To keep immune cells from reacting to inappropriate triggers or dragging out their response for too long, they’re equipped with sundry molecular safeguards called immune checkpoints that nearby immune cells can flip on. As important as these checkpoints are, many tumors take advantage of them, throwing on these molecular brakes to dampen the antitumor immune response. A common form of immunotherapy called immune checkpoint blockade, or checkpoint inhibition, seeks to counter this kind of immunosuppression by physically blocking the immune checkpoint molecules so they can’t relay inhibitory signals. But while this treatment is successful in some patients, it fails in many others, and scientists don’t entirely know why—or more importantly, how to overcome the tumor’s immunosuppression. Key clues to solving this mystery are coming from an unexpected source: the brain-dwelling parasite Toxoplasma gondii.
The idea of tackling cancer with a brain parasite arose in the 1960s and 1970s when scientists observed that Toxoplasma infection boosted murine immune resistance to several infections and diseases, including cancers. In the decades since, evidence that Toxoplasma infections could aid in cancer treatment has been mounting. And a study published in November in the Journal for ImmunoTherapy of Cancer found that treating mice with a checkpoint inhibitor while injecting Toxoplasma directly into a tumors shrank both the injected tumor and tumors in other parts of the body better than when the mice were treated with checkpoint inhibitors alone.
The findings are “really intriguing, [and] potentially important down the road, because there’s been a lot of interest and hype and commercialization of checkpoint blockade therapy,” says David Bzik, an immunoparasitologist at Dartmouth’s Geisel School of Medicine who has spent the past decade exploring the immunology of Toxoplasma infections. He adds that these results may help make checkpoint inhibitor therapy more effective in patients who don’t initially respond to it.
But experts in the field, including the new study’s authors, note that challenges remain, particularly with regards to actually implementing a live parasite–based therapy. Still, they say that even if such a treatment doesn’t pan out, Toxoplasma may ultimately end up helping cancer patients.
“I think you can use this system to establish principles that may lead to better therapies,” says Christopher Hunter, an immunoparasitologist at the University of Pennsylvania who was not involved with the study. “If you can understand what this parasite is doing that other immunotherapies aren’t doing, then maybe you can leverage that knowledge to develop new anticancer therapies.”
A brain parasite to treat cancer
Toxoplasma gondii is a single-celled eukaryotic parasite that can only replicate inside host cells. It’s often found in soil or water—shed from cat feces—or in meat from infected animals. Unlike some pathogens that only infect certain cell types within select organisms, “Toxoplasma is a pretty ubiquitous parasite,” says Pascale Guiton, a microbiologist who studies Toxoplasma at California State University, East Bay, and was not involved with the new study. “It can infect virtually all warm-blooded animals, and can infect virtually all nucleated cells. After entering a host cell, the parasite forms a protective vacuole that houses the replicating parasites until they’re ready to burst out of the host cell—killing it in the process—and infecting new cells to replicate in. Once it infects an animal, Guiton notes, the parasite usually hunkers down in skeletal muscle and brain cells for the rest of the host’s life.
According to Guiton, one-third of the world’s human population is believed to be infected with the parasite. For most people, Toxoplasma is fairly innocuous and asymptomatic; however, for the immunocompromised, pregnant people, and developing fetuses, the infection can be fatal, as there is currently no vaccine or treatment for chronic toxoplasmosis.
Using a potentially dangerous parasite to fight cancer may seem like throwing a patient out of the frying pan and into the fire, but the idea of using pathogens to treat cancer dates back over a century.
Around the turn of the twentieth century, New York–based cancer surgeon William Coley made a surprising discovery while reading through old patient records: seven years earlier, a terminal cancer patient had contracted a bacterial infection. This may seem unremarkable, except for the fact that the patient, who should have succumbed to cancer years before, was still alive and doing well. Coley hypothesized that something about the bacterial infection had caused the tumor to shrink, so he started to experiment by injecting his own cancer patients with living or dead bacteria. If patients survived the infection (not all did), they often saw their tumors shrink and their prognoses improve. Eventually, Coley standardized his treatment into a vaccine comprised of dead bacteria known as Coley’s toxin. By injecting patients with it, he could induce cancer-killing inflammation without risking infection with live pathogens.
Over time, though, Coley’s toxin fell out of favor with doctors as a cancer treatment. “A lot of people tried to reproduce Coley’s work, and they didn’t get it working very well,” says Steven Fiering, a tumor immunologist at Dartmouth’s Geisel School of Medicine. He adds that the advent of radiation as a cancer treatment quickly superseded Coley’s toxin in popularity. But the general idea of boosting the immune system to fight cancer persisted.
