Clostridium as a Cancer Therapy: Difference between revisions

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Clostridium is one of the largest prokaryotic genera and consists of a diverse range of obligatory anaerobic bacteria. There are over 100 species of Clostridium, including common free-living bacteria and pathogens. Clostridium bacteria are Gram-positive and rod-shaped. All of the members of the genus, with the exception of Clostridium perfringens, are motile and flagellated and form oval or spherical endospores that distend from the cell.
Clostridium is one of the largest prokaryotic genera and consists of a diverse range of obligatory anaerobic bacteria. There are over 100 species of Clostridium, including common free-living bacteria and pathogens. Clostridium bacteria are Gram-positive and rod-shaped. All of the members of the genus, with the exception of Clostridium perfringens, are motile and flagellated and form oval or spherical endospores that distend from the cell.


Most Clostridium species are nonpathogenic, and many are harmless and can be found in the soil. Some species have applications in bioremediation and wastewater treatment. Nonpathogenic species include Clostridium acetobutylicum and Clostridium beijerinckii, both of which have industrial applications. These solventogenic clostridia are used in acetone, butanol and isopropanol fermentation.
The ability of Clostridium bacteria to produce endospores that are highly resistant to extreme environmental conditions including high temperature, disinfectants, and low-energy radiation, makes many pathogenic Clostridium bacteria a major health concern and of clinical significance. Several pathogens-producing potent toxins part of this genera include C. tetani, C. botulinum, C. difficile, and C. perfringens. C. tetani is the causative agent of tetanus, and Clostridium botulinum is related to food poisoning and causes botulism. Other members of the Clostridium genus include Clostridium perfringens which can infect wounds and cause gas gangrene, and Clostridium difficile which can cause diarrhea and more serious intestinal conditions such as colitis. Clostridium difficile is one of the most common causes of infection of the large bowel. Another species, Clostridium novyi has been associated with an outbreak of serious illness and death amongst intravenous drug users. Despite pathogenic properties, research has shown that Clostridium novyi seems to be very promising in the fight against cancer as a bacteriolytic anti-cancer agent. It has been well known that severe bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated.


The ability of Clostridium bacteria to produce endospores that are highly resistant to harsh environmental conditions and can withstand high temperature, disinfectants, and low-energy radiation, makes many pathogenic Clostridium bacteria a major health concern and of clinical significance. Several pathogens-producing potent toxins part of this genera include C. tetani, C. botulinum, C. difficile, and C. perfringens. C. tetani is the causative agent of tetanus, and Clostridium botulinum is related to food poisoning and causes botulism. Other members of the Clostridium genus include Clostridium perfringens which can infect wounds and cause gas gangrene, and Clostridium difficile which can cause diarrhea and more serious intestinal conditions such as colitis. Clostridium difficile is one of the most common causes of infection of the large bowel. Another species, Clostridium novyi has been associated with an outbreak of serious illness and death amongst intravenous drug users. Despite pathogenic properties, research has shown that Clostridium novyi seems to be very promising in the fight against cancer as a bacteriolytic anti-cancer agent. It has been well known that severe bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated.
==Microbial Applications in Cancer Therapy==
Cancer can be characterized by uncontrolled cell growth forming tumors that are composed of necrotic, hypoxic, and/or well-oxygenated regions. However, most conventional anticancer therapies target the well-vascularized component of tumors and rely on tumor vasculature or oxygen transport, leaving behind hypoxic cells that can potentially regenerate the tumor after treatement. Hypoxic regions in tumors are a major cause of treatment failures. Hypoxic regions are especially difficult to treat, as they are more resistant to systemic anticancer agents and radiotherapy. Oxygen is a required effector of radiation-induced cell death, and the dose required to kill hypoxic cells through radiation is up to 3 times higher compared with the same amount in well-oxygenated cells. Despite the non-specificity of conventional cancer treatments towards hypoxic regions, the use of anaerobic bacteria has been recently studied in its ability to target oxygen-poor tumors that facilitate the growth of anaerobic bacteria.  
===History===
The introduction of bacterial applications in cancer therapy is not an entirely novel idea as studies have shown that bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated. Using bacteria to treat cancer was first suggested by William B. Coley, who in 1891, injected Streptococcus pyogenes into a patient with terminal cancer. He thought that the induced infection would have the side effect of shrinking the malignant tumor and was successful as he continued to inject more than 1000 cancer patients with dead bacteria or bacterial products over the next forty years. However, with the evolution of radiotherapy, chemotherapy, and other modern cancer treatments, the idea of using bacteria in cancer therapies were disregarded due to the inability to produce a viable anticancer agent, in part because of poor reproducibility and unacceptable toxicity.
Since Coley’s original work, a variety of anaerobic bacteria have been considered for cancer therapy.
Cancer can be characterized by uncontrolled cell growth forming tumors that are composed of necrotic, hypoxic, and/or well-oxygenated regions. However, most conventional anticancer therapies target the well-vascularized component of tumors and rely on tumor vasculature or oxygen transport, leaving behind hypoxic cells that can potentially regenerate the tumor after treatement. Hypoxic regions in tumors are a major cause of treatment failures. Hypoxic regions are especially difficult to treat, as they are more resistant to systemic anticancer agents and radiotherapy. Oxygen is a required effector of radiation-induced cell death, and the dose required to kill hypoxic cells through radiation is up to 3 times higher compared with the same amount in well-oxygenated cells. Despite the non-specificity of conventional cancer treatments towards hypoxic regions, the use of anaerobic bacteria has been recently studied in its ability to target oxygen-poor tumors that facilitate the growth of anaerobic bacteria.  
===Applications of Clostridium===
Due to their anaerobic nature, species of Clostridium bacteria have been recognized for their ability to selectively target and lyse tumor cells growing in hypoxic environments marked by uncontrolled growth and low oxygen levels. Intravenous administration of nonpathogenic bacterial strains or spores from nonpathogenic strains of Clostridium bacteria may be used to target hypoxic or necrotic solid tumors. With Clostridium-directed tumor therapy, advantages include tumor selectivity and safety of using nonpathogenic clostridia in cancer treatment.


