Is it reliable to use parasites to treat tumors?

Studies by multiple research groups have shown that infection with Toxoplasma gondii can enhance anti-tumor immune responses in mice. Can single-cell parasites really be developed into safe and effective therapeutic drugs?

By Annie Melchor

Compiled by Yu Tao (Indiana University School of Medicine)

We all know that the human body has a complex and efficient immune system that can not only resist the invasion of pathogens, but also kill "bad" cells in the cradle. At the same time, the human body is also equipped with a brake system called "checkpoints" for the immune system, which can prevent the immune response from being accidentally activated or lasting too long. The cunning tumor cells have learned to "step on the brakes" - they can activate the brake system and suppress the immune system's attack, so that they can be domineering in the patient's body.

Figure 1. The relationship between the immune system, tumor cells, and the brake system. 丨Adapted from the Internet

In recent years, scientists have found many brake proteins on immune cells and developed corresponding antibodies that can bind to these brake proteins and inhibit brake signals. When these antibodies are injected into tumor patients, the inhibitory effect of tumor cells on immune cells can be relieved and the activity of immune cells can be reactivated, thus achieving an anti-tumor effect. This is the "tumor immune checkpoint blockade therapy" that has emerged in recent years.

However, as more and more clinical trials are conducted, doctors have found that blocking therapy does not work well for some types of tumor patients, but the reasons are unknown. Unexpectedly, a parasite in the brain seems to have brought a glimmer of hope to solve this problem.

Crazy Ideas

Using parasites to fight cancer may sound dangerous and crazy, but similar ideas have been around for more than a century.

Around the turn of the 20th century, there was a cancer surgeon in New York named William Coley. While reading old medical records, he made an amazing discovery: a terminal cancer patient who would have died seven years ago was still alive today. An inconspicuous record in the medical record caught his attention: the patient had been infected with a bacteria. At that time, such a trivial matter would not attract the attention of doctors. But Dr. Coley made a very bold guess: Could it be that this bacterial infection cured the patient's cancer?

So he immediately began to experiment, injecting his cancer patients with live or inactivated bacteria. After being infected with bacteria, some patients' tumors shrank and they survived, but not all patients were so lucky. After a long period of experimentation, Dr. Coley finally invented a cancer treatment vaccine containing inactivated bacteria, which can induce an inflammatory response after injection, enough to kill tumor cells. Because the vaccine contains inactivated bacteria, it does not pose any risk of bacterial infection.

This inactivated bacteria is called "Coley toxin".

Figure 2. Left: The infection caused by Coley's toxin liquefied the tumor of the patient on the left within a few days; Right: The patient on the right received 63 injections before the tumor shrank. Some tumor patients died. It can be seen that the effect of Coley's toxin is unpredictable. (Source: Cancer Research Institute/Proceedings of the Royal Society of Medicine 01/1910/3 (Surg Sect): 1-48)

Over time, however, Coley's toxins fell out of favor with doctors. "A lot of people tried to replicate Coley's work, but it didn't work very well," says Steven Fiering, a tumor immunologist at Dartmouth's Geisel School of Medicine. "Radiation therapy quickly replaced Coley's toxins. But the overall idea of ​​boosting your own immune system to fight cancer has never been ignored."

Brain parasites to treat cancer

Toxoplasma gondii is a parasite smaller than a cell. It can infect host cells, multiply inside them and produce offspring. Cats are the only definitive hosts of Toxoplasma. Toxoplasma produces a large number of oocysts in cats, which flow into the soil or water with cat feces and infect a large number of hosts. In fact, Toxoplasma can infect almost all warm-blooded animals and exist for a long time in the skeletal muscles and brains of infected animals.

Figure 3. It is easy to be infected with Toxoplasma gondii by contacting cat feces. Source: Internet

Although Toxoplasma has adopted a clever immune evasion strategy when infecting the host, it can also trigger a strong immune response at certain stages of its life cycle. In the early stages of infection, Toxoplasma replicates rapidly in the form of "tachyzoites". At this time, if the host dies or is eaten by a predator, Toxoplasma will be killed when passing through the predator's digestive tract and cannot infect a new host because Toxoplasma is not yet safely in the cyst.

Therefore, in order to ensure that the host is not killed in the tachyzoite stage, Toxoplasma will trigger a strong immune response in the host in the early stages of infection to control its own replication and amplification level. Specifically, after the tachyzoites invade the cells, they rapidly multiply in large numbers and burst out of the cells, causing cell death and triggering systemic and local inflammation. Generally, after a few weeks, Toxoplasma that survives the initial inflammatory outbreak will hide in long-lived cells in skeletal muscle or the brain.

