer.

In oral cancer patients, Porphyromonas gingivalis (P.gingivalis), F. nucleatum and

Streptococcus species were found predominantly compared to healthy subjects (Bi et al., 2019).

While, in pancreatic cancer patients, P. gingivalis, G. adiacens were found prevalent compared

with control (Bi et al., 2019). In oral cancer DNA mutations are introduced by microbes and

their metabolites via stimulating fibroblasts and immune cells leading to the production of

reactive oxygen species. Similarly, in pancreatic cancer, bacteria have been implicated in causing

inflammation, immune response evasion and microRNA regulation (Shirazi et al., 2019).

Besides the fact that bacterial prevalence can be used as a biomarker in several cancer types,

genetically engineered bacteria can also facilitate the precise detection of various cancer types.

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More recently tumors targeting bioluminescent bacteria are created which can be imaged by high

resolution, in vivo imaging methods, for example bioluminescence imaging, Magnetic resonance

imaging and positron emission tomography. One example of genetically engineered bacteria in

cancer detection is genetically modified Salmonella, VNP20009, that expresses the herpes

simplex thymidine kinase reporter gene. This bioengineered bacterial strain was able to

selectively colonize within murine tumor models and effectively sequestered a radiolabeled

nucleoside analogue, 20-fluoro-1- b-D-arabino-furanosyl-5-iodouracil, which is detected by

positron emission tomography (Janku et al., 2021). Similarly, E. coli Nissle 1917 was imaged

with radiolabeled pyrimidine nucleoside analogues that are selectively phosphorylated and

trapped in the bacteria. Magnetotactic bacteria such as Magnetospirillum magneticum were also

used to detect tumors in mice models where the bacterial growth conditions were manipulated to

produce magnetite particles (around ~25 nm diameter) and tumor targeting ability was confirmed

using 64Cu-labeled bacteria and positron emission tomography (Wrobel and Ahmed, 2019).

2.2.5 Bacteria as Anti-Cancer Agents through Enhancing Human Immunity

2.2.5.1 Activating Inflammasome Pathways

The ΔppGpp Salmonella typhimurium strain activates inflammasome pathways by damaging the

signals released from cancer cells. This phenomenon significantly increases the amount of

inflammatory cytokine IL-1β, TNF-α and Il18 in tumors, which results in drastic tumor growth

suppression (Sedighi et al., 2019). IL-1β is the proinflammatory cytokine that plays a pivotal role

in immunity against pathogens. The IL-1β is secreted by LPS (lipopolysaccharides) during the

activation of toll like receptor (TLR4) and inflammasomes, which then causes the damage to

cancer cells (Song et al., 2018). The cancer cells are also damaged when bacteria activate

inflammasomes in BMDM, which is involved in phagocytosis of damaged cancer cells by

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macrophages. Therefore, the ΔppGpp Salmonella typhimurium shows therapeutic efficacy for

cancer through involving the inflammasome pathway (Song et al., 2018).

2.2.5.2 CD4, CD25 and CD8 Anti-Tumor Effectors T cell Responses

Anaerobic bacteria such as E. Coli, which are capable of engulfing the solid tumors, are

indirectly involved in clearance of some tumor cells (e.g. CT26) through infectious-defense

mechanism. Once these bacteria invade the host, they stimulate the initiation of the defense

mechanism of the host, which results in the production of lymphocytes T cells. The produced

lymphocytes T cells are significantly involved in anti-tumor activity. During the induction phase

of bacterial infection, CD8+ T cells are the only effectors responsible for tumor clearance;

whereas, in the memory phase the clearance also involves CD8+ and CD4+ T cells (Baindara

and Mandal, 2020). CD8+ T is reported to take part in the clearance of the original tumor after

bacterial infection. Similarly, the anti-tumor effectors T cells (CD4+ and CD8+) have the

potential to block the formation of a new set of tumors. The CD8+ T cell is said to have the

additional ability to eradicate even the already established tumors. Furthermore, the lymphocytes

(CD4 and CD25) and cytokines are reported to be the novel therapies for colon cancer in humans

(Baindara and Mandal, 2020). Their role in host immune response on carcinogenesis is

invaluable. The regulatory cells (like CD4 and CD25) are capable of reducing the severity of

inflammatory bowels and lower the risk of colon cancer (Saltzman, 2021). In addition, the

introduction of regulatory cells into chronically infected mice with established cancer showed the

reduction of the severity of colitis, epithelial dysplasia, and cancer (Saltzman, 2021).