In particular, Clostridium novyi is a highly mobile spore-forming organism that is extremely sensitive to oxygen and has never been shown to germinate in areas of tumors outside of the hypoxic regions. The bacteria are exquisitely sensitive to oxygen but its spores are stable to oxygen as well as to harsh conditions. Due to its highly motile nature, the bacteria can easily disperse itself throughout the tumor. Injection of C novyi takes advantage of the hypoxic nature of rapidly growing tumors and can serve as a selective tumor killer overlooked by conventional cancer therapies.


==Anticancer Properties==
==Current Forms of Cancer Treatments==
Species of Clostridium bacteria are notable for their ability to selectively target and lyse tumor cells growing in hypoxic environments due to their anaerobic nature. Hypoxia is a major cause of resistance to ionizing radiation and the dose required to kill hypoxic cells is up to 3 times higher compared with the same amount of well-oxygenated cells. Cancerous tumors marked by uncontrolled growth and low oxygen levels that can provide a niche for anaerobic bacteria such as Clostridium, and intravenous administration of spores from nonpathogenic strains of Clostridium bacteria can be used to target hypoxic or necrotic solid tumors. With Clostridium-directed tumor therapy, advantages include tumor selectivity and safety of using nonpathogenic clostridia in cancer treatment.


==Current Forms of Cancer Treatments==
===Traditional cancer therapies===
<b>Traditional cancer therapies <br></b>
Current and traditional cancer therapies involve regulating of cell division and growth most commonly at the genetic level. Conventional methods involve delivering tumor suppressor genes to cancer cells, delivering genes that activate toxic products to kills tumor cells and their neighbors, introducing defective genes that cause cell death, and directly attacking tumor cells and harnessing the immune responses to tumor antigens. However, the primary problem with such traditional methods is the lack of tumor specificity. Disadvantages of both viral and non-viral gene delivery methods include but are not limited to low transfection transduction levels, immune responses that may hinder repeated administrations of the vector, potential random integrations from retroviruses that could have carcinogenic effects, and a preferential infection of only non-dividing cells. Most drugs and current treatments are effective against rapidly dividing tumor cells but not against hypoxic regions in which cell proliferation is decreased.  
Current and traditional cancer therapies involve regulating of cell division and growth most commonly at the genetic level. Conventional methods involve delivering tumor suppressor genes to cancer cells, delivering genes that activate toxic products to kills tumor cells and their neighbors, introducing defective genes that cause cell death, and directly attacking tumor cells and harnessing the immune responses to tumor antigens. However, the primary problem with such traditional methods is the lack of tumor specificity. Disadvantages of both viral and non-viral gene delivery methods include but are not limited to low transfection transduction levels, immune responses that may hinder repeated administrations of the vector, potential random integrations from retroviruses that could have carcinogenic effects, and a preferential infection of only non-dividing cells. Most drugs and current treatments are effective against rapidly dividing tumor cells but not against hypoxic regions in which cell proliferation is decreased.  


