Raquel Rodrigues Science Reviews - Biology, 2023, 2(2), 30 - 39

Using spore-forming bacteria to treat cancer: recent

advancements in clostridial-based therapies



Raquel Rodrigues, PhD

Independent Researcher, Aveiro, Portugal; raquel.sof.rodrigues@gmail.com

https://orcid.org/0000-0002-2903-3258

https://doi.org/10.57098/SciRevs.Biology.2.2.3

Received June 17, 2023. Accepted July 21, 2023.

Abstract: The use of bacteria in cancer therapy has emerged as a promising approach, offering unique

advantages for targeted treatment and immunotherapy. This review paper explores some recent advances in

the application of bacteria, particularly Clostridium species, in cancer therapy. For instance, the Clostridial-

Directed Enzyme Prodrug Therapy (CDEPT) utilises non-pathogenic strains of Clostridium as carriers for

targeted delivery of anti-cancer drugs to solid tumour cells. This strategy aims to minimise systemic side effects

associated with traditional chemotherapy. Additionally, Clostridium novyi and Clostridium sporogenes present

oncolytic properties and have shown potential for tumour regression in preclinical models. The engineering of

these bacteria to produce cytokines, such as interleukin-12 (IL-12) and interleukin-2 (IL-2), further enhances their

therapeutic potential by activating the immune system to target cancer cells. Clinical trials in humans have

demonstrated the feasibility and safety of C. novyi-based therapies, and early results indicate potential efficacy

in tumour regression. Overall, this review provides valuable insights into the multifaceted roles of bacteria,

particularly Clostridium species, in cancer therapy, emphasizing their potential as targeted therapeutics and

immunomodulators for improved cancer treatment outcomes.

Keywords: Spores, Clostridium sporogenes, Clostridium novyi, cancer therapy, CDEPT, hypoxia, solid tumour

Introduction

Cancer is a challenging disease that continues to

pose a significant threat to human health, affecting

a substantial number of individuals each year. Ac-

cording to projections from the American Cancer

Society, an estimated 1,958,310 new cancer cases

and 609,820 cancer-related deaths were anticipated

in the United States for 2023 (Siegel, Miller, Wagle,

& Jemal, 2023). Through the dedicated efforts of the

scientific community, remarkable progress has been

achieved in the realm of cancer therapeutics, lead-

ing to notable reductions in mortality rates (Siegel

et al., 2023). Nonetheless, the complex nature of can-

cer presents a formidable challenge, as different

cancer types exhibit diverse characteristics and fea-

tures that hinder the development of universally

applicable treatments or cures. Particularly, solid



The article was awarded free publication and an honorarium.

tumours pose a significant obstacle due to the for-

mation of necrotic tissue, rendering them resistant

to conventional treatment (Tharmalingham &

Hoskin, 2019). Addressing this challenge entails ex-

ploring alternative approaches, such as the targeted

delivery of spores of anaerobic bacteria to these tu-

mours. This approach capitalises on the presence of

hypoxic regions within solid tumours, where oxy-

gen supply is deficient.

Bacterial endospores represent one of the most re-

silient life forms found on Earth, enabling bacteria

to withstand extreme conditions including temper-

ature variations, desiccation, radiation, disinfect-

ants, and, in the case of Clostridium, oxygen (Setlow,

2007). These extraordinary structures feature multi-

ple protective layers, including membranes, coat,

cortex, and, in certain instances, an exosporium, all

of which shield the core containing DNA (Setlow,

Science Reviews - Biology, 2023, 2(2), 30 - 39 Raquel Rodrigues

2007). Such formidable defence mechanisms ensure

their long-term survival, potentially spanning mil-

lions of years (Cano & Borucki, 1995; Kennedy,

Reader, & Swierczynski, 1994). However, a trade-

off of spore formation is the metabolic dormancy

imposed on the cells. Yet, when confronted with

conditions conducive to cellular viability, spores

can undergo germination, reactivating metabolic

processes (Setlow, 2003).

Over the years, numerous spore-forming bacteria

have been identified, predominantly belonging to

the taxonomic classes Bacilli and Clostridia within

the Firmicutes phylum (Galperin et al., 2012). Nota-

bly, the main distinction between these classes lies

in their aerobic requirements, with Clostridia typi-

cally classified as anaerobic bacteria, while Bacilli

encompass both obligate and facultative aerobes

(Collins et al., 1994; Ludwig, Schleifer, & Whitman,

2015). While numerous spore-forming species are

typically associated with pathogenicity, it is im-

portant to acknowledge that certain species within

this group are benign and exhibit intriguing appli-

cations. Notably, some of these species have

demonstrated the capacity to produce solvents of

commercial significance, such as Clostridium aceto-

butylicum, Clostridium stercorarium, and Clostridium

thermocellum (Lamed & Zeikus, 1980; Napoli,

Olivieri, Russo, Marzocchella, & Salatino, 2010;

Tran et al., 2012). Additionally, Clostridium celluloly-

ticum has been investigated for its ability to effi-

ciently degrade cellulose (Desvaux, Guedon, &

Petitdemange, 2000). This review focuses on the uti-

lisation of Clostridia in the treatment of diseases,

specifically cancer (Andryukov, Karpenko, & Lya-

pun, 2021).