Delivering immune-stimulating treatments directly into tumors has come to be known as in situ vaccination. According to Fiering, the vaccination acts as an adjuvant to jumpstart an antitumor immune response that has often been weakened by a number of self-protection strategies tumor cells deploy. Fiering says the idea is to break the local immunosuppression at the tumor site and to generate tumor-specific T cells that can recognize and kill metastasized tumors elsewhere in the body even before clinicians can detect them.
Other than Coley’s toxin, researchers have explored in situ vaccination strategies such as injecting the tumor with attenuated Listeria monocytogenes, the bacteria that causes the foodborne disease listeriosis, or nanoparticles coated in or filled with pathogen-derived antigens. For instance, in 2016, Fiering’s group demonstrated that nanoparticles packed with a killed plant pathogen called cowpea mosaic virus could suppress metastatic tumor growth in multiple mouse cancer models, including ovarian, colon, and breast cancer. And a therapy called T-Vec, based on an oncolytic virus injected into a tumor, is in use to treat advanced melanoma.
One reason to look at Toxoplasma in addition to these other in situ vaccination contenders is the type of immune response it elicits. “Pathogens come in different flavors,” explains Hunter. “We need different types of immunity to fight different types of infections.”
Unlike the type of immunity that’s optimized for fighting bacteria or parasitic worms, the mechanisms the immune system uses to fight intracellular infections such as Toxoplasma are the same pathways you want to engage to fight cancer, Hunter says, including a strong T cell response and cytokines such as interleukin-12 and interferon-gamma.
“When [Toxoplasma is] introduced into tumors, it really drives the exact responses that you want to see to eliminate them,” says Bzik. “You reawaken that immunity and you reverse the immune suppression.”
Furthermore, although the parasite employs clever immune evasion strategies, Toxoplasma is also known for triggering the immune system in a counterintuitive attempt at self-preservation. That’s because the parasite needs its host to survive long enough for it to reach the stage in its lifecycle where it’s ready to be passed on. At the beginning of a Toxoplasma infection, the parasite exists in a quickly replicating form called a tachyzoite. Tachyzoites invade cells, replicate, and then burst out, causing cell death and massive systemic and local inflammation. After a few weeks, parasites that survive the initial burst of inflammation hide out in long-lived cell types like those in skeletal muscle and the brain. Here, the parasites transition to a slowly replicating form called a bradyzoite, and construct a wall of sugars to protect them from host immune defenses. Occasionally a parasite-filled cyst will rupture, causing local inflammation, but otherwise, the parasite can survive in cyst form for the rest of the host’s life. Then, if tissues containing the cysts are ingested by a new host, Toxoplasma can spread.
If the host dies when the parasite is in the tachyzoite form, the parasite won’t be transmitted, because this form dies in the digestive tract. It’s only when the parasite is safely housed inside a cyst that Toxoplasma is stable enough to spread when its host is consumed. To ensure both its progeny and its host survive long enough, Toxoplasma actually reins in its own replication during the early stages of infection by triggering a robust immune response (albeit not so robust that the tachyzoites are all destroyed).
Something else that makes Toxoplasma attractive for in situ vaccination is its versatility in invading different cells and tissue types. The parasite isn’t picky about invading tumor tissue and can be tested in multiple model organisms.
In 2010, in a bid to modify Toxoplasma to use as a vaccine against toxoplasmosis, Bzik and his lab reported knocking out a key enzyme for synthesizing pyrimidines in order to create a strain of Toxoplasma that couldn’t replicate. The parasites could grow normally in medium supplemented with uracil, but couldn’t replicate in a mammalian host where the RNA base wasn’t freely available to scavenge. Then, in 2013, Bzik, Fiering, and their colleagues found that infection with this strain significantly boosted the number and activity of tumor-infiltrating T cells in a mouse model of ovarian cancer. In fact, just injecting T cells from infected, tumor-bearing mice were enough to significantly suppress tumor growth in other cancer-struck but uninfected mice. Other studies showed the parasite was also effective in mouse models of pancreatic cancer and melanoma.