<b>Bacteria-directed cancer therapies <br></b>
===Bacteria-directed cancer therapies===
The introduction of bacterial applications in cancer therapy is not an entirely novel idea as studies have shown that bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated. Bacteria-directed cancer treatment has advantages over the traditional cancer therapies. Some nonrecombinant bacteria already exert inherent antitumor activities, and recombinant bacteria can be used as vectors to produce protein of therapeutic interest in tumor environments as an alternative to gene therapy. Through the bacterial-directed protein delivery system, there will be an efficient distribution of vector throughout the tumor mass with sufficient transfection levels and transient gene expression. This method prevents the random insertion of foreign DNA into the genome and the transfection of tumor cells with therapeutic genes that may instead enhance antitumor properties. The bacteria can then be inactivated at any moment during therapy by administering antibiotics.
 
Difficulties in delivering anticancer chemotherapeutics to the poorly vascularized, hypoxic regions of tumors can be overcome with bacteria-based tumor specific therapies using anaerobic bacteria. Anaerobic bacteria can specifically target the hypoxic or necrotic regions of the tumors and either directly exert innate oncolytic effects or act as a vector in introducing locally expressed therapeutic proteins.
Difficulties in delivering anticancer chemotherapeutics to the poorly vascularized, hypoxic regions of tumors can be overcome with bacteria-based tumor specific therapies using anaerobic bacteria. Anaerobic bacteria can specifically target the hypoxic or necrotic regions of the tumors and either directly exert innate oncolytic effects or act as a vector in introducing locally expressed therapeutic proteins.
Bacteriolytic anti-cancer therapies use attenuated bacterial strains that selectively proliferate within tumors. An advantage of using bacteria to treat cancer is that bacteria can be modified relatively easily such as by eliminating its toxicity or equipping it with other therapeutic agents. In addition, some nonrecombinant bacteria may already exert inherent antitumor activities, and recombinant bacteria can be used as vectors to produce protein of therapeutic interest in tumor environments as an alternative to gene therapy. Using bacteria as a therapeutic agent can induce a strong immune response against tumor cells themselves.
Through the bacterial-directed protein delivery system, there will be an efficient distribution of vector throughout the tumor mass with sufficient transfection levels and transient gene expression. This method prevents the random insertion of foreign DNA into the genome and the transfection of tumor cells with therapeutic genes that may instead enhance antitumor properties. The bacteria can then be inactivated at any moment during therapy by administering antibiotics.


==Research and Studies==
==Research and Studies==
Clostridium novyi is a highly mobile spore-forming organism that is extremely sensitive to oxygen. Injection of C novyi takes advantage of the hypoxic nature of rapidly growing tumors and thus serves as a selective tumor killer.
To create a nonpathogenic strain of the target bacteria, researchers at Johns Hopkins University engineered the bacteria to get rid of its toxin gene. This way, once injected into the host tissue, the host would survive, but the cancerous tumors would be targeted.
When mice were injected with the engineered nonpathogenic strain of C. novyi, the bacteria were found sporulating within the tumors within 16 hours with no migration to other healthy organs of the body. 100 million spores can be injected into a cancer patient causing mass proliferation of the bacteria in the malignant tumor but no harm to the host.
It was found that pathogenic species from the genus Clostridium may be responsible for contamination of the materials used for illicit drug use involving injection. C. novyi type A has been targeted as one of these species in an outbreak that involved high mortality rates among drug users who injected heroin extravascularly. 16S rDNA sequence data was used to identify C. novyi as the pathogen responsible for these mortalities.
The 2.55 Mb genomic sequence of C. novyi has recently been determined. From this determination, a new type of transposition and 139 genes which do not have homologs in other bacteria have been identified. Knowing C. novyi’s genomic sequence has allowed detection of transcripts in vitro and infections of tumors in vivo. From this, it was found that C. novyi-NT spores contain mRNA, and that the spores’ transcripts are very different from those transcripts found in the bacterium’s vegetative form.
[[
|thumb|300px|right|Effect of C. novyi-NT plus external beam radiation on various transplanted tumor models after the indicated treatments. a, HCT116; b, HuCC-T1; c, B16; d, HT29. Irradiated mice received 2 Gy/day for 5 days. Spores were administered after the third dose of radiation. Tumor growth curves are color-coded: light blue, untreated control; purple, C. novyi-NT spores alone; green, radiation (10 Gy) alone; red, radiation (10 Gy) plus C. novyi-NT spores. Each group contained at least six mice. Student's t test was performed by using the linear regression slope for each individual mouse. Each arm was compared with the combination of C. novyi-NT plus 10 Gy, and all were statistically significant in a–c. The P values for comparison between irradiation alone and irradiation plus C. novyi-NT were as follows: a, P < 0.0001; b, P < 0.05; and c, P < 0.05. Chetan Bettegowda et al. PNAS 2003;100:15083-15088
Copyright © 2003, The National Academy of Sciences[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC299912/bin/pq2436598003.jpg]]]
Previous attempts to treat advanced cancers using sterile filtrates of C. histolyticum produced proteolytic enzymes that degraded cancerous tissues without affecting normal tissue. Spores germinated in tumors after inoculation of C. histolyticum spores into mice with transplanted sarcomas and observed lysis of tumor tissues.
Intravenously injected spores of nonpathogenic strains of Clostridium species in rodent tumor models with transplanted tumors resulted in softening of tumors and tumor cell death primarily in the tumor center. However, tumor regrowth occurred overtime from the remaining outer rim of viable cells. To specifically attack the remaining cancer cells at the tumor periphery, combination therapies have been studied to improve the therapeutic outcomes.