The link between spore formers and cancer goes

back to at least 1947: when mice sarcomas were in-

fected with Clostridium histolyticum, tumour tissue

was lysed, albeit not completely (Parker, Plummer,

Siebenmann, & Chapman, 1947). More recently, ad-

vances in genetic engineering allowed improve-

ments and oncolysis can be enhanced. This is im-

portant because when the outer rim of the tumour

is not fully eliminated, tumour regrowth frequently

occurs (Minton et al., 1995).

This review will focus on Clostridium sporogenes and

Clostridium novyi, two anaerobic spore-formers that

have been extensively researched for their cancer-

treatment capabilities. The combined use of these

species with immunotherapy will also be explored.

Clostridium sporogenes

as a versatile tool for

targeted cancer therapy

C. sporogenes is rod-shaped, anaerobic, produces en-

dospores, and can be found in a variety of environ-

ments, including soil and human/animal intestines.

This species is safe, being classified as a harmless

hazard group I organism by the UK Advisory Com-

mittee on Dangerous Pathogens and as a harmless

biosafety level 1 organism by the American Type

Culture Collection (Kubiak et al., 2015).

Extensive research has been conducted on C. sporo-

genes in the context of Clostridial-Directed Enzyme

Prodrug Therapy (CDEPT). CDEPT encompasses

the utilisation of non-pathogenic strains of Clostrid-

ium as carriers for targeted delivery of anti-cancer

drugs to solid tumour cells (Kubiak & Minton, 2015;

Minton et al., 1995). The development of CDEPT

stemmed from the need to mitigate the undesired

side effects often associated with conventional can-

cer treatments. Traditional chemotherapeutic

agents are frequently designed to disrupt crucial

cellular processes like DNA replication, mitosis, or

cell proliferation, and may inadvertently impact

healthy cells (Karnofsky, 1968). To overcome these

challenges, the concept of a prodrug was devised,

involving the delivery of a biologically inert com-

pound to the tumour site, which is subsequently ac-

tivated into a highly cytotoxic drug (Rautio et al.,

2008). CDEPT employs the administration of a pro-

drug in conjunction with a prodrug-converting en-

zyme (PCE) (Kubiak & Minton, 2015; Minton et al.,

1995). Ideally, the conversion of the prodrug occurs

solely within the tumour microenvironment, spar-

ing healthy tissues from harm.

One notable advantage of the CDEPT strategy lies

in its utilisation of obligate anaerobes as vectors for

delivering the prodrug-converting enzyme to hy-

poxic regions within tumours. Hypoxia, character-

ized by reduced oxygen levels, is a common feature

observed in tumour tissues, often attributed to in-

adequate blood supply caused by the distance be-

tween certain tumour regions and blood vessels.

This poses challenges for conventional treatment

modalities such as radiotherapy and chemotherapy

(Weinmann, Belka, & Plasswilm, 2004). Administra-

tion of Clostridium spores to cancer patients has

been reported to induce tumour regression, primar-

ily owing to their oncolytic properties, while selec-

tively targeting hypoxic regions, as spores germi-

nate exclusively in poorly oxygenated areas (Möse

& Möse, 1964; Thiele, Arison, & Boxer, 1964).

Raquel Rodrigues Science Reviews - Biology, 2023, 2(2), 30 - 39

Consequently, spores derived from Clostridium spe-

cies have been extensively investigated as potential

vehicles for delivering the prodrug-converting en-

zyme to hypoxic tumour tissues (Heap et al., 2014)

Figure 1: Representation of the CDEPT therapy. Spores of an engineered strain of Clostridium are administered intravenously to

a cancer patient. The engineered strain contains a gene encoding a prodrug-converting enzyme (PCE). Then, a prodrug is also

delivered to the patient. In the tumour, the spores can germinate and express the PCE, which converts the prodrug into a toxic

derivative.

C. sporogenes has been extensively studied as the

prodrug-converting enzyme delivery vector, as part

of the CDEPT therapy. The successful germination

of C. sporogenes exclusively in necrotic tumours has

been demonstrated but researchers are still testing

the ideal PCE-prodrug combination.

Prodrug-converting enzymes, such as nitroreduc-

tases (NTR), hold significant potential in the field.

Notably, enzymes like NfsB, HsoNTR, and

NmeNTR exhibit the ability to activate the prodrug

CB1954 (5-(aziridin-l-yl)-2,4- dinitrobenzamide).

NmeNTR, a nitroreductase from Neisseria meningit-

idis, can catalyse the reduction of the 4-nitro group

found in the anti-tumour prodrug CB1954, resulting

in the formation of its 4-hydroxylamine derivative.