Veterinarian Hany Elsheikha at the University of Nottingham and his colleagues in China took these findings a step further in the new paper by showing that infection with a replication-attenuated strain of Toxoplasma (different from the strain Bzik’s lab developed) sensitized various types of tumors to checkpoint inhibition, leading to an influx of CD8+ T cells and natural killer cells that killed the cancer cells and shrank the tumor much more effectively than the checkpoint inhibitor alone. The dual therapy was only effective when the mice were treated with living parasites—injection with heat-killed parasites had no effect on tumor shrinking. The researchers also found that when they gave the mice two tumors, treating the animals systemically with the checkpoint inhibitor but only injecting one of the tumors with the parasite, the other tumor still shrank.
Bzik says he finds the results intriguing, particularly in the context of treating later-stage cancers. He notes that the unfortunate reality of cancer therapy is that many patients are not diagnosed until their cancer has already metastasized, making it nearly impossible to treat or remove all the tumors. But with this new observation that the dual therapy also attacks distal tumors in mice, it might be possible to target a primary tumor with a treatment that would also hit metastasized tumors.
Despite some promising results, Fiering says he ended up shifting away from using Toxoplasma as an in situ cancer vaccine a few years ago because of a formidable technical hurdle. Since Toxoplasma can only grow and reproduce inside host cells, growing it in culture requires a single layer of cells in a flask or petri dish that the parasites can infect and reproduce inside. The parasites can be harvested out of the host cells and frozen for long-term storage, then thawed and revived in a flask of fresh cells, but it takes a few days for the parasite numbers to bounce back to a useful titer.
According to Fiering, this just wouldn’t be feasible in the clinic. He recalls talking with clinicians at Dartmouth about the potential for using Toxoplasma as an immunotherapy. “‘We need something that we can take out of the freezer or the refrigerator or off the shelf and inject into the patient,’” he remembers them saying, not something that has to be recovered from live cell culture every few days. It’s not impossible to make it work, he says, but it’s also not practical from a clinical standpoint.
Technical challenges aside, all of the researchers who spoke with The Scientist were doubtful that a therapy involving injecting patients with a live parasite—especially one that is particularly dangerous in immunosuppressed patients—would ever take hold in the clinic, even with an attenuated version.
Toxoplasma as a tool
A Toxoplasma-based treatment was never Elsheikha and colleagues’ plan, however. “We’re not promoting this as a therapeutic,” says Elsheikha. Instead, he says, the goal of their research is to figure out what exactly it is about Toxoplasma infection that was so useful at overcoming resistance to checkpoint inhibition and shrinking tumors.
Rather than simply a pathogen, Elsheikha sees Toxoplasma as a powerful tool to understand fundamental biological mechanisms. He wondered if Toxoplasma’s ability to force target cells to change their normal biology could be harnessed and exploited in an innovative way. “This is exactly what we have done in our study,” he says.
Another huge upside of Toxoplasma is its genetic tractability. “Unlike a lot of other model organisms, Toxoplasma has a long history of scientists being able to genetically modify it,” says Hunter. “It’s relatively easy to genetically manipulate compared to lots of other parasites.” He says this makes it easy for immunologists and microbiologists to use the parasite as a tool to answer questions about the immune system, such as by knocking out certain genes to nail down which pathways are important for antitumor immunity. For instance, he points to work his group has done showing how the cytokine interleukin-27 (IL-27) inhibits the immune response during Toxoplasma infection, which has led to phase 1 clinical trial blocking IL-27 in advanced solid tumors.
According to Guiton, the results of Elsheikha’s recent study can tell us what type of immune response is needed to fight those tumors, and from there, the researchers can start to work backward to answer the big question: “How do we recapitulate that response in the absence of a full-blown parasite?”
One clue comes from the group’s observation that dead parasites didn’t elicit the same reaction, suggesting that it may be a protein secreted by the parasite that ramps up the immune response, rather than proteins decorating its surface.
Additionally, understanding how the infection boosted the checkpoint blockade therapy is key from a cancer biology perspective, says Bzik. “There’s all these FDA-approved treatments using checkpoint blockade. But the clinicians don’t know why some patients—and even lots of patients, in certain cancers—don’t respond,” he says, adding that if researchers could figure out how the infection overcame the tumor’s immunosuppression, they’d likely find ways to improve checkpoint blockade therapy.
Guiton says it’s not unheard of to exploit parasites for therapeutic benefit, “but the reason why we cannot exploit them even more is because we don’t have enough understanding of the biology.” That lack of insight stems in part from the lack of funding for basic parasitology research, especially in countries that aren’t heavily affected by parasites, she says.
“For us to be able to explore these parasites for other ends like treating cancer,” she says, “we really need to study them and understand what’s going on.”