C. sporogenes was transformed with the E. coli codA gene encoding cytosine deaminase. When spores of this recombinant strain were administered to mice bearing SCCVII tumors followed by 5-fluorocytosine injection, a significant antitumor effect was observed. C. sporogenes was also engineered to produce nitroreductase by combining it with the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid administered 4 h after injection of spores. These recombinants showed a fourfold increase in tumor colonization and in combination with the respective prodrugs, complete tumor reduction was observed.  
===Intratumoral injection of Clostridium novyi-NT spores ===
Recently, a Johns Hopkins team has since revisited the idea of using microbes to attack cancer. In a study recently published in Science, they used C. noyvi by removing the α-toxin-producing genes to make it safer for internal use. The new bacteria strain is called C. noyvi-NT (C. noyvi-non-toxic). As a non-toxic strain of bacteria, C. noyvi-NT can thus grow within a tumor without harming the host. Looking at the bacteria’s genomic sequence, C. novyi-NT spores contain mRNA and the spore transcripts are distinct from those in vegetative forms of the bacterium.
Roberts et al.(2014) injected a single dose of intravenous C. novyi-NT spores directly into mice and rabbits bearing transplanted tumors.  They observed an antitumor response including localized tumor necrosis, intense inflammatory responses, and complete responses in 25 to 30% of the treated animals. Based on the resultant data obtained from the experiment, intravenously injected C. novyi-NT spores were also evaluated in spontaneously occurring canine tumors. For this experiment, each dog received at least one single intratumoral injection of 1 × 108 C. novyi-NT spores into one target tumor. Dogs received up to four cycles of treatment with a 1-week interval between cycles. Treated dogs were followed for at least 90 days after the first intratumoral injection. The injections of C. novyi-NT spores were well-tolerated in dogs bearing spontaneous solid tumors, and side effects were expected symptoms associated with bacterial infections mild in nature. Responses were observed in 37.5% of the 16 dogs, but complete responses were observed in only 3 of the dogs. Overall, improved survival rates of the cancerous animals were observed, and the intratumoral injection of C. novyi-NT spores was further tested in one human patient.
A phase 1 investigational study was initiated in human patients with solid tumors that were refractory to standard therapy or lacked standard therapy. The first patient enrolled in this trial was a 53-year-old female diagnosed with advanced retroperitoneal leiomyosarcoma. The patient was treated with an intratumoral injection of 10, 000 C. novyi-NT spores, and resulted in a rapid and robust local antitumor response. Extensive tumor necrosis of residual tumor cells was observed with reduced tumor tissue within and surrounding the bone.  


Administration of C. novyi-NT spores to nude mice with various human xenografts worked in combination with different amounts of radiotherapy. The best result was observed with brachytherapy, as a single dose of spores combined with this treatment resulted in 100% cure in mice. When immunocompetent tumor-bearing mice were treated with C. novyi-NT spores alone, only 30% of the animals exhibited complete tumor regression.  
These results collectively support the further development of intratumoral injections of C. novyi-NT spores as a therapeutic for patients with locally advanced, non-resectable cancers and the viability of C. noyvi-NT bacteria as a form of cancer therapy.  