Importantly, this converted form of the drug

demonstrates substantially increased toxicity com-

pared to the prodrug itself One downside of the

NmeNTR/CB1954 combination is that the toxic 4-

hydroxylamine has limited ability to diffuse into

neighbouring cancer cells that are not necrotic tis-

sue and, consequently, are not colonised by the en-

gineered C. sporogenes

An alternative prodrug, PR-104 (dinitrobenzamide

mustard), can be converted into the PR-104A me-

tabolite, which in turn can be further reduced to

generate genotoxic drugs PR-104H (hydroxyla-

mine) and PR-104M (amine). These metabolites ex-

hibit the ability to induce DNA damage and inter-

fere with cell division, thereby exerting negative ef-

fects on cellular processes In a recent study pub-

lished in 2021, the NmeNTR/PR-104A prodrug

converting enzyme (PCE)/prodrug combination

was investigated, revealing superior activation of

PR-104A by NmeNRT compared to CB1954 Moreo-

ver, the authors enhanced PCE activity by modulat-

ing the enzyme-encoding gene with an alternative

promoter. Exclusive germination of C. sporogenes

within necrotic tissue was also demonstrated, along

with the inhibition of tumour growth upon intrave-

nous administration of genetically engineered C.

sporogenes spores alongside the prodrug PR-104.

The incorporation of the tumour vascular disrupt-

ing agent 5,6-dimethylxanthenone-4-acetic acid (va-

dimezan), which was shown to increase tumour ne-

crotic fraction, further augmented tumour growth

inhibition and increased median survival time. Va-

dimezan was administered 60 minutes post-spore

injection to ensure delivery of C. sporogenes spores

to the tumour microenvironment before necrosis in-

duction. Importantly, the ability of NmeNRT to me-

tabolize the PET imaging agent EF5 offers

Science Reviews - Biology, 2023, 2(2), 30 - 39 Raquel Rodrigues

additional advantages to this therapeutic approach,

enabling non-invasive imaging of therapeutic gene

expression and tumour colonisation

The utilisation of β-lactamases as prodrug-convert-

ing enzymes in CDEPT therapy was proposed in

2006 This class of enzymes can cleave cephalosporin

prodrugs, generating two active compounds. One

notable advantage of employing β-lactamases as

PCE is their natural occurrence in certain Clostrid-

ium species, such as Clostridium butyricum This in-

herent presence obviates the necessity for genetic

modifications of bacterial strains, streamlining the

implementation of the therapy.

C. sporogenes has undergone extensive evaluation

within the scope of CDEPT; however, this bacterial

species has also found application in other ap-

proaches to cancer treatment. Notably, C. sporogenes

possesses the ability to produce methionine γ-lyase

(MGL), an enzyme capable of catalysing the γ-elim-

ination of L-methionine (MET). As MET plays a piv-

otal role in the growth of malignant tumour cells,

MGL has been investigated for its potential anti-

cancer properties. In a study conducted in 2019, the

MGL gene from C. sporogenes was expressed in E.

coli strain BL21. Subsequent testing of MGL, both as

a standalone treatment and in combination with an-

other compound, involved A549 human lung can-

cer cells. Encouragingly, when combined with dox-

orubicin (DOX), MGL demonstrated cytotoxicity

and exhibited tumour growth inhibition (Pokrov-

sky et al., 2019).

Clostridium novyi

: oncolytic properties and ther-

apeutic applications

C. novyi has emerged as a compelling subject of in-

vestigation for its remarkable oncolytic properties.

Upon colonisation of tumours, this species appears

to produce extracellular lipases, which have been

hypothesised to exert cytotoxic effects against tu-

mour cells (Bettegowda et al., 2006).

In a study conducted in 2001, C. novyi ATCC 19402

was compared to 25 other anaerobic strains encom-

passing three distinct genera, namely Bifidobacte-

ria, Lactobacilli, and Clostridia (Dang, Bettegowda,

Huso, Kinzler, & Vogelstein, 2001). Due to its re-

markable capacity to infiltrate and disperse

throughout tumour tissue while sparing healthy tis-

sue, C. novyi was selected by the authors for further

investigation, as it demonstrated the ability to in-

duce tumour cell death. To ensure safety, a non-

toxic strain (C. novyi-NT) was engineered by elimi-

nating the alpha toxin gene. To enhance the efficacy

of the treatment and target both hypoxic and non-

hypoxic regions within tumours, a combination

bacteriolytic therapy (COBALT) approach was de-

veloped, employing spores of C. novyi-NT in con-

junction with D10 (an agent disrupting tumour vas-

culature) and MMC (a DNA-damaging agent). In

vivo experiments using mouse models with xeno-

grafts of HCT116 colorectal cancer cells demon-

strated that this therapy resulted in tumour regres-

sion, albeit potentially accompanied by a phenome-

non known as tumour lysis syndrome, toxicity re-

sulting from the rapid destruction of large tumours

(Dang et al., 2001). Notably, the observed toxicity

appeared to be attributed to germinated cells rather

than spores and was found to be proportional to

both tumour size and spore dosage (Diaz et al.,

2005). Nevertheless, the toxicity could be managed

through the administration of the antibiotic

imipenem after tumour colonisation (although this

compromised the efficacy of the therapy), or

through systemic hydration to counteract fluid loss

during infection. Furthermore, it was observed that

90% of intravenously injected spores were cleared

from circulation within 14 days (Diaz et al., 2005).

