Black-sheep immune cells activated to eliminate tumors in laboratory mice

Elevated levels of immune cells, called neutrophils, in tumors have been associated with poor outcomes in people with cancer. But a study led by researchers at Stanford Medicine have found that, in mice, these cells can be transformed from black sheep into potent cancer fighters through an injection directly into the tumor site.

The treatment eliminated existing tumors and reduced the number and size of subsequent metastases in mice with cancers of the skin, lung, breast and colon. It also activated human neutrophils to kill human cancer cells in a laboratory dish, suggesting the approach could be useful in treating people with many types of cancers.

The researchers injected three agents simultaneously into the tumors: one that summons neutrophils to the tumor, another that amps up neutrophils’ cell-killing capacity, and a third that helps them locate and latch onto cancer cells. In initial studies, two treatments, given two days apart, eliminated melanoma tumors in seven out of eight mice, and the mice remained cancer free until the end of the study five months later. In contrast, untreated animals experienced uncontrollable tumor growth and were euthanized within one month.

“These are striking findings,” said Edgar Engleman, MD, a professor of pathology and of medicine at the Stanford School of Medicine. “Mice are not people, obviously, but it’s very rare to see complete eradication of existing tumors. We believe this treatment has the potential to be useful in humans, and we are working toward bringing it into the clinic.”

Engleman is the senior author of the research, which was published online Jan. 26 in Cancer Cell. Postdoctoral scholar Ian Linde, PhD, is the first author of the study.

An (almost) symbiotic relationship

Researchers in Engleman’s laboratory have been working for decades to understand how the immune system responds to cancer. It’s a delicate push and pull, they’ve found.

“Cancer and the immune system have an intimate, almost symbiotic relationship,” Engleman said.

Although the immune system can be a potent destroyer of cancerous cells, many cancers have found sneaky ways to exploit its safety valves, which prevent the mistaken destruction of healthy cells as well as autoimmune disorders. A cancer cell might disguise itself as a healthy cell, or it might secrete substances or harbor molecules on its surface that tell the immune system to stand down rather than attack. Many current cancer immunotherapies focus on unleashing the immune system’s powers by overriding these suppressive signals.

Neutrophils are part of what’s known as the innate immune system, which recognizes broad categories of common threats but is unable to identify and attack specific invaders. (In contrast, the antibodies pumped out by B cells or the specialized receptors on the surfaces of T cells are fine-tuned to recognize unique protein structures called antigens on pathogens that spell trouble.) Neutrophils can also regulate other arms of the immune system to maintain the delicate balance between a response that’s overly strong or too weak.

“We have not yet learned why neutrophils can have different and seemingly opposing functions,” Engleman said. “They are effective killers of bacteria and of cells infected with viruses. But they can also suppress the immune response, and when they infiltrate tumors, the outcome is almost always bad.”

An unexpected finding

Linde was injecting tumors in laboratory mice with various combinations of molecules called cytokines that Engleman’s laboratory had previously shown modulated the immune response to cancers.

“We didn’t set out to study neutrophils per se,” Engleman said. “But Ian came to me and said, ‘Neutrophils are infiltrating the tumors and the tumor cells are dying.’ We really didn’t expect that. But he repeated the experiments, and I became increasingly enthusiastic when I saw how potent and reproducible the results were.”

Linde tested a combination of a cytokine called tumor necrosis factor that draws neutrophils to the tumor, an antibody called CD40 that activates their killing abilities and another antibody that binds to the surface of tumor cells on mice with several types of cancers. Of the treated animals, tumors in 7 of 10 mice with melanoma, 8 of 10 with mammary cancer, 8 of 10 with colon cancer and 4 of 10 with lung cancer disappeared, and the animals lived for at least 60 days without detectable tumors. In contrast, no mice pre-treated with an antibody that eliminates neutrophils lived to the end of the experiment — indicating that the cancer-killing activity was due to the presence of neutrophils.

Linde then tested the ability of the treatment, which the researchers termed neutrophil activating therapy, to prevent cancer cells from migrating to other parts of the body. He found that injecting the activating therapy into a primary melanoma tumor inhibited the ability of other melanoma cells to establish themselves and grow in the animals’ lungs (a common site for melanoma metastasis).

“Our theory is that the neutrophils kill the injected tumor, then the dead cancer cells and their antigens enter lymph nodes and activate T cells to seek out and kill distant metastases,” Engleman said.

Further research showed that the neutrophils kill tumor cells by stimulating the production of molecules called reactive oxygen species, which damage DNA and proteins and can cause cell death.

More research is needed before the neutrophil activating therapy can be tested in people with cancer. But Linde showed that human neutrophils exposed to the three components of the combination therapy are stimulated to kill human cancer cells in a lab dish.

“This suggests that we might be able to use activated neutrophils as a cell therapy,” Engleman said. “We could remove a patient’s neutrophils and activate them in the laboratory before giving them back to the patient. This is completely different from current T cell–based cell therapies, and because there is no overlap, we might be able to use them in combination to great effect.”

Engleman is hopeful that clinical trials in humans could be launched soon.

“Our results go against the current belief that neutrophils are pro-tumor,” Engleman said. “But this shouldn’t be a shock, because in some cases neutrophils can be very effective killer cells.”

A researcher from the University of Pennsylvania also contributed to the work.

Engleman and Linde have filed a patent application based on the research.

The study was funded by the National Institutes of Health (grants U54 CA209971, R01 CA222969, R01 CA233958, R01 AI085596, R01 AI117410, R01 AI146162, F31 CA196029, F30 CA196145, T32 HL120824 and F32 CA189408).