Clostridia is still currently under investigation for cancer therapy uses in humans. Recently, Clostridium species have been engineered  to produce proteins of interest in antitumor therapy. Although Clostridia is not yet completely efficient to sufficiently control tumor growth, recent combination treatments have improved in preclinical antitumor responses. Combination of clostridial spores administration and vascular targeting agents increase tumor colonization and concentrations of degradative enzymes and therapeutic proteins in the tumor and also reduce the probability of incomplete tumor cell death or tumor regrowth. Ongoing studies are searching for the best combinations or recombinant cancer therapies using a bacterial approach.
===Recombination cancer therapies===
Bacteria may also enhance the therapeutic effects of radiation by killing tumor regions resistant to radiation therapy when used in conjunction with radiation treatment. Studies have been done using recombination bacterial cancer therapies.
Bettegowda et. al (2006) compared the effects of C. novyi-NT on colorectal tumor tissue when administered alone and in conjunction with conventional cancer therapies of different amounts of radiotherapy. When C. novyi-NT spores were intravenously injected into mice bearing human colorectal cancer xenografts, germination occurred exclusively within the tumors. The bacteria were dispersed throughout the tumor and tumor tissue showed a significant reduction overtime following the intravenous injection. However, a rim of viable tumor was almost always left after treatment with the spores (Fig. 2c), limiting the efficacy of the therapeutic response.
====Combination with External Beam Radiation====
To determine whether the combination of both C. novyi-NT and irradiation could eliminate both the well vascularized and poorly vascularized regions of these tumors, the tumor-bearing mice were treated with a protocol using five daily doses of external beam radiation, with spores administered between the third and fourth doses. The spores significantly enhanced the effect of radiation in most of these tests, and this combination therapy produced remarkable tumor shrinkage evident on gross inspection as well as microscopically (Fig. 3a). There was complete or nearly complete lysis of all tumor cells independent of size with tumors ranging from 50 to 1,000 mm3.
In addition, other tumor models were tested to determine whether C. novyi-NT could enhance the effects of radiation administered by external beam. C. novyi-NT spores significantly enhanced the effects of radiation in the human biliary cancer and the mouse melanoma B16. The human colorectal cancer cell line HT-29 and the human lung cancer Calu-3 were found to be unresponsive to C. novyi-NT spores when used alone, and in conjunction with radiation. Although C. novyi-NT substantially improved the effects of external beam irradiation as safe doses of radiation, a small number of residual tumor cells eventually proliferated, resulting in tumor recurrence including in models most sensitive to the combination therapy.
====Combination with Brachytherapy====
A single dose of spores combined with brachytherapy resulted in 100% cure in mice, defined by 3 months without evidence of disease.
====Combination with RAIT====
In addition the effects of C. novyi-NT given in combination with RAIT (radioactive immunotherapy) was also studied. RAIT is used to treat systemic disease and is dependent on the vascularization and oxygen delivery of radiolabeled compounds to tumor cells. As both are compromised in poorly vascularized and hypoxic tumors, C. novyi-NT was studied to see if it could enhance the effects of RAIT. The experiment found that the combination therapy was effective whether the spores were administered before or after injection of the labeled antibody. Two of 14 mice treated with both RAIT and spores combination therapy were cured after a follow-up of 6 months. Mice treated with RAIT alone, on the other hand, showed excessive tumor growth.
Clostridia continues to be under investigation for cancer therapy uses in humans. Although Clostridia is not yet completely efficient to sufficiently and quantitatively control tumor growth, recent combination treatments have improved in preclinical antitumor responses. Combination of clostridial spores administration with current cancer therapies reduce the probability of incomplete tumor cell death or tumor regrowth. Ongoing studies are searching for the best combinations or recombinant cancer therapies using a bacterial approach. In addition, Clostridium species have been engineered to produce proteins of interest in antitumor therapy.


==Other bacterial treatments==
==Other bacterial treatments==
Other anerobic bacterial species under investigation for cancer therapy include Bifodobacterium, Streptococcus pyogenes, and Salmonella Typhimurium . Streptococcus pyogenes and Salmonella typhimurium have been used with some success in clinical trials.
Other anaerobic bacterial species under investigation for cancer therapy include Bifodobacterium, Streptococcus pyogenes, and Salmonella Typhimurium. Streptococcus pyogenes and Salmonella typhimurium have been used with some success in clinical trials.
Phase 1 clinical trials of S. typhimurium in both dogs and human patients have demonstrated that safe administration and tumor specificity, with limited efficacy. Genetically modified strains of the bacterium such as incorporating cytosine deaminase, have been developed to enhance efficacy with S. typhimurium therapy.  