Due to its inherent oncolytic properties, C. novyi of-

fers a promising avenue for treating certain tu-

mours as a monotherapy, without the need for ad-

ditional treatment combinations. A recent study

conducted in 2022 explored the use of a novel non-

toxic strain of C. novyi for the treatment of breast tu-

mours in mice (Abedi Jafari, Abdoli, Pilehchian, So-

leimani, & Hosseini, 2022). Through intratumoral

injection of spores, complete remission was ob-

served in all mice (n = 8) with tumour sizes up to

1000 mm

. However, larger tumours demonstrated

a diminished response to the treatment. The authors

postulated that for smaller tumours, the bacteria

primarily targeted hypoxic regions within the tu-

mour, while the immune system response played a

significant role in eliminating non-hypoxic cells. In

contrast, administration of C. novyi alone was insuf-

ficient to entirely eradicate larger tumours (Abedi

Jafari et al., 2022).

In addition to its applications in colorectal and

breast cancer, C. novyi-NT has also been investi-

gated as a potential treatment for glioblastoma, a

form of brain cancer (V. Staedtke et al., 2022). Re-

searchers directed their focus towards managing

Raquel Rodrigues Science Reviews - Biology, 2023, 2(2), 30 - 39

host inflammatory responses, which can lead to oe-

dema and elevated intracranial pressure. Such re-

sponses pose additional challenges by impeding the

dissemination of C. novyi-NT throughout the tu-

mour, hindering the lysis of tumour cells located in

the outer rim. To address this issue, the authors em-

ployed neutrophil depletion achieved through the

administration of the 1A8 antibody before spore de-

livery. Neutrophil depletion not only facilitated im-

proved tumour clearance but also enhanced the

safety of the procedure by mitigating toxicity. En-

couraging results were observed in rabbit models

treated with this therapy, with 70% of animals ex-

hibiting no tumour recurrence (V. Staedtke et al.,

2022).

In another study, orthotopically implanted glioblas-

toma rat models were utilised to assess the efficacy

of C. novyi-NT treatment. Intravenous injection of C.

novyi-NT spores led to tumour regression and a sig-

nificant increase in survival time (Verena Staedtke

et al., 2015). Although treatment-associated side ef-

fects such as brain oedema and increased intracra-

nial pressure were observed, their impact could be

controlled with the administration of steroids and

antibiotics. Furthermore, combining C. novyi-NT

treatment with a liposomal formulation of the DNA

intercalating agent doxorubicin resulted in im-

proved tumour clearance. Importantly, the authors

demonstrated that spore germination occurred ex-

clusively within tumours and not in hypoxic re-

gions induced by stroke or myocardial infarctions

(Verena Staedtke et al., 2015).

While the majority of studies involving C. novyi-NT

have employed mouse, rat, or rabbit models with

induced tumours, efforts have been made to bridge

the gap between animal models and human pa-

tients by treating naturally occurring tumours in ca-

nines (Roberts et al., 2014). In a study involving 16

dogs with solid tumours, one to four cycles of intra-

tumoral injections of C. novyi-NT spores were ad-

ministered. Two dogs were excluded from evalua-

tion as their tumours were surgically removed be-

fore day 21, which was set as the time point for re-

sponse assessment. Among the 14 evaluated dogs

on day 21, three demonstrated a complete response

to treatment, three showed a partial response, five

exhibited stable disease, and disease progression

was observed in three dogs (Roberts et al., 2014).

Moreover, a human patient with retroperitoneal

leiomyosarcoma was treated using intratumoral

spore injections, which resulted in a successful anti-

tumour response (Roberts et al., 2014).

Promising strides have been made in exploring the

safety and efficacy of C. novyi in early clinical trials

involving human patients. In a phase I clinical trial

involving 22 patients, C. novyi-NT was delivered

through intratumoral injection, and spore germina-

tion was observed in 10 patients (Janku et al., 2021).

The most common adverse effects reported were

pain at the injection site, fever, and fatigue. No seri-

ous complications or deaths were reported, and any

significant toxicities encountered were manageable.

Notably, tumour shrinkage was reported in nine

patients. Treatment with C. novyi stimulated a tran-

sient systemic cytokine response and enhanced sys-

temic tumour-specific T-cell responses. Overall, the

authors concluded that the treatment was both fea-

sible and safe (Janku et al., 2021).

Finally, notable progress has been achieved in the

development of imaging tools to monitor the colo-

nisation of tumours by C. novyi-NT. A study utilised

intratumoral injection of iron-oxide labelled C. no-

vyi-NT into mice with orthotopically implanted

pancreatic tumours, allowing the tracking of bacte-

ria using magnetic resonance imaging (MRI)

(Zheng et al., 2015). This in vivo imaging technique

enables the monitoring of tumour colonisation and

distribution of bacteria, facilitating the optimisation

of injection procedures and enabling both intra-pro-

cedural and post-procedural monitoring. Addition-

ally, the authors demonstrated that C. novyi-NT in-

duced tumour shrinkage in a mouse model of pan-

creatic carcinoma (Zheng et al., 2015).

Harnessing bacteria for immunotherapy: enhanc-

ing immune response against cancer cells

Utilising bacteria for cancer treatment offers a sig-

nificant advantage in addition to their oncolytic

properties: they can serve as immune system mod-

ulators, directing immune cells to target cancer

cells. C. sporogenes, for instance, has been genetically

engineered to produce cytokines, thereby activating

the immune system, and promoting the elimination

of tumour cells.

