Self-destructing bacteria to fight cancer

Earlier this year, it was discovered that the superbug Pseudomonas aeruginosa causes infection by blowing itself up, releasing virulence factors into the environment in the process. Now, US scientists have created their own self-destructing bacteria — one which they hope to deploy in the fight against cancer.

“In synthetic biology, one goal of therapeutics is to target disease sites and minimise damage,” said Jeff Hasty, a professor at University of California San Diego, who wondered if a genetic ‘kill’ circuit could be engineered to control a population of bacteria in vivo. “We also wanted to deliver a significant therapeutic payload to the disease site.”

With this in mind, Professor Hasty and his team at UC San Diego decided to synchronise a strain of Salmonella to release bursts of cancer drugs when a colony self-destructs within the tumour environment. The concept of using bacteria to deliver cancer drugs in vivo was promising because conventional chemotherapy doesn’t always reach the inner regions of a tumour, but bacteria can colonise there.

“One of the difficulties in treating cancer is the fact that tumours often have poor blood supply, meaning that it is difficult for chemotherapy drugs to reach them,” commented Dr Tom Williams from Macquarie University’s synthetic biology consortium. “On the other hand, poor blood supply means that tumours don’t receive much oxygen. This can provide a means of distinguishing tumour cells from normal healthy tissue so that cancer treatments can be targeted and have fewer side effects.”

Writing in the journal Nature, the study authors noted, “The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body. It is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbour disease and thus provide a natural platform for the development of engineered therapies.”

The researchers hypothesised that such therapies could “benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents” in situ. They thus began by observing cycling of the bacterial population that limits overall growth while simultaneously enabling production and release of encoded cargo — a gene that drives production of a therapeutic.

A schematic of the microfluidic device used to co-culture engineered bacteria and cancer cells (top). This device was used to test if the bacteria could release toxic proteins to kill cancer cells via synchronised lysis. The bacteria and cancer cells are localised to grow in fluidically connected compartments. Cancer cells were made to adhere inside the main channel, while the bacteria were trapped in smaller growth chambers connected to the sides of the channel. The two images (bottom) show live co-culture of engineered bacteria and cancer cells via time lapse fluorescence microscopy, immediately before and after the synchronised lysis event. The bacteria release a toxic protein and cancer cell death is evident after lysis. Image credit: Jeff Hasty, UC San Diego.

“In this paper, we describe a circuit that contains a gene that codes for a small molecule that can diffuse between cells and can turn on genes,” said lead author and UC San Diego PhD student Omar Din. “Once the population grows to a critical size — a few thousand cells — there’s a high enough concentration of that molecule present in the cells to cause mass transcription of the genes behind the promoter.”

The molecule, AHL, coordinates gene expression across a colony of bacterial cells. Once on, the genes driven by the promoter are also activated, including the AHL-producing gene itself. The more AHL accumulates, the more it is produced. And because AHL is small enough to diffuse between cells and turn on the promoter in neighbouring cells, the genes activated by it would also be produced in high amounts. This leads to a phenomenon known as quorum sensing, used by bacteria to communicate with each other about the size of their population and regulate gene expression accordingly. Din used quorum sensing to synchronise the cells and then added a kill gene that causes cells to break open (lyse) when a bacterial colony grows to a threshold. Only a few cells remain to repopulate the colony.

In order to find the right drug for delivery by the bacteria, the researchers tested three different therapeutic proteins that had been shown to shrink tumours. The tests showed that the proteins were most effective when combined. The scientists placed the genes responsible for these proteins in the circuit, along with the lysis gene, then conducted experiments that showed enough protein was produced to kill cancer cells.

The bacterial therapy was transferred to UC San Diego alumnus and postdoctoral researcher Tal Danino, based at the Massachusetts Institute of Technology (MIT), for testing in an animal model. The bacteria were first injected into mice with a grafted subcutaneous tumour, resulting in a decrease in tumour size. Danino then used a more advanced mouse model with liver metastases, where bacteria were fed to the mice.

“We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour activity along with a marked survival benefit over either therapy alone,” the researchers said. And while this new approach has not yet cured any mice, the therapy has led to around a 50% increase in life expectancy — though it is difficult to anticipate how this would translate to humans.

The experiments thus establish a proof-of-principle for using the tools of synthetic biology to engineer tumour-targeting bacteria to deliver therapeutic proteins in vivo. The next possible steps include investigating the natural presence of bacteria in tumours and then engineering these bacteria for use in vivo and using multiple strains of bacteria to form a therapeutic community.

“Additionally, we are currently investigating methods for maintaining the circuit inside bacteria,” said Din. “Since the proteins produced by the circuit put a burden on the bacteria, the bacteria are prone to mutate these genes.

“Additionally, there is a selection pressure to get rid of the plasmids which harbour the genes comprising the circuit. Thus, one of our future research aims is to identify strategies for stabilising the circuit components in bacteria and decreasing their susceptibility to mutations.”

While further work will be required to make this therapy ready for application in humans, fellow scientists have been quick to voice their support. Dr Williams, for instance, said the treatment “represents a creative and promising weapon in the ongoing fight against cancer”. Cancer genomics pioneer Bert Vogelstein, director of the Ludwig Center at Johns Hopkins, added that the paper describes “a highly innovative strategy” which is “just the kind of new, forward-thinking approach that we desperately need if we are to more effectively combat cancer”.

But perhaps the highest praise has come from Jim Collins, a professor at MIT and co-founder of the field of synthetic biology, who said the work by Professor Hasty and his team is “a brilliant demonstration of how theory in synthetic biology can lead to clinically meaningful advances”.

“Over a decade ago, during the early days of the field, Jeff developed a theoretical framework for synchronising cellular processes across a community of cells,” said Collins. “Now his team has shown experimentally how one can harness such effects to create a novel, clinically viable therapeutic approach.”

Top image caption: This image shows a motherboard image micropatterned using programmable probiotic bacteria. The bright lines are composed of dots made of bacteria. Researchers delivered artificial genetic circuits into the bacteria which allow the microbes to kill cancer cells in three different ways. Image courtesy of Vik Muniz and Tal Danino under CC BY-NC-ND 3.0