==References==
==References==

Revision as of 03:46, 30 March 2015

This student page has not been curated.
A photomicrograph of Clostridium botulinum bacteria.The bacterium C. botulinum causes the rare, but serious paralytic illness Botulism. [1]


Clostridium is one of the largest prokaryotic genera and consists of a diverse range of obligatory anaerobic bacteria. There are over 100 species of Clostridium, including common free-living bacteria and pathogens. Clostridium bacteria are Gram-positive and rod-shaped. All of the members of the genus, with the exception of Clostridium perfringens, are motile and flagellated and form oval or spherical endospores that distend from the cell.

The ability of Clostridium bacteria to produce endospores that are highly resistant to extreme environmental conditions including high temperature, disinfectants, and low-energy radiation, makes many pathogenic Clostridium bacteria a major health concern and of clinical significance. Several pathogens-producing potent toxins part of this genera include C. tetani, C. botulinum, C. difficile, and C. perfringens. C. tetani is the causative agent of tetanus, and Clostridium botulinum is related to food poisoning and causes botulism. Other members of the Clostridium genus include Clostridium perfringens which can infect wounds and cause gas gangrene, and Clostridium difficile which can cause diarrhea and more serious intestinal conditions such as colitis. Clostridium difficile is one of the most common causes of infection of the large bowel. Another species, Clostridium novyi has been associated with an outbreak of serious illness and death amongst intravenous drug users. Despite pathogenic properties, research has shown that Clostridium novyi seems to be very promising in the fight against cancer as a bacteriolytic anti-cancer agent. It has been well known that severe bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated.

Microbial Applications in Cancer Therapy

Cancer can be characterized by uncontrolled cell growth forming tumors that are composed of necrotic, hypoxic, and/or well-oxygenated regions. However, most conventional anticancer therapies target the well-vascularized component of tumors and rely on tumor vasculature or oxygen transport, leaving behind hypoxic cells that can potentially regenerate the tumor after treatement. Hypoxic regions in tumors are a major cause of treatment failures. Hypoxic regions are especially difficult to treat, as they are more resistant to systemic anticancer agents and radiotherapy. Oxygen is a required effector of radiation-induced cell death, and the dose required to kill hypoxic cells through radiation is up to 3 times higher compared with the same amount in well-oxygenated cells. Despite the non-specificity of conventional cancer treatments towards hypoxic regions, the use of anaerobic bacteria has been recently studied in its ability to target oxygen-poor tumors that facilitate the growth of anaerobic bacteria.

History

The introduction of bacterial applications in cancer therapy is not an entirely novel idea as studies have shown that bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated. Using bacteria to treat cancer was first suggested by William B. Coley, who in 1891, injected Streptococcus pyogenes into a patient with terminal cancer. He thought that the induced infection would have the side effect of shrinking the malignant tumor and was successful as he continued to inject more than 1000 cancer patients with dead bacteria or bacterial products over the next forty years. However, with the evolution of radiotherapy, chemotherapy, and other modern cancer treatments, the idea of using bacteria in cancer therapies were disregarded due to the inability to produce a viable anticancer agent, in part because of poor reproducibility and unacceptable toxicity. Since Coley’s original work, a variety of anaerobic bacteria have been considered for cancer therapy. Cancer can be characterized by uncontrolled cell growth forming tumors that are composed of necrotic, hypoxic, and/or well-oxygenated regions. However, most conventional anticancer therapies target the well-vascularized component of tumors and rely on tumor vasculature or oxygen transport, leaving behind hypoxic cells that can potentially regenerate the tumor after treatement. Hypoxic regions in tumors are a major cause of treatment failures. Hypoxic regions are especially difficult to treat, as they are more resistant to systemic anticancer agents and radiotherapy. Oxygen is a required effector of radiation-induced cell death, and the dose required to kill hypoxic cells through radiation is up to 3 times higher compared with the same amount in well-oxygenated cells. Despite the non-specificity of conventional cancer treatments towards hypoxic regions, the use of anaerobic bacteria has been recently studied in its ability to target oxygen-poor tumors that facilitate the growth of anaerobic bacteria.

Applications of Clostridium

Due to their anaerobic nature, species of Clostridium bacteria have been recognized for their ability to selectively target and lyse tumor cells growing in hypoxic environments marked by uncontrolled growth and low oxygen levels. Intravenous administration of nonpathogenic bacterial strains or spores from nonpathogenic strains of Clostridium bacteria may be used to target hypoxic or necrotic solid tumors. With Clostridium-directed tumor therapy, advantages include tumor selectivity and safety of using nonpathogenic clostridia in cancer treatment.