In one study, C. sporogenes ATCC 3584 was engi-

neered to secrete interleukin-12 (IL-12) (Zhang et

al., 2014). The authors demonstrated that the bacte-

ria successfully survived and secreted IL-12 within

the tumour environment. This resulted in immune

cell activation and destruction of tumour cells in a

murine model of EMT6 mammary carcinoma. The

Science Reviews - Biology, 2023, 2(2), 30 - 39 Raquel Rodrigues

treatment achieved a 14.3% cure rate with no appar-

ent toxicity (Zhang et al., 2014).

Similarly, C. sporogenes NCIMB 10696 was modified

to produce murine interleukin-2 (IL-2) (Kubiak, Bai-

ley, Dubois, Theys, & Lambin, 2021). The study em-

ployed an attenuated strain, C. sporogenes-NT,

which lacked the streptolysin S operon to reduce

bacterial-induced haemolysis. IL-2 plays a vital role

in activating and proliferating T cells. Given that C.

sporogenes germinates exclusively within hypoxic

tumours, IL-2 production is limited to the tumour

microenvironment, guiding the immune system's

response against cancer. The researchers demon-

strated that C. sporogenes-NT could successfully col-

onise tumours in mouse models, secrete IL-2 in

vitro, and induce T cell proliferation (Kubiak et al.,

2021). Furthermore, interleukin-2 expression was

also achieved in C. acetobutylicum DSM792, as

demonstrated in a study by Barbé et al. (2005).

ELISA assays confirmed the secretion of IL-2 by

C. acetobutylicum, and T-cell proliferation assays

validated the biological activity of the cytokine

(Barbé et al., 2005).

In a recent 2023 study, multifunctional porous mi-

crospheres (MPMs) were utilised to enhance the de-

livery and retention of C. novyi-NT spores within

the tumour microenvironment (Bae et al., 2023). The

results indicated that this delivery approach did not

impair spore germination within a simulated tu-

mour microenvironment and showcased its poten-

tial for image-guided cancer immunotherapy (Bae

et al., 2023).

Conclusion

Bacterial-based cancer therapies hold immense

promise, particularly due to the ability of anaerobic

bacteria to specifically target the challenging hy-

poxic and necrotic regions of tumours that tradi-

tional treatments like chemotherapy or radiother-

apy struggle to reach. In recent years, significant

progress has been made, largely driven by advance-

ments in genetic engineering techniques. Substan-

tial improvements have been achieved not only in

therapy efficacy but also in safety. Encouraging re-

sults from animal models and early-stage clinical

trials underscore the potential of these approaches.

As with other therapies, the adaptation of bacterial-

based treatments to specific cancer types, tumour

sizes, and individual patient characteristics is likely

to be crucial. Clostridium species have demonstrated

tumour shrinkage across various cancers, including

mammary/breast cancer (mouse model) (Abedi

Jafari et al., 2022; Minton et al., 1995), Ehrlich carci-

noma (mouse model) (Möse & Möse, 1964), sarcoma

(mouse model), melanoma (hamster model) (Thiele

et al., 1964), colon carcinoma (mouse model) (Dang

et al., 2001; Diaz et al., 2005; Heap et al., 2014), lung

carcinoma (mouse model) (Mowday et al., 2022;

Pokrovsky et al., 2019), glioblastoma (mouse model)

(V. Staedtke et al., 2022), glioblastoma (rat model)

(Verena Staedtke et al., 2015), pancreatic cancer

(mouse model) (Zheng et al., 2015), sarcoma (dog

study) (Roberts et al., 2014), retroperitoneal leiomy-

osarcoma (human patient) (Roberts et al., 2014), and

solid tumours in human patients (Janku et al., 2021).

While oncolysis has been achieved with Clostridium

injections alone (either intravenous or intra-

tumoral), combination therapies incorporating bac-

teria with other agents have shown greater success

in achieving complete tumour destruction. This is

due to the frequent resistance of tumour rims to

Clostridium-induced lysis, leading to tumour re-

growth.

While this review focused primarily on C. novyi and

C. sporogenes, other Clostridial species are being ex-

plored for their oncolytic properties. For example,

C. butyricum has exhibited the ability to inhibit the

growth of colorectal tumours in mice when com-

bined with 5-fluorouracil (5-FU) chemotherapy,

and it has also shown potential in enhancing the ef-

ficacy of immunotherapy (anti-PD-1) (Xu, Luo,

Zhang, Li, & Lee, 2023).

The diverse array of bacterial strains, coupled with

the possibility of combining them with other agents

or treatments, offers oncologists the opportunity to

tailor the most suitable therapy for their patients.

Furthermore, the administration of antibiotics or

other anti-infective agents can effectively eliminate

the Clostridium species after treatment, providing an

additional safety measure to prevent infections and

allowing the interruption of treatment in the event

of adverse effects (Mowday et al., 2022).

In conclusion, the use of bacteria, specifically Clos-

tridium species, holds significant promise for cancer

therapy. These bacteria offer the advantage of tar-

geted drug delivery, oncolytic properties, and im-

munomodulation, contributing to improved treat-

ment outcomes and reduced side effects. Further re-

search and clinical trials are necessary to refine and

validate these approaches, to advance bacterial-

based therapies into mainstream cancer treatments.

