Dying Cells Push the Mouse Immune System into Killing Tumors

Every day, billions of our cells die quietly and in an orderly fashion by activating a cell suicide program known as apoptosis. But others, often when they're infected with viruses, opt for a messier, more violent form: necroptosis, which harnesses the immune system to attack and kill the body's own cells.

In recent years, biologists have begun to investigate whether activating necroptosis in cancer cells could similarly coax the immune system into attacking tumors. Now, researchers show that injecting cells undergoing necroptosis into mice's tumors directs killer T cells to attack the malignancies and slow their growth. In addition, they find that making tumor cells produce a necroptosis-inducing enzyme is enough to kickstart the tumor-stunting process—a strategy the authors think could boost the efficacy of existing immunotherapies. The results were published in Science Immunology.

“It adds more evidence [that targeting] this type of cell death in cancer's a good thing: it will elicit anti-tumor immunity,” remarks Stephen Tait, a cancer cell biologist at the Cancer Research Center UK’s Beatson Institute who has collaborated with one of the study’s authors in the past but wasn’t involved in the new project. “It gives a strong rationale for targeting or activating this pathway as a cancer therapy.”

To investigate the effects of necroptotic cells on tumors, doctoral student Annelise Snyder at the University of Washington along with her colleagues engineered cancer cells in which the RIPK3 protein—an enzyme that triggers the necroptotic pathway—was activated. She injected the cells into mouse melanoma or adenocarcinoma tumors the animals carried on their flanks. Over the following week, the team observed the animals’ tumor growth slowing down—a process that later results revealed was dependent on the presence of killer T cells. Importantly, these animals survived significantly longer than mice that had received injections of cancer cells that had apoptosis-inducing enzymes activated instead.

In the first experiment, the injected necroptotic cells were derived from the same cell line as the tumors, and carried the same cell surface antigens. But to the researchers' surprise, they witnessed the same slowdown of tumor growth when they instead injected necroptotic cells from a healthy fibroblast cell line into the tumors. That necroptotic cells don't have to carry any tumor antigen to have this effect was a remarkable finding to senior author Andrew Oberst, a cancer immunologist at the University of Washington.

To him, it suggests that the necroptotic cells don’t direct T cells to target tumors by displaying particular antigens, but rather by secreting particular cytokines and chemokines that activate local T cells that reside around the tumor and are already primed to attack those cancer cells, but need an external nudge to become active. “Location is everything,” Oberst tells The Scientist. “Making this change to the tumor microenvironment by introducing necroptotic cells is really what allows this to work.”

His lab's previous research has shown that the RIPK3 enzyme can drive cells to generate inflammatory chemokines and cytokines during cell death, which can stimulate T killer cells.

Further experiments seemed to support the importance of cytokines and chemokines in stimulating the immune system. For instance, when the researchers treated the necroptotic cells to curtail the production of those cytokines, and then injected them into tumors, they didn't see the slowdown in growth they had observed in earlier experiments. This was surprising because it was long thought that necroptosis itself provokes the immune system. However, “it’s not actually the death itself, the bursting of the cell, that is the key event,” he adds. “If we just induce cell lysis, we don't see the same effect.”

“It's a really well done and elegant study,” notes Dmitri Krysko, a cancer biologist at the Cancer Research Institute in Ghent, Belgium, who wasn’t involved in the study, in an email. That necroptotic cancer cells produce an immune response was already known—as his lab and others have shown—but the mechanisms involved were largely unexplored, he says.

Triggering necroptosis is considered an attractive strategy over inducing apoptosis because many tumors develop ways to block or evade apoptosis, he writes. However, “it is important to stress that many cancers often develop necroptosis resistance as well.” For instance, several cancer cell lines and cancers such as breast cancer and acute myeloid leukemia are known to lose RIPK3 expression, he adds.

In an additional experiment, Oberst and his team explored a way to get around this problem: if they could manage to deliver the RIPK3 enzyme directly into the tumor cells, then even cancer cells that have lost the ability to produce the enzyme or other components of the necroptotic pathway would still release the immune-stimulating cytokines and chemokines. They genetically engineered a virus to express a specific form of the gene for RIPK3 such that it would be constantly active. Once injected into melanoma tumors, the vector had a similar tumor-controlling effect as they had observed in earlier experiments. In addition, when they administered the virus in combination with an immune checkpoint blockade—an immunotherapy that works by inhibiting molecules that suppress the immune system—this led to longer-lasting tumor clearance.

“That's quite a big finding,” Tait notes, “because we've been talking about this in the field, targeting necroptosis in different ways, but actually it's very difficult to figure out a way in which that can be done in tumor cells.” The authors show “very nicely” that this viral approach might be one way to induce activation of RIPK3 in the cells. “The only thing is, we’re not sure if systematic activation of RIPK3 would have any unwanted toxic effects.”

Oberst suggests the viral delivery approach may be an interesting avenue to explore in further research. “Of course, there’s a really long way to go from really simple tumor models that we used to anything that would be directly clinically applicable.”

A.G. Snyder et al., “Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity,” Science Immunology, doi:10.1126/sciimmunol.aaw2004, 2019.