Here, the parasite transforms into a slow-replicating form, the bradyzoite, and builds a "wall" of sugar molecules to protect itself from the host's immune system (see the cyst in Figure 4). Although occasionally, the parasite-filled cysts will rupture and cause local inflammation, most parasites can spend the rest of their lives in these cysts. After this, if a predator ingests the tissue containing the cysts, the parasite can successfully infect a new host.

Figure 4. Different forms of Toxoplasma gondii. a. Tachyzoites, b. Schematic diagram of the internal structure of tachyzoites, c. Cysts, which are filled with a large number of bradyzoites, which are similar in shape to tachyzoites, but smaller in size. 丨Image source:
https://www.jotscroll.com/forums/11/posts/204/toxoplasma-gondii-life-cycle-parasite-transmission-diagnosis-test.html

"About a third of the world's population may have been infected with the parasite," said Pascale Guiton, a microbiologist and expert on Toxoplasma at California State University, East Bay. "For most people, infection with Toxoplasma tends not to cause symptoms. However, for immunocompromised individuals, pregnant women and developing fetuses, infection with Toxoplasma can be fatal. There is currently no vaccine or treatment for chronic toxoplasmosis."

In the 1960s and 1970s, scientists discovered that Toxoplasma infection can enhance the immune resistance of mice to cancer and various infections. In the following decades, there has been increasing evidence that Toxoplasma infection may help treat cancer. A study published in the Journal for Immuno Therapy of Cancer in November 2021 found more direct evidence: injecting Toxoplasma directly into mouse tumors can greatly promote the efficacy of immune checkpoint blockade therapy.

In situ tumor vaccination

The treatment method of injecting immunostimulants (such as Coley toxins and Toxoplasma gondii) directly into the tumor is called in situ vaccination. According to Fiering, tumor cells often deploy a series of self-protection strategies to weaken the body's anti-tumor immune response, and in situ vaccination can reactivate the immune response suppressed by the tumor, similar to an adjuvant. It can break the local immunosuppression of the tumor and activate or recruit more tumor-specific T cells. These T cells can not only kill local tumor cells, but also kill tumor cells that have metastasized to other parts in the early stages-at this stage, clinicians usually find it difficult to detect signs of tumor metastasis.

In addition to Coley's toxins, doctors have tried many other in situ vaccines, such as attenuated Listeria, or nanoparticles that encapsulate/carry pathogen antigens. In 2016, Flin's research team found in a mouse model that nanoparticles containing inactivated bean mosaic virus (a plant pathogen) can inhibit the growth of various tumors such as ovarian cancer, colon cancer, and breast cancer. The oncolytic virus T-Vec, which was approved by the FDA in recent years, has also shown good results in the treatment of advanced melanoma.

Figure 5. Schematic diagram of oncolytic virus injection | Source:
https://www.drugtargetreview.com/article/76602/developing-an-effective-vaccine-for-breast-cancer/

Compared to many other in situ vaccination options, the type of immune response triggered by Toxoplasma gondii is unique. This is why cancer scientists continue to be interested in Toxoplasma gondii. Christopher Hunter, an immunoparasitologist at the University of Pennsylvania, explains: "Each pathogen has its own characteristics, and our bodies will produce different types of immune responses to different pathogens. The mechanisms by which the immune system responds to intracellular infection with Toxoplasma gondii are the same as the mechanisms by which the body fights some cancers. For example, both can trigger strong T cell responses and induce cytokines such as interleukin 12 and interferon gamma."

In addition, using Toxoplasma as an in situ vaccine for tumors has a huge advantage: they can infect a variety of cells and tissues, are not picky about tumor types, and scientists can test them in a variety of model organisms.

“When Toxoplasma gondii are introduced into a tumor, they actually stimulate the anti-tumor immunity that you would hope to see,” says David Bzik, a parasite immunologist at the Geisel School of Medicine at Dartmouth. “It reawakens immunity that had been suppressed by the tumor.”

Ideas become reality

In 2010, Bzik's laboratory knocked out a key enzyme that synthesizes pyrimidine in Toxoplasma, creating a Toxoplasma that cannot replicate. It can grow and replicate normally in a culture medium supplemented with uracil, but cannot replicate in host animals. Like vaccines made from non-replicable viral vectors, this defective strain can be used as a vaccine to prevent Toxoplasma infection.

In 2013, the laboratories of Bzik and Fiering jointly found that injecting this defective Toxoplasma into an ovarian cancer mouse model can significantly increase the number and activity of tumor-infiltrating T cells. They also found that if these activated T cells are extracted and injected into other tumor-bearing mice, they can also exert anti-tumor effects. Some other studies have also found that defective Toxoplasma can also play a good role in pancreatic cancer and melanoma mouse models.