The continued exploration of bacteria in cancer

Raquel Rodrigues Science Reviews - Biology, 2023, 2(2), 30 - 39

therapy opens new avenues for innovation and the

potential to transform the landscape of cancer treat-

ment in the future.

References

Abedi Jafari, F., Abdoli, A., Pilehchian, R., Soleimani, N., & Hosseini, S. M. (2022). The oncolytic ac-

tivity of Clostridium novyi nontoxic spores in breast cancer. Bioimpacts, 12(5), 405-414.

https://doi.org/10.34172/bi.2021.25

Andryukov, B. G., Karpenko, A. A., & Lyapun, I. N. (2021). Learning from Nature: Bacterial Spores

as a Target for Current Technologies in Medicine (Review). Sovrem Tekhnologii Med, 12(3), 105-122.

https://doi.org/10.17691/stm2020.12.3.13

Bae, G.-H., Ryu, Y.-H., Han, J., Kim, S. H., Park, C. G., Park, J.-H., . . . Park, W. (2023). Multifunctional

porous microspheres encapsulating oncolytic bacterial spores and their potential for cancer immuno-

therapy. Biomaterials Science, 11(13), 4652-4663. https://doi.org/10.1039/D3BM00635B

Barbé, S., Van Mellaert, L., Theys, J., Geukens, N., Lammertyn, E., Lambin, P., & Anné, J. (2005). Se-

cretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as

a tool for anti-tumor treatment. FEMS Microbiology Letters, 246(1), 67-73.

https://doi.org/10.1016/j.femsle.2005.03.037.

Bettegowda, C., Huang, X., Lin, J., Cheong, I., Kohli, M., Szabo, S. A., . . . Zhou, S. (2006). The genome

and transcriptomes of the anti-tumor agent Clostridium novyi-NT. Nature Biotechnology, 24(12),

1573-1580. https://doi.org/10.1038/nbt1256.

Cano, R., & Borucki, M. (1995). Revival and identification of bacterial spores in 25- to 40-million-year-

old Dominican amber. Science, 268(5213), 1060-1064. Retrieved from http://science.science-

mag.org/content/sci/268/5213/1060.full.pdf. https://doi.org/10.1126/science.7538699

Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., . . .

Farrow, J. A. E. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and

eleven new species combinations. International Journal of Systematic and Evolutionary Microbiol-

ogy, 44(4), 812-826. Retrieved from http://ijs.microbiologyresearch.org/content/jour-

nal/ijsem/10.1099/00207713-44-4-812. https://doi.org/10.1099/00207713-44-4-812

Dang, L. H., Bettegowda, C., Huso, D. L., Kinzler, K. W., & Vogelstein, B. (2001). Combination bacte-

riolytic therapy for the treatment of experimental tumors. Proceedings of the National Academy of

Sciences, 98(26), 15155-15160. Retrieved from https://www.pnas.org/con-

tent/pnas/98/26/15155.full.pdf. https://doi.org/10.1073/pnas.251543698

Desvaux, M., Guedon, E., & Petitdemange, H. (2000). Cellulose catabolism by Clostridium celluloly-

ticum growing in batch culture on defined medium. Applied and Environmental Microbiology, 66(6),

2461-2470. Retrieved from http://aem.asm.org/content/66/6/2461.abstract.

https://doi.org/10.1128/aem.66.6.2461-2470.2000

Diaz, L. A., Jr., Cheong, I., Foss, C. A., Zhang, X., Peters, B. A., Agrawal, N., . . . Huso, D. L. (2005).

Pharmacologic and toxicologic evaluation of C. novyi-NT spores. Toxicological Sciences, 88(2), 562-

575. https://doi.org/10.1093/toxsci/kfi316.

Galperin, M. Y., Mekhedov, S. L., Puigbo, P., Smirnov, S., Wolf, Y. I., & Rigden, D. J. (2012). Genomic

determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific

genes. Environmental Microbiology, 14(11), 2870-2890. https://doi.org/10.1111/j.1462-

2920.2012.02841.x

Gu, Y., Patterson, A. V., Atwell, G. J., Chernikova, S. B., Brown, J. M., Thompson, L. H., & Wilson, W.

R. (2009). Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated

Science Reviews - Biology, 2023, 2(2), 30 - 39 Raquel Rodrigues

dinitrobenzamide mustard PR-104A. Molecular Cancer Therapeutics, 8(6), 1714-1723.

https://doi.org/10.1158/1535-7163.MCT-08-1209.

Heap, J. T., Theys, J., Ehsaan, M., Kubiak, A. M., Dubois, L., Paesmans, K., . . . Minton, N. P. (2014).

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors

in vivo. Oncotarget, 5(7), 1761-1769. Retrieved from http://www.impactjournals.com/oncotar-

get/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=1761.

https://doi.org/10.18632/oncotarget.1761

Helsby, N. A., Ferry, D. M., Patterson, A. V., Pullen, S. M., & Wilson, W. R. (2004). 2-Amino metabo-

lites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. British

journal of cancer, 90(5), 1084-1092. https://doi.org/10.1038/sj.bjc.6601612.

Janku, F., Zhang, H. H., Pezeshki, A., Goel, S., Murthy, R., Wang-Gillam, A., . . . Gounder, M. M.

