When you get right down to it, bacteria are just plain cool. They can self-replicate, evolve in the span of several days, exchange DNA with neighbours and enemies, survive in the most inhospitable conditions, and occupy such a broad range of hosts and environments that they constitute the largest biomass on the planet. Scientists have long recognized the utility of these prokaryotes, and over the years, they have become an indispensable tool for advancing scientific research and, more importantly, improving human health.
Like so many others, my family has not been immune to the effects of cancer. I lost one uncle in 2011 after a long battle with lung cancer, and another to an aggressively metastatic cancer last April. And so when I came across an article that lauded engineered bacteria as the perfect solution in the ongoing quest for an oncological panacea, I was determined to learn more.
The War on Cancer
It is a story we know all too well. A close friend or family member is diagnosed with cancer. They scour through the list of treatment options: chemotherapy, surgery, radiation, stem cell transplant, immunotherapy, active surveillance. They go in for multiple rounds of treatment. They enter remission. They relapse. [pullquote]Cancer remains the leading cause of death in Canada, predicted to claim the lives of approximately 1 in 4 men and women.[/pullquote]Treatments that appeared to work the first time are less effective the second time. They succumb to the disease, and the family is left to grieve and to wonder why cancer remains the leading cause of death in Canada, predicted to claim the lives of approximately 1 in 4 men and women.
The deadliness of cancer comes from the variation and disinhibition inherent in its development. Malignant cancer cells can arise as a result of spontaneous or environmentally-induced mutations in any number of tumour suppressor genes or proto-oncogenes. These include an extensive list of genes that regulate the cell cycle, DNA repair, immune surveillance, cell signalling, apoptosis, autophagy and more. As these mutated cells grow and divide, they upregulate genes required to mediate angiogenesis, resulting in the formation of blood vessels that provide oxygen and nutrients to the growing tumour. Some of the tumour cells are identified as aberrant and destroyed by the immune system, but this process can in turn select for those cancer cells that are able to evade the immune response. Eventually, some of the tumour cells break off from the primary site and migrate through the new vasculature or the lymphatic system to take root in the lungs, lymph nodes, bone or liver, forming the secondary tumours or metastases that prove fatal if left untreated. Current treatment options include local, targeted solutions like surgery, radiation and heat ablation, most effective when the tumour is sizeable enough and concentrated within a single location in the body. Systemic treatments like chemotherapy can then be used in combination with local techniques to remove the remaining cancer cells and prevent neoplastic growth.
The nature of cancer cells generates two main impediments to effective treatment. The first problem is one of specificity. While cancer cells accumulate mutations that change their function in significant ways, they ultimately remain similar to healthy cells, which is a significant obstacle when developing effective therapies. Many conventional treatments involving delivery of cytotoxic materials to cancer cells are not optimal due to their toxicity to the host. This issue has been addressed in recent years through the development of preliminary targeted therapies that take advantage of certain cell surface markers that are overexpressed on tumour cells compared to healthy tissue. These tumour antigens are already being targeted to great effect with various forms of immunotherapy (see article by Michael Le).
The second issue is selectivity. Removal of solid tumours is limited as a treatment option by the fact that secondary tumours are likely to form before the primary site has even reached sufficient mass for clinical detection, making it difficult to guarantee complete eradication of the malignant cells. Furthermore, the disorganized nature of tumour vasculature results in inconsistent blood flow throughout the tumour, preventing adequate distribution of chemotherapeutic drugs and generating regions with abnormally low oxygen pressure, which increases the radioresistance of the surrounding cancer cells. This is where bacteria come in.
From Cancer to Culture
The concept of using bacteria to treat cancer originated in the late 19th century, when the German physicians W. Busch and F. Fehleisen observed that cancer patients who developed erysipelas, an inflammation of the dermis caused by Streptococcus pyogenes, experienced significant tumour regression and remission. Similar observations made by American researcher Dr. William B. Coley prompted him to begin treating terminal sarcoma patients with live, laboratory-grown streptococcal cultures, and later with inoculations containing heat-killed Streptococcus pyogenes and the nosocomial pathogen Serratia marcescens. These treatments were very successful for the time and “Coley’s Toxin” remained a promising cancer therapy for several decades.
Although the direct injection of pathogens into an ailing individual may seem counterintuitive, bacteria are remarkably well suited to address the dual challenges of specificity and selectivity posed by cancer. Part of this utility derives from the ability of anaerobic bacteria to colonize the hypoxic regions of tumours that are otherwise difficult to treat. In the case of obligate anaerobes, inability to grow in the presence of oxygen ensures that bacterial infection remains limited to the hypoxic and necrotic cancer cells while leaving healthy cells intact. This tumour-colonizing behaviour has been identified in a number of bacterial species, including the pathogens Salmonella, Bacillus, and Listeria and the commensal gut microbe Bifidobacterium. Experiments conducted in the late 90s by Vogelstein et al. with the obligate anaerobe Clostridium novyi revealed specific bacterial colonization of murine B16 melanoma tumours in vivo, resulting in the death of the infected cancer cells. By using an attenuated strain of C. novyi that lacks the lethal alpha toxin (C. novyi–NT), researchers were able to decrease the mortality from infection while preserving oncolysis, and clinical trials with this strain are currently underway. One important caveat to these treatments is that malignant cells found in the oxygenated areas of the tumour remain impervious to infection and are sufficient to seed tumour re-growth, leading to relapse. Furthermore, while attenuated bacterial strains may reduce the systemic effects associated with infection, such modifications may fail to sufficiently curb toxicity to host cells while also resulting in diminished oncolytic activity.