In particular, Clostridium novyi is a highly mobile spore-forming organism that is extremely sensitive to oxygen and has never been shown to germinate in areas of tumors outside of the hypoxic regions. The bacteria are exquisitely sensitive to oxygen but its spores are stable to oxygen as well as to harsh conditions. Due to its highly motile nature, the bacteria can easily disperse itself throughout the tumor. Injection of C novyi takes advantage of the hypoxic nature of rapidly growing tumors and can serve as a selective tumor killer overlooked by conventional cancer therapies.

Current Forms of Cancer Treatments

Traditional cancer therapies

Current and traditional cancer therapies involve regulating of cell division and growth most commonly at the genetic level. Conventional methods involve delivering tumor suppressor genes to cancer cells, delivering genes that activate toxic products to kills tumor cells and their neighbors, introducing defective genes that cause cell death, and directly attacking tumor cells and harnessing the immune responses to tumor antigens. However, the primary problem with such traditional methods is the lack of tumor specificity. Disadvantages of both viral and non-viral gene delivery methods include but are not limited to low transfection transduction levels, immune responses that may hinder repeated administrations of the vector, potential random integrations from retroviruses that could have carcinogenic effects, and a preferential infection of only non-dividing cells. Most drugs and current treatments are effective against rapidly dividing tumor cells but not against hypoxic regions in which cell proliferation is decreased.

Bacteria-directed cancer therapies

Difficulties in delivering anticancer chemotherapeutics to the poorly vascularized, hypoxic regions of tumors can be overcome with bacteria-based tumor specific therapies using anaerobic bacteria. Anaerobic bacteria can specifically target the hypoxic or necrotic regions of the tumors and either directly exert innate oncolytic effects or act as a vector in introducing locally expressed therapeutic proteins. Bacteriolytic anti-cancer therapies use attenuated bacterial strains that selectively proliferate within tumors. An advantage of using bacteria to treat cancer is that bacteria can be modified relatively easily such as by eliminating its toxicity or equipping it with other therapeutic agents. In addition, some nonrecombinant bacteria may already exert inherent antitumor activities, and recombinant bacteria can be used as vectors to produce protein of therapeutic interest in tumor environments as an alternative to gene therapy. Using bacteria as a therapeutic agent can induce a strong immune response against tumor cells themselves. Through the bacterial-directed protein delivery system, there will be an efficient distribution of vector throughout the tumor mass with sufficient transfection levels and transient gene expression. This method prevents the random insertion of foreign DNA into the genome and the transfection of tumor cells with therapeutic genes that may instead enhance antitumor properties. The bacteria can then be inactivated at any moment during therapy by administering antibiotics.

Research and Studies

Intratumoral injection of Clostridium novyi-NT spores

Recently, a Johns Hopkins team has since revisited the idea of using microbes to attack cancer. In a study recently published in Science, they used C. noyvi by removing the α-toxin-producing genes to make it safer for internal use. The new bacteria strain is called C. noyvi-NT (C. noyvi-non-toxic). As a non-toxic strain of bacteria, C. noyvi-NT can thus grow within a tumor without harming the host. Looking at the bacteria’s genomic sequence, C. novyi-NT spores contain mRNA and the spore transcripts are distinct from those in vegetative forms of the bacterium. Roberts et al.(2014) injected a single dose of intravenous C. novyi-NT spores directly into mice and rabbits bearing transplanted tumors. They observed an antitumor response including localized tumor necrosis, intense inflammatory responses, and complete responses in 25 to 30% of the treated animals. Based on the resultant data obtained from the experiment, intravenously injected C. novyi-NT spores were also evaluated in spontaneously occurring canine tumors. For this experiment, each dog received at least one single intratumoral injection of 1 × 108 C. novyi-NT spores into one target tumor. Dogs received up to four cycles of treatment with a 1-week interval between cycles. Treated dogs were followed for at least 90 days after the first intratumoral injection. The injections of C. novyi-NT spores were well-tolerated in dogs bearing spontaneous solid tumors, and side effects were expected symptoms associated with bacterial infections mild in nature. Responses were observed in 37.5% of the 16 dogs, but complete responses were observed in only 3 of the dogs. Overall, improved survival rates of the cancerous animals were observed, and the intratumoral injection of C. novyi-NT spores was further tested in one human patient. A phase 1 investigational study was initiated in human patients with solid tumors that were refractory to standard therapy or lacked standard therapy. The first patient enrolled in this trial was a 53-year-old female diagnosed with advanced retroperitoneal leiomyosarcoma. The patient was treated with an intratumoral injection of 10, 000 C. novyi-NT spores, and resulted in a rapid and robust local antitumor response. Extensive tumor necrosis of residual tumor cells was observed with reduced tumor tissue within and surrounding the bone.