(2021). Intratumoral injection of Clostridium novyi-NT spores in patients with treatment-refractory

advanced solid tumors. Clinical Cancer Research, 27(1), 96-106. https://doi.org/10.1158/1078-

0432.CCR-20-2065.

Karnofsky, D. A. (1968). Mechanisms of action of anticancer drugs at a cellular level. Ca: A Cancer

Journal For Clinicians, 18(4), 232-234. https://doi.org/10.3322/canjclin.18.4.232

Kennedy, M. J., Reader, S. L., & Swierczynski, L. M. (1994). Preservation records of micro-organisms:

evidence of the tenacity of life. Microbiology, 140(10), 2513-2529. Retrieved from http://mic.microbi-

ologyresearch.org/content/journal/micro/10.1099/00221287-140-10-2513.

https://doi.org/10.1099/00221287-140-10-2513

Kubiak, A. M., Bailey, T. S., Dubois, L. J., Theys, J., & Lambin, P. (2021). Efficient secretion of murine

IL-2 from an attenuated strain of Clostridium sporogenes, a novel delivery vehicle for cancer immu-

notherapy. Frontiers in microbiology, 12. Retrieved from https://www.frontiersin.org/arti-

cles/10.3389/fmicb.2021.669488. https://doi.org/10.3389/fmicb.2021.669488

Kubiak, A. M., & Minton, N. P. (2015). The potential of clostridial spores as therapeutic delivery ve-

hicles in tumour therapy. Research in Microbiology, 166(4), 244-254. Retrieved from http://www.sci-

encedirect.com/science/article/pii/S0923250814002551.

https://doi.org/10.1016/j.resmic.2014.12.006

Kubiak, A. M., Poehlein, A., Budd, P., Kuehne, S. A., Winzer, K., Theys, J., . . . Minton, N. P. (2015).

Complete genome sequence of the nonpathogenic soil-dwelling bacterium Clostridium sporogenes

strain NCIMB 10696. Genome Announcements, 3(4), e00942-00915. Retrieved from http://ge-

nomea.asm.org/content/3/4/e00942-15.abstract. https://doi.org/10.1128/genomeA.00942-15

Lamed, R., & Zeikus, J. G. (1980). Ethanol production by thermophilic bacteria: relationship between

fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and

Thermoanaerobium brockii. Journal of Bacteriology, 144(2), 569-578. Retrieved from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC294704/.

Ludwig, W., Schleifer, K., & Whitman, W. B. (2015). Bacilli class. nov. In Bergey's Manual of System-

atics of Archaea and Bacteria (pp. 1-1).

Minton, N. P., Mauchline, M. L., Lemmon, M. J., Brehm, J. K., Fox, M., Michael, N. P., . . . Brown, J.

M. (1995). Chemotherapeutic tumour targeting using clostridial spores. FEMS Microbiology Reviews,

17(3), 357-364. https://doi.org/10.1111/j.1574-6976.1995.tb00219.x.

Möse, J. R., & Möse, G. (1964). Oncolysis by clostridia. I. Activity of Clostridium butyricum (M-55)

and other nonpathogenic clostridia against the Ehrlich carcinoma. Cancer Research, 24(2 Part 1), 212-

216. Retrieved from http://cancerres.aacrjournals.org/content/24/2_Part_1/212.abstract.

Mowday, A. M., Dubois, L. J., Kubiak, A. M., Chan-Hyams, J. V. E., Guise, C. P., Ashoorzadeh, A., . . .

Patterson, A. V. (2022). Use of an optimised enzyme/prodrug combination for Clostridia directed

Raquel Rodrigues Science Reviews - Biology, 2023, 2(2), 30 - 39

enzyme prodrug therapy induces a significant growth delay in necrotic tumours. Cancer Gene Ther-

apy, 29(2), 178-188. https://doi.org/10.1038/s41417-021-00296-7.

Napoli, F., Olivieri, G., Russo, M. E., Marzocchella, A., & Salatino, P. (2010). Butanol production by

Clostridium acetobutylicum in a continuous packed bed reactor. Journal of Industrial Microbiology

& Biotechnology, 37(6), 603-608. https://doi.org/10.1007/s10295-010-0707-8.

Parker, R. C., Plummer, H. C., Siebenmann, C. O., & Chapman, M. G. (1947). Effect of histolyticus

infection and toxin on transplantable mouse tumors. Proceedings of the Society for Experimental Bi-

ology and Medicine, 66(2), 461-467. Retrieved from https://jour-

nals.sagepub.com/doi/abs/10.3181/00379727-66-16124. https://doi.org/10.3181/00379727-66-

16124

Pokrovsky, V. S., Anisimova, N. Y., Davydov, D. Z., Bazhenov, S. V., Bulushova, N. V., Zavilgelsky,

G. B., . . . Manukhov, I. V. (2019). Methionine gamma lyase from Clostridium sporogenes increases

the anticancer efficacy of doxorubicin on A549 cancer cells in vitro and human cancer xenografts. In

R. M. Hoffman (Ed.), Methionine Dependence of Cancer and Aging: Methods and Protocols (pp. 243-

261). New York, NY: Springer New York.