2.2.5.3 The TNF-α Innate Immune System in Bacteria-Based Tumor Necrosis

The tumor necrosis factor (TNF- α) has the potential to damnify the vascular endothelial cells.

TNF-α plays the significant part in the formation of the large haemorrhaging area that appears

19
within the tumor. Systematic administering of Salmonella enterica serovar Typhimurium to mice

models indicated that haemorrhaging increases the flushing of bacteria into the solid tumor

resulting in necrosis (Gharaibeh and Jobin, 2019). Moreover, the activeness of the innate

immune system of the host, especially neutrophilic granulocytes, is proportional to the area of

necrotic. Neutrophiles function to separate the bacteria-containing necrotic region from the

bacteria that migrate into the tumor from viable tumor cells. The depletion of host neutrophils

increases the number of bacteria in the tumor and increases the ability of bacteria to migrate into

vital tumor tissue. Thus, the complete eradication of the established tumors could be attained

with the increasing size of necrosis. Similarly, the depletion of host neutrophils amplifies the

bacteria-mediated tumor therapy (Wei et al., 2018).

2.2.6 Bacteria as Anti-Cancer Agents through Released Substances

2.2.6.1 Bacteriocins

Bacteriocins are cationic peptides that are synthesized by almost all groups of bacteria

ribosomally. Bacteriocins are non-immunogenic, biodegradable and contain cancer cellspecific

toxicities. The bacteriocins have the potential to serve as synergistic agents to conventional

cancer drugs (Kim et al., 2021). Cancer cell membranes predominantly carry negative charge;

thus bacteriocins preferentially bind to cancer cell membranes than to the normal cell

membranes, which are neutral in charge and selective for binding of bacteria (Hradicka et al.,

2020). Colicins, the bacteriocin secreted from Enterobacteriaceae such as Escherichia coli

(E.coli), are known to have anti-cancer activities against a variety of human tumor cell lines in

vitro, including breast cancer, colon cancer, bone cancer and uteri cell line HeLa (human cervical

adenocarcinoma) (Hradicka et al., 2020). Microcin E492, part of Colicins from Klebsiella

pneumoniae, was found to induce apoptosis in some human malignant cell lines such as HeLa,

20
Jurkat (T cell derived from acute T cell leukemia), RJ2.25 (a variant of Burkitt’s lymphoma), and

colorectal carcinoma cells, with no effect on normal cells (Wang et al., 2018). Pediocin isolated

from Pediococcus acidilactici K2a2-3 was reported to have cytotoxic activities against HT29

(human colon adenocarcinoma) and HeLa cell lines [39]. Similarly, Nisin (the bacteriocins from

Lactobacillus lactis) possesses cytotoxic effect on MCF-7 (human breast adenocarcinoma cell

line), HepG2 (liver hepatocellular carcinoma), and HNSCC (head and neck squamous cell

carcinoma), both in vitro and in vivo (Wang et al., 2018). Conversely, Nisin is nontoxic and safe

to humans, WHO has approved it for human consumption. Furthermore, partially purified

bacteriocins produced by certain bacteria such as Pseudomonas aeruginosa have shown anti-

cancer activities (WHO, 2020). Pyocin, the bacteriocin produced by more than 90% of

Pseudomonas aeruginosa strains showed lethal effect on the L6OT mice fibroblast cell line

(WHO, 2020). Likewise, purified and partially purified pyocin S2 showed the cytotoxicity effect

on tumor cell line HepG2 and Im9 (Human immunoglobulin-secreting cell line derived from

multiple myeloma) with no effect on normal cell line HFFF (Human fetal foreskin fibroblast)

(Hradicka et al., 2020).