These results collectively support the further development of intratumoral injections of C. novyi-NT spores as a therapeutic for patients with locally advanced, non-resectable cancers and the viability of C. noyvi-NT bacteria as a form of cancer therapy.

Recombination cancer therapies

Bacteria may also enhance the therapeutic effects of radiation by killing tumor regions resistant to radiation therapy when used in conjunction with radiation treatment. Studies have been done using recombination bacterial cancer therapies. Bettegowda et. al (2006) compared the effects of C. novyi-NT on colorectal tumor tissue when administered alone and in conjunction with conventional cancer therapies of different amounts of radiotherapy. When C. novyi-NT spores were intravenously injected into mice bearing human colorectal cancer xenografts, germination occurred exclusively within the tumors. The bacteria were dispersed throughout the tumor and tumor tissue showed a significant reduction overtime following the intravenous injection. However, a rim of viable tumor was almost always left after treatment with the spores (Fig. 2c), limiting the efficacy of the therapeutic response.

Combination with External Beam Radiation

To determine whether the combination of both C. novyi-NT and irradiation could eliminate both the well vascularized and poorly vascularized regions of these tumors, the tumor-bearing mice were treated with a protocol using five daily doses of external beam radiation, with spores administered between the third and fourth doses. The spores significantly enhanced the effect of radiation in most of these tests, and this combination therapy produced remarkable tumor shrinkage evident on gross inspection as well as microscopically (Fig. 3a). There was complete or nearly complete lysis of all tumor cells independent of size with tumors ranging from 50 to 1,000 mm3. In addition, other tumor models were tested to determine whether C. novyi-NT could enhance the effects of radiation administered by external beam. C. novyi-NT spores significantly enhanced the effects of radiation in the human biliary cancer and the mouse melanoma B16. The human colorectal cancer cell line HT-29 and the human lung cancer Calu-3 were found to be unresponsive to C. novyi-NT spores when used alone, and in conjunction with radiation. Although C. novyi-NT substantially improved the effects of external beam irradiation as safe doses of radiation, a small number of residual tumor cells eventually proliferated, resulting in tumor recurrence including in models most sensitive to the combination therapy.

Combination with Brachytherapy

A single dose of spores combined with brachytherapy resulted in 100% cure in mice, defined by 3 months without evidence of disease.

Combination with RAIT

In addition the effects of C. novyi-NT given in combination with RAIT (radioactive immunotherapy) was also studied. RAIT is used to treat systemic disease and is dependent on the vascularization and oxygen delivery of radiolabeled compounds to tumor cells. As both are compromised in poorly vascularized and hypoxic tumors, C. novyi-NT was studied to see if it could enhance the effects of RAIT. The experiment found that the combination therapy was effective whether the spores were administered before or after injection of the labeled antibody. Two of 14 mice treated with both RAIT and spores combination therapy were cured after a follow-up of 6 months. Mice treated with RAIT alone, on the other hand, showed excessive tumor growth. Clostridia continues to be under investigation for cancer therapy uses in humans. Although Clostridia is not yet completely efficient to sufficiently and quantitatively control tumor growth, recent combination treatments have improved in preclinical antitumor responses. Combination of clostridial spores administration with current cancer therapies reduce the probability of incomplete tumor cell death or tumor regrowth. Ongoing studies are searching for the best combinations or recombinant cancer therapies using a bacterial approach. In addition, Clostridium species have been engineered to produce proteins of interest in antitumor therapy.

Other bacterial treatments

Other anaerobic bacterial species under investigation for cancer therapy include Bifodobacterium, Streptococcus pyogenes, and Salmonella Typhimurium. Streptococcus pyogenes and Salmonella typhimurium have been used with some success in clinical trials. Phase 1 clinical trials of S. typhimurium in both dogs and human patients have demonstrated that safe administration and tumor specificity, with limited efficacy. Genetically modified strains of the bacterium such as incorporating cytosine deaminase, have been developed to enhance efficacy with S. typhimurium therapy.

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Edited by Anh Tran, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2014.