Rautio, J., Kumpulainen, H., Heimbach, T., Oliyai, R., Oh, D., Jarvinen, T., & Savolainen, J. (2008).

Prodrugs: design and clinical applications. Nat Rev Drug Discov, 7(3), 255-270.

https://doi.org/10.1038/nrd2468

Roberts, N. J., Zhang, L., Janku, F., Collins, A., Bai, R. Y., Staedtke, V., . . . Zhou, S. (2014). Intratumoral

injection of Clostridium novyi-NT spores induces antitumor responses. Sci Transl Med, 6(249),

249ra111. https://doi.org/10.1126/scitranslmed.3008982

Setlow, P. (2003). Spore germination. Current Opinion in Microbiology, 6(6), 550-556. Retrieved from

http://www.sciencedirect.com/science/article/pii/S1369527403001371.

https://doi.org/10.1016/j.mib.2003.10.001

Setlow, P. (2007). I will survive: DNA protection in bacterial spores. Trends in Microbiology, 15(4),

172-180. Retrieved from http://www.sciencedirect.com/science/article/pii/S0966842X07000261.

https://doi.org/10.1016/j.tim.2007.02.004

Siegel, R. L., Miller, K. D., Wagle, N. S., & Jemal, A. (2023). Cancer statistics, 2023. Ca: A Cancer Jour-

nal For Clinicians, 73(1), 17-48. Retrieved from https://acsjournals.onlineli-

brary.wiley.com/doi/abs/10.3322/caac.21763. https://doi.org/10.3322/caac.21763

Staedtke, V., Bai, R.-Y., Sun, W., Huang, J., Kibler, K. K., Tyler, B. M., . . . Riggins, G. J. (2015). Clos-

tridium novyi -NT can cause regression of orthotopically implanted glioblastomas in rats. Oncotar-

get, 6(8). Retrieved from https://www.oncotarget.com/article/3627/text/.

Staedtke, V., Gray-Bethke, T., Liu, G., Liapi, E., Riggins, G. J., & Bai, R. Y. (2022). Neutrophil depletion

enhanced the Clostridium novyi-NT therapy in mouse and rabbit tumor models. Neurooncol Adv,

4(1), vdab184. https://doi.org/10.1093/noajnl/vdab184

Tharmalingham, H., & Hoskin, P. (2019). Clinical trials targeting hypoxia. Br J Radiol, 92(1093),

20170966. https://doi.org/10.1259/bjr.20170966

Thiele, E. H., Arison, R. N., & Boxer, G. E. (1964). Oncolysis by clostridia. III. Effects of clostridia and

chemotherapeutic agents on rodent tumors. Cancer Research, 24(2 Part 1), 222-233. Retrieved from

http://cancerres.aacrjournals.org/content/24/2_Part_1/222.abstract.

Tirandaz, H., Hamedi, J., & Marashi, S.-A. (2006). Application of β-lactamase-dependent prodrugs in

clostridial-directed enzyme therapy (CDEPT): A proposal. Medical Hypotheses, 67(4), 998-999. Re-

trieved from https://www.sciencedirect.com/science/article/pii/S0306987706003409.

https://doi.org/10.1016/j.mehy.2006.05.008

Science Reviews - Biology, 2023, 2(2), 30 - 39 Raquel Rodrigues

Tran, H. G., Desmet, T., Saerens, K., Waegeman, H., Vandekerckhove, S., D'hooghe, M., . . . Soetaert,

W. (2012). Biocatalytic production of novel glycolipids with cellodextrin phosphorylase. Bioresource

Technology, 115, 84-87. Retrieved from http://www.sciencedirect.com/science/arti-

cle/pii/S0960852411013733. https://doi.org/10.1016/j.biortech.2011.09.085

Weinmann, M., Belka, C., & Plasswilm, L. (2004). Tumour hypoxia: impact on biology, prognosis and

treatment of solid malignant tumours. Oncology Research and Treatment, 27(1), 83-90. Retrieved

from http://www.karger.com/DOI/10.1159/000075611. https://doi.org/10.1159/000075611

Xu, H., Luo, H., Zhang, J., Li, K., & Lee, M. H. (2023). Therapeutic potential of Clostridium butyricum

anticancer effects in colorectal cancer. Gut Microbes, 15(1), 2186114.

https://doi.org/10.1080/19490976.2023.2186114

Zhang, Y. L., Lü, R., Chang, Z. S., Zhang, W. Q., Wang, Q. B., Ding, S. Y., & Zhao, W. (2014). Clostrid-

ium sporogenes delivers interleukin-12 to hypoxic tumours, producing antitumour activity without

significant toxicity. Lett Appl Microbiol, 59(6), 580-586. https://doi.org/10.1111/lam.12322

Zheng, L., Zhang, Z., Khazaie, K., Saha, S., Lewandowski, R. J., Zhang, G., & Larson, A. C. (2015).

MRI-monitored intra-tumoral injection of iron-oxide labeled Clostridium novyi-NT anaerobes in pan-

creatic carcinoma mouse model. PLoS ONE, 9(12), e116204. https://doi.org/10.1371/jour-

nal.pone.0116204.

The study did not involve humans or animals

Conflict of Interest statement

The author declares no conflict of interest.