2.2.6.2 Phenazine 1,6-Di-Carboxylic Acid (PDC)

Multiple phenazine metabolites such as phenazine 1- carboxylic acid (PCA) and Phenazine 1,6-

di-carboxylic acid (PDC) are derived from bacteria strains (e.g. Pseudomonus aeruginosa). The

PDC phenazine was first isolated from Streptomyces species; it was demonstrated to be the

potential agent for controlling metabolism and biofilm formation in Candida albicans

(Chowdhury et al., 2020). Compared to other phenazine metabolites, PDC showed a

substantially broader spectrum of cytotoxicity effect towards a number of cancer cells of

different origins, including HT29, HeLa, and MCF7 cell lines, with less activity on DU145

21
(Human prostate cancer cell lines) (Chowdhury et al., 2019). Bacteria as anti-cancer agents

through biofilms Biofilm is a primitive form of multicellular life that provides bacteria with

tolerance strength against antibiotics and host defense mechanisms. Biofilms are common to

opportunistic bacterial pathogens such as Salmonella tyhimurium, and they (the biofilms) are

decisive in the pathogenesis of chronic infectious diseases (Chowdhury et al., 2020). Salmonella

tyhimurium and some other pathogens are known to cause severe haemorrhage within the tumor.

Once the haemorrhae is activated, it induces the production of T cells that are very significant in

biofilm induction (Chowdhury et al., 2019). Notwithstanding the etiopathogenesis of biofilms

and its protective role that allows bacteria to escape from the host defense system, recent

discoveries have revealed the potential ability and efficacy of biofilms in cancer therapy. Anti-

cancer drugs cause the induction of biofilm formation during cancer treatment, which results in

metastasis distraction (Samec et al., 2020). Similarly, the formation of bacteria biofilm on cancer

cells during the SOS response results in metastasis disruption. Thus bacteria biofilm shows

potential usefulness in cancer treatment. Bacteria biofilm can affect colon cancer development

and progression through modifying cancer metabolome to produce a regulator of cellular

proliferation (Samec et al., 2020). Also, the bacterial macromolecules necessary for biofilm

formation such as proteins and DNA coat cancer cells to block metastasis (Zainodin et al., 2018).

For example, polysaccharides released by Streptococcus agalactiae inhibit adhesion of cancer

cells to endothelial cells, an essential step in cancer metastasis. Furthermore, the potential

application of iron oxide nanowires from a biofilm waste produced by bacteria (Mariprofundus

ferroxydans) as a new multifunctional drug carrier for cancer therapy and cancer hyperthmia.

While the above hypotheses avow for the potential of bacterial biofilm in cancer therapy, the

evidence is relatively insufficient to build-up the case. However, the efficacy of bacteria biofilm

22
for metastasis distraction calls for the further examination and investigation of the anticancer

ability of bacteria biofilm (Zainodin et al., 2018).

