Salmonella directs brain tumors to self destruct
Biomedical engineers at Duke University have recruited an unlikely ally in the fight against the deadliest form of brain cancer — a strain of salmonella that usually causes food poisoning. The new approach produces a 20 percent survival rate in the rat model tested, typically few live.
Clinicians sorely need new treatment approaches for glioblastoma, the most aggressive form of brain cancer. The blood-brain barrier, a protective sheath separating brain tissue from its blood vessels, makes it difficult to attack the disease with drugs. It's also difficult to completely remove glioblastoma through surgery, as even tiny remnants inevitably spawn new tumors.
Even with the best care available, median survival time for glioblastoma is 15 months. Only 10 percent of patients survive five years after diagnosis.
The Duke team decided to pursue an aggressive treatment, turning to the bacteria Salmonella typhimurium. With a few genetic tweaks, engineers turned the bacterium into a cancer-seeking missile that produces self-destruct orders deep within tumors. Tests in rats with extreme cases of glioblastoma showed a 20 percent survival rate over 100 days — roughly equivalent to 10 human years — with tumors going into complete remission. Results appeared online on December 21, 2016, in the journal Molecular Therapy - Oncolytics.
"Since glioblastoma is so aggressive and difficult to treat, any change in the median survival rate is a big deal. And since few survive a glioblastoma diagnosis indefinitely, a 20 percent effective cure rate is phenomenal and very encouraging."
Jonathan Lyon post doctoral student working with Ravi Bellamkonda and Vinik Dean, Duke's Pratt School of Engineering, currently transitioning his laboratory to Duke from Georgia Tech, where much of the work was completed.
Previous studies have shown, quite accidentally, that the presence of bacteria can cause the immune system to recognize and begin attacking tumors. However, follow-up clinical trials with genetically detoxified strains of S. typhimurium have since proven ineffective by themselves.
To use common intestinal bacteria as tumor-seeking missiles, Lyon and Bellamkonda along with lead Nalini Mehta, selected a detoxified strain of S. typhimurium deficient in the enzyme purine — forcing bacteria to find purine elsewhere.
Tumors happen to be an excellent source of purine, causing bacteria to flock to them in droves.
Duke engineers then made a series of genetic tweaks so that bacteria would produce two proteins — Azurin and p53 — instructing cells to commit suicide in the presence of low oxygen levels. Because cancerous cells multiply rapidly, the environment within tumors is low in oxygen.
"A major challenge in treating gliomas is that the tumor is dispersed with no clear edge, making them difficult to completely surgically remove. So designing bacteria to actively move and seek out these distributed tumors, and express their anti-tumor proteins only in hypoxic, purine rich tumor regions is exciting.
"And because their natural toxicity has been deactivated, they don't cause an immunological response. At the doses we used in the experiments, they were naturally cleared once they'd killed the tumors, effectively destroying their own food source."
Ravi Bellamkonda PhD, and Vinik Dean, Duke University, Pratt School of Engineering, Durham, North Carolina, USA and corresponding author of the paper.
Researchers tested the modified bacteria by injecting them directly into the rats' brains. While this may sound like an extreme delivery option, the first course of action usually performed with glioblastoma is to surgically remove the primary tumor, if possible, leaving the opportunity to directly deliver therapeutics.
The treatment worked in 20 percent of the rats, causing complete tumor regression and extending their lives by 100 days, which translates to roughly 10 human years.
In the 80 percent that did not survive, however, the treatment didn't change the length of their survival. After testing for common signs of resistance to the anti-tumor compounds and finding none, researchers concluded the ineffectiveness was likely due to inconsistencies in the bacteria's penetration, or to the aggressive tumor growth outpacing the bacteria. But every rat showed initial signs of improvement after treatment.
"It might just be a case of needing to monitor the treatment's progression and provide more doses at crucial points in the cancer's development.
"However, this was our first attempt at designing such a therapy, and there is some nuance to the specific model we used, thus more experiments are needed to know for sure."
Jonathan Lyon PhD
Researchers now plan to program their bacteria to produce different drugs that cause stronger reactions in the tumors. These will be more difficult to implement, however, as other drugs are not as specific to tumor cells as those used in this study, making potential side effects more of a concern.
Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these barriers, bacterial carriers that are actively motile, and programmed to migrate and localize to tumor zones were designed. These carriers can induce apoptosis via hypoxia-controlled expression of a tumor suppressor protein p53 and a pro-apoptotic drug, Azurin. In a xenograft model of human glioblastoma in rats, bacterial carrier therapy conferred a significant survival benefit with 19% overall long-term survival of >100 days in treated animals relative to a median survival of 26 days in control untreated animals. Histological and proteomic analyses were performed to elucidate the safety and efficacy of these carriers showing an absence of systemic toxicity, and a restored neural environment in treated responders. In the treated non-responders, proteomic analysis revealed competing mechanisms of pro-apoptotic and drug resistant activity. This bacterial carrier opens a versatile avenue to overcome diffusion barriers in glioblastoma by virtue of its active motility in extracellular space, and can lead to tailored therapies via tumor-specific expression of tumoricidal proteins.
"Bacterial Carriers for Glioblastoma Therapy." Nalini Mehta, Johnathan G. Lyon, Ketki Patil, Nassir Mokarram, Christine Kim, Ravi V. Bellamkonda. Molecular Therapy - Oncolytics, 2016. DOI: 10.1016/j.omto.2016.12.003
This research was supported by the Ian's Friends Foundation, Ann Rankin Cowan, Children's Healthcare of Atlanta and Georgia Research Alliance.
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A fluorescent stained image of a tumor marking bacterial nanocarriers in pink,
cancer cell nuclei in blue, and human mitochondria (another indicator of tumor cells) in green.
Image Credit: Duke University