Hany Elsheikha, a veterinarian at the University of Nottingham, and collaborators in China further confirmed Bzik's findings in their new paper. They used a replication-weakened strain of Toxoplasma gondii (different from Bzik's strain) in multiple tumor-bearing mouse models and found that this strain can also promote the invasion of cytotoxic T cells and natural killer cells into tumors and enhance the effects of immune checkpoint blockade therapy. In other words, the combination of replication-weakened strains and immune checkpoint inhibitors can shrink tumors more effectively than using inhibitors alone. However, the combination therapy is only effective when using live parasites, and the use of heat-killed parasites has no effect at all. At the same time, in a mouse model with two tumors, the replication-weakened strain was injected into one of the tumors and combined with treatment, and the other tumor in the mouse was significantly reduced! This shows that this therapy may also have therapeutic potential for distant metastatic tumors.

Bizik believes that this study has important implications, especially for patients with advanced cancer. Many patients are not diagnosed until the cancer has metastasized, and it is almost impossible to remove all the tumors at this time. But Bizik said that this study gives us new hope that combination therapy can also have a significant therapeutic effect on distant tumors, so that we can target metastatic tumors while treating the original tumor.

Of course, things won’t always go so smoothly. Using Toxoplasma as an in situ tumor vaccine faces a huge technical hurdle.

Since vaccines can only use living Toxoplasma, and Toxoplasma can only grow and reproduce inside host cells, it is also necessary to rely on cell culture when culturing Toxoplasma in the laboratory, which greatly increases the difficulty and cost of obtaining Toxoplasma. In fact, after resuscitating fresh cells in the laboratory, it takes several days of culture to obtain Toxoplasma of sufficient titer, which is not feasible in ordinary clinics. Filin once discussed the potential of using Toxoplasma as an immunotherapy with clinicians at Dartmouth: "We need something that can be taken out of the refrigerator and injected directly into the patient, rather than something that must be recovered from live cell culture every few days. Even if this is theoretically possible, it is impractical from a clinical perspective."

Technical challenges aside, many scientists are skeptical that injecting patients with live parasites (even attenuated strains) — especially ones that are potentially threatening to immunosuppressed patients — can really find a place in the clinic.

Toxoplasma as a tool

In fact, a Toxoplasma-based treatment has never been El Sheha's goal. "We will not promote it as a treatment," he said. "Our research goal is to figure out the mechanism of Toxoplasma infection and activation of immune response." He sees Toxoplasma as more than just a pathogen, but as a powerful tool for understanding basic biological mechanisms. Since Toxoplasma can force target cells to change their normal biological properties, he wondered if this could be used creatively - "This is exactly what we are trying to figure out in our research."

Toxoplasma has another huge advantage: its genetic tractability.

Toxoplasma has a long history of genetic modification. Unlike many other model organisms, it is relatively easy to manipulate the genes of Toxoplasma, and immunologists and microbiologists can easily use Toxoplasma as a tool to solve immune system problems. Hunter discovered the mechanism by which interleukin 27 suppresses immune response during Toxoplasma infection, and a phase 1 clinical trial based on this research result is already underway. The research team is looking forward to seeing whether they can relieve tumor immunosuppression by blocking interleukin 27 in advanced solid tumors.

El Sheha’s recent findings may tell us what type of immune response is needed to fight tumors. From here, researchers will next need to figure out how to stimulate such an immune response in tumors without using live Toxoplasma gondii.

The current study also found an important clue, that is, dead parasites do not cause the same reaction. This means that it may be a protein secreted by live Toxoplasma that enhances the anti-tumor immune response, while the constitutive protein on its surface does not have such a function. If we can identify this secreted protein, we are not far from our goal.

In addition, from the perspective of cancer biology, it is of utmost importance to understand how infection promotes immune checkpoint blockade therapy. "All current FDA-approved treatments use immune checkpoint inhibitors," said Bzik. "But scientists and clinicians don't know why immune checkpoint blockade therapy doesn't work as well in some patients (actually most patients) or in certain types of cancer. If researchers can figure out how "infection" overcomes tumor immunosuppression, they may find ways to improve efficacy."

Guiton added: "Using parasites to treat diseases is not unheard of, but we can't use it widely at present because we don't understand parasite biology well enough. This is partly due to insufficient government funding for basic parasitology research, especially in countries that are less affected by parasites. If we want to help people by exploring these parasites, such as curing more cancer patients, we really need to study them in depth."

This article is authorized to be translated from TheScientist.com. The original title is Turning Toxoplasma Against Cancer. The pictures are added by the translator. The original text can be viewed by clicking "Read Original" at the end of the article.

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