From Culture to Cure
The key to overcoming this barrier may lie in genetically modifying bacteria to enhance existing cancer therapies. In the case of chemotherapy, bacteria can be made to express any number of drugs or pro-drug converting enzymes, which can then be delivered directly to the tumour. This combination bacteriolytic therapy (COBALT) results in greater selectivity of cancer cells and fewer off-target effects on the host. For example, using the C. novyi–NT strain in combination with liposome-encapsulated microtubule destabilizers leads to specific release of the anti-microtubule agents at the tumour site through the activity of bacterial liposomase. These agents then damage the tumour vasculature, increasing the volume of the hypoxic region and consequently increasing the area in which C. novyi–NT spores are able to germinate. Similar techniques can be used to enhance cancer cell responsiveness to radiation therapy.
[pullquote]In the case of obligate anaerobes, inability to grow in the presence of oxygen ensures that bacterial infection remains limited to the hypoxic and necrotic cancer cells while leaving healthy cells intact.[/pullquote]
One of the most promising bacterial therapies currently being pursued harnesses both innate and adaptive mechanisms of immunity. As a facultative anaerobe, Salmonella is able to colonize both aerobic and non-aerobic environments. This circumvents the problem of resistant oxygenated tumour cells, but also leaves the healthy cells open to infection. While several attenuated versions of Salmonella have been approved for clinical use, adverse effects from generalized infection remain a concern. On its own, Salmonella has been shown to kill cancer cells in mice through upregulation of the transmembrane protein Connexion 43; this protein induces formation of gap junctions between tumour cells and DCs, leading to improved antigen presentation and an enhanced T cell anti-tumour response despite pre-existing immune evasion. In an attempt to increase the specificity of Salmonella for tumour cells and decrease off-target effects on the host, Massa et al. modified the AT14 mutant of S. typhimurium to express a fusion protein consisting of the bacterial transmembrane protein OmpA and the anti-hCD20 antibody clone 2G9. They observed that cell surface expression of this clone increased specific targeting and invasion of tumour cells in vitro while preventing infection of cells that did not express the specific tumour antigen. When these bacteria were injected into mice with tumours expressing CD20, the result was not only specific targeting of tumour cells, with increased penetrance and invasion, but also decreased invasion of healthy cells, even in typical Salmonella reservoirs such as the liver. The therapeutic potential of this treatment is further heightened by its capacity to be uncoupled from the immune response entirely, as evidenced by the ability of 2G9-Salmonella bearing the ganciclovir-converting enzyme Herpes Simplex Virus tyrosine kinase (HSV-TK) to induce specific killing of CD20+ lymphoma cells in NOD/SCID mice. The ability to eradicate tumours in the absence of an immune system is an important consideration, as many patients in the advanced stages of cancer are immunocompromised.
Conclusion
With an aging population and improved early detection of neoplasia, rates of cancer diagnosis are increasing, and so too is awareness of the need for novel and creative solutions to address the limitations of current cancer treatments. Interdisciplinary scientific research will be critical in this endeavour (see article by Joseph Manion), and this is exemplified in the ongoing transformation of bacteria into the ultimate weapon against cancer.
References
- Begley, G. (2011, March 26). TEDxConejo- Dr. Glenn Begley: The Complex Biology of Cancer (or Why Haven’t We Cured It Yet?) [Video File]. Retrieved from: https://www.youtube.com/watch?v=TpALjMJEb50.
- “Cancer 101”. Canadian Cancer Society. Retrieved from: http://www.cancer.ca/en/cancer-information/cancer-101/what-is-cancer/?region=on
- Coley, WB. (1910). The Treatment of Inoperable Sarcoma by Bacterial Toxins (the Mixed Toxins of the Streptococcus erysipelas and the Bacillus prodigiosus). Proceedings of the Royal Society of Medicine. 3(Surg Sect):1-48.
- Dang, LH. et al. (2001). Combination bacteriolytic therapy for the treatment of experimental tumors. PNAS. 98(26): 15155-15160.
- Dunn, GP., et al. (2002). Cancer immunoediting: from immune-surveillance to tumor escape. Nature Immunology. 3 (11): 991-998.
- Eshel, BJ. (2013). Engineering Trojan-horse bacteria to fight cancer. Blood. 122: 619-620.
- Forbes, NS. (2010). Engineering the perfect (bacterial) cancer therapy. Nature Reviews Cancer. 10: 785-794.
- Lee, C. (2012). Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives. Applied Microbiology and Biotechnology. 93: 517-523.
- Linnebacher, M. et al. (2012). Bacterial immunotherapy of gastrointestinal tumors. Langenbecks Archives of Surgery. 397: 557-568.
- Madigan, MT. (2009). Brock Biology of Microorganisms 12th Edition. San Francisco, CA; Pearson Benjamin Cummings, Inc.
- Massa, PE. et al. (2013). Salmonella engineered to express CD20-targeting antibodies and a drug-converting enzyme can eradicate human lymphomas. Blood. 122: 705-714.
- Patyar, S. et al. (2010). Bacteria in cancer therapy: a novel experimental strategy. Journal of Biomedical Science. 17: 21-29.
Kieran Manion
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