Table 1. A Comprehensive List of Bacteria and Bacterial Genera Used As Therapeutic Agents in
The Treatment Of Several Cancers.
Genus Bacteria Cancer Therapeutic Effects
Breast Prevented and inhibited breast cancer bone
Pancreatic metastasis
cancer Extended survival with frequent total regression
Lung cancer of autochthonous and orthotopic tumors
Bone tumor Induced apoptosis and Inhibited cancer growth
Prostate cancer Reduced tumor size and bone to lung metastasis
Killed cancer cells via inducing apoptosis and
Melanoma necrosis, and inhibited cancer metastasis
S. Promoted apoptosis and autophagy of tumor
Salmonella typhimurium cells
Colon cancer Inhibited the development of tumor and activates
immune response
Brain cancer Reduced tumor growth, induction of apoptosis
via p53 link mechanisms
Cervical cancer Suppressed tumor growth`
Gastrointestinal Significantly reduced tumor growth and was
cancer more effective compared to Imatinib
Stomach cancer Regressed tumor growth via inducing cell cycle
arrest
Adenocarcino Caused tumor regression and activated innate
ma immune response
C. novyi Brain tumor Caused dramatic destructions of cancer cells and
Leiomyosarco significantly increased survival rate
C.
ma Reduced the tumor within and surrounding the
sporogenes
Colon cancer bone
Lung cancer Suppressed tumor growth via inhibiting cell
C.
perfringens Prostate cancer proliferation significantly
Ovarian cancer Inhibited tumor growt
Increased cellular death
Colon cancer
Cytolysis of tumor cells via binding to claudin
family proteins
Clostridium High cytotoxicity towards colon cancer cells
Breast cancer Reduced tumor volume and increased survival
23
 Breast cancer Cytotoxic to cancer cells and induced caspase-3
Prostatic cancer and -7 dependent apoptosis
Colon cancer Inhibited tumor growth and shrank tumor size
Inhibited cancer cell proliferation and migration
C. botulinum via p53 linked mechanisms
Pancreatic Reduced tumor size via induction of apoptosis
cancer
Melanoma Improved immunotherapy and killed cancer cells
Lung cancer via apoptosis
Prostate cancer Improved immune and immunotherapies
L. efficacies and reduced tumor growth
monocytogen Significantly regressed the tumor via exerting
Listeria es immunogenic effect
Pancreatic Increased cure rate and prolonged the survival
cancer
B. bifidum Lung cancer Inhibited tumor growth via activation of tumor-
Colon cancer specific IL-12 and IFN-γ, lymphocyte
Colon cancer proliferation, and CD8+ cytolytic responses
B. longum Prostate cancer Increased expression of tumor suppressor
Liver cancer miRNAs
Breast cancer
Regressed weight, volume, growth, and
microvessel density of tumor
Significantly inhibited tumor growth
Inhibited tumor growth and activated innate
immunity
Bifidobacteriu Selectively localized in tumor and Suppressed
m cancer growth
Lung cancer Selectively localized in these tissue however
Melanoma anti-tumor effects were not checked
Head and Induced apoptosis and consequently inhibited
Neck cancer tumor growth
B. breve Colon Significantly ameliorated the disease activity
cancer index, restored colon length, and inhibited
progress of tumors to higher stages and grades.
L. reuteri Gastric cancer Inhibited cell proliferation in dose-dependent
melanoma manner
L. casei Colon cancer Cancer incidence was reduced from 30 to 70 %
Gastric cancer and prolonged the survival rate Inhibited cancer
development via inducing anti-proliferative and
immunomodulatory effect
Induced apoptosis in cancer cells by inactivating
Lactobacillus NF-κB promoter activity
Lung cancer Significantly reduced tumor growth and
activated innate immunity
H & N cancer Induced apoptosis in cancer cells through a
Lactococcus L. lactis
24
 Melanoma calpain-dependent pathway
Lung cancer Selectively localized in tumor hypoxic
microenvironment
Significantly lowered tumor size via secreting
biologically active IL-17A cytokine
Colon cancer Inhibited proliferation, migration and induced
apoptosis in cancer cells
Colon cancer Inhibited proliferation and migration of cancer
Melanoma cells
Lung cancer Selectively localized and remarkably restrained
tumor growth
Selectively targeted cancer cells and induced
Escherichia E. coli apoptosis
Pancreatic Reduced tumor growth and Increased median
cancer survival rate
Liver cancer Inhibited tumor growth and up regulated
caspase-3

Source. (Davar et al., 2021).

CHAPTER THREE

3.0 Conclusion

Multidimensional role of bacteria in human health is what makes them indispensable for human

life. Majority of these tiny organisms are located in the gastrointestinal, respiratory and

urogenital tracks. Several infections in these vital systems have previously been connected to the

bacterial pathogenesis. However, the direct involvement of bacteria in Emerging experimental

evidence has indicated that bacteria can directly manipulate host cells’ normal physiology by

virtue of various mechanisms such as inflammation, alteration in cell signaling, invasion and

immune evasion, induction of DNA damage, and production of onco-proteins that have direct

mitogenic or mutagenic effects. Recent development in the understanding of tumor

microenvironment and technological advancements has turned this enemy into an ally. Studies

using bacteria for cancer treatment and detection have shown noticeable effects. Therapeutic

25
abilities of bioengineered bacteria such as high specificity, selective cytotoxicity to cancer cells,

responsiveness to external signals and control after ingestion have overcome the challenges faced

by conventional cancer therapies and highlighted the bacterial based therapy as an ideal approach

to treat cancer.

3.1 Recommendation

Further investigations are essential to increase our knowledge in this area and to determine the

role bacteria in cancer. Moreover, reviewing all the research achievements can help scientists

reach a consensus and make this method more efficient in future applications. Further

investigations are essential to increase knowledge in this area and determine the advantages and

disadvantages of bacteria-based strategies for cancer treatment.

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