Cancer cells are adept at escaping death. One of the ways in which cancer cells prevent death from fatal attacks by cytotoxic T lymphocytes (CTLs) is by efficiently repairing damage to their cell membranes.
With vivid live-cell videographic micro-imagery, a new study published in the journal Science “ESCRT-mediated membrane repair protects tumor-derived cells against T cell attack“, uncovers mechanistic insights into how cancer cells escape death, which could increase the effectiveness of current immunotherapies for cancer.
Senior author Ira Mellman, PhD, vice president of oncology research at Genentech, said, “This study shows cancer cells use a generalized membrane repair mechanism to protect themselves against immune attack. Expected and unexpected at the same time. Evolutionarily, this may help cells guard against unwanted or ‘accidental’ killing under healthy conditions, but in cancer, it may explain at least a portion of inherent resistance to immunotherapy. Understanding and solving these mechanisms will contribute to more effective therapeutic approaches.”
CTLs zero in on abnormal cells such as virus-infected cells and tumor cells, killing them by shooting dual bullets: perforin and granzymes. These are protein toxins. Perforin punctures tiny holes in the outer membrane of the targeted aberrant cell. Granzymes, which are proteolytic enzymes, pass through the openings and induce programmed cell death (apoptosis). The mechanism by which cancer cells escape this deadly immune attack was unclear until now.
Mellman said his team was inspired to take on this investigation “to understand precisely what transpires between a CTL and its target at the moment of cell killing. Remarkably, despite decades of interest in the problem, this precise issue has remained unexplored.”
Another realization that intrigued Mellman’s team stemmed from their imaging and functional studies: not all cell contacts were productive. “We wondered whether cells had intrinsic mechanisms that could protect them against CTL attack,” Mellman said. “This brought us to ESCRTs [Endosomal Sorting Complexes Required for Transport], which have for years been implicated in membrane repair.”
Alex Ritter, PhD, a postdoctoral researcher in Mellman’s laboratory in the department of cancer immunology at Genentech, and his colleagues combined high-resolution imaging data with functional analysis in live cells to demonstrate cancer cells overcoming CTL attack through a class of proteins called ESCRTs, which repair openings created by perforin in the target cell’s outer membrane. This efficient repair mechanism delays or prevents the entry of killer granzymes into the cancer cell.
“We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release,” the authors noted.
To exploit this newfound mechanism for therapeutic advantage, the investigators reasoned, inhibition of the target cell’s ESCRT-mediated pore-repairing strategy should make it easier for CTLs to kill them.
As earlier studies have shown extinguishing the ESCRT pathway for extended periods can itself kill cells, the researchers used two alternative approaches to inhibit the ESCRT pathway in target cells: they used CRISPR to knock out the gene Chmp4b, a component of the ESCRT pathway that sorts engulfed cell-surface receptors into vesicles. They also over-expressed a dominant mutant form of a phosphorylating enzyme, VPS4, in the ESCRT pathway.
A killer T cell engages and destroys a monstrous ovarian cancer cell. The red waves in the cancer cell emanating from the site of T cell attachment indicate release of toxic proteins that the T cell uses to destroy its target (Alex Ritter).
Adopting these methods in cancer cells grown in culture, the researchers showed that preventing the ESCRT pathway from repairing perforin pores in the cell membrane, increased the susceptibility of these cancer cells to be killed by CTLs.
Michael Dustin, PhD, professor of molecular immunology at the University of Oxford, who was not involved in the current study, told GEN: “The work highlights a striking correlative imaging technology for investigating a key moment in T-cell–mediated killing and a new mechanism of tumor immune evasion. A potential limitation of targeting ESCRT machinery in tumors is that it’s a double-edged sword that can suppress or accelerate tumor growth.”
“This work applies innovative imaging and molecular intervention to uncover an important, previously unappreciated aspect of perforin-pore removal and membrane repair. Of particular interest for improving immunotherapy, the authors identified the ESCRT machinery as a potential target, which in their defined model increased the kill rate 2- to 3-fold. Thus, interfering with the ESCRT membrane repair complex may open a new type of targeting approach to prolong the damage caused by individual cytotoxic T cells,” said Peter Friedl, PhD, professor of medicine and chair of microscopical imaging of the cell at Radbound University in the Netherlands, who was also not involved in the current study.
Friedl added, “The mechanisms by which prolonged perforin effects death induction is improved will require further work, yet the considerable gain of efficiency after ESCRT inhibition strongly indicates that the additive dose of lethal hit delivery can be modulated by targeted approaches. Inhibiting ESCRT-mediated membrane repair may also be of relevance for other cytotoxic effector activities of the immune system, such as attack by natural killer cells. I can see this direction of increasing CTL-mediated membrane vulneration [wounding] as a future avenue for anti-cancer therapies.”
In a commentary on the study in Science, Norma Andrews, PhD, a professor of cell biology at the University of Maryland, said defining the relationship of membrane damage with processes of pinching off (exocytosis) or sucking in (endocytosis) loaded membrane-bound vesicles from the cell membrane and the ESCRT repair pathway, would clarify how these processes induce membrane healing. This, in turn, could reveal druggable mechanisms, as in a recent finding where therapeutic expression of a lysosomal enzyme called acid sphingomyelinase increased muscle repair.
In future experiments, Mellman intends to functionally dissect the ESCRT complex with the aim of identifying therapeutic targets.
