A technique that transforms immune cells into cancer-seeking bloodhounds may overcome a roadblock that has hampered immunotherapy for solid tumors, according to a new study by Stanford Medicine researchers.
The approach equips certain types of immune cells with proteins on their surfaces that can recognize byproducts of cancer cells' abnormal metabolism diffusing in the spaces between cells and stimulates the immune cells to migrate toward the tumor.
It differs from another common immunotherapy, called CAR-T cell therapy, in that CAR-T cells are engineered to have receptors that recognize a protein tethered to the surface of a cancer cell, rather than small molecules released into the extracellular spaces.
Arming CAR-T cells with specific metabolite-sensing receptors markedly increased the therapies' effectiveness.
"We found that when we equip immune cells with receptors that sense metabolites released by cancer cells, they can sense the tumor, migrate toward it, infiltrate it and control tumor growth, which markedly enhances the survival of mice with human breast and ovarian cancers," said Livnat Jerby , PhD, assistant professor of genetics.
Jerby is the senior author of the research, which was published March 23 in Nature Immunology. Postdoctoral scholar Young-Min Kim, PhD, is the lead author of the study.
An innovative therapy has limits
CAR-T cell therapy has transformed the treatment of several blood cancers since it was first approved by the Food and Drug Administration in 2017 for the treatment of acute lymphoblastic leukemia. But it's been less successful in patients with solid tumors. The thought among the CAR-T research community has been that the CAR-T cells, which are prone to excessive signaling, become exhausted before they can eliminate solid tumors. Additionally, unlike in blood cancers, it is difficult to identify molecular targets on solid tumors that are found only on the cancer cells and not on normal tissue.
"There have been many studies trying to overcome T cell exhaustion," Jerby said. "But our study supports and was driven by the notion that the problem with treating solid tumors is also a spatial issue. Too few T cells are getting into the tumor."
In short, if CAR-T cells can't get to the cancer cells, their cancer cell killing potential goes untapped. Jerby and her lab members wanted to know if it is possible to tweak cancer-killing immune cells so they can migrate more successfully into solid tumors.
Chemokines, a subset of proteins called cytokines, are known to attract immune cells to solid tumors as well as sites of infection, inflammation and injury; they generally play an important role in immune cell migration. The researchers first suspected that engineering immune cells to express chemokine receptors could enhance their migration to solid tumors, but they also wanted to examine whether additional genes could be used for this purpose.
To approach the question agnostically, they compared the level of different RNA molecules (the genetic material that serves as information for protein production) between breast cancer tumors and blood samples from 22 breast cancer patients — looking for genes that were expressed more highly in immune cells present in the tumor compared with those circulating in the blood. They also mined a database of RNA expression levels in a specific type of immune cell called a natural killer, or NK, cell in more than 700 patients and 24 types of cancer.
After identifying 256 candidate genes for further study, the researchers used a genetic engineering technique called CRISPR to individually activate each of the genes in human NK cells grown in the laboratory and infused these cells into mice bearing human breast or ovarian cancers. They then removed the tumors from the animals and analyzed how many NK cells had infiltrated the tumors.
A 'natural killers' plot twist
"We basically let these NK cells compete against one another to identify the genes that drive migration to and into the tumors," Jerby said. "Surprisingly, we didn't see many chemokine receptors among the winners. What came up were receptors that recognize bioactive, chemoattracting metabolites that have not been studied nearly as much in the context of cell engineering and tumor immunology. And we saw the same hits, time after time in different model systems with different screens and different experimental settings. It was quite striking."
In contrast to chemokines, which are proteins, chemoattracting metabolites are small molecules, fats or ions that can attract various cell types, including immune cells, to specific locations in the body.
Jerby and her colleagues found that NK cells that had been engineered to express one of six genes were consistently better at infiltrating breast and ovarian tumors in the animals, migrating specifically to cancer cells and the factors they release. Because the genes they identified encode proteins belonging to a class of receptors called G-protein coupled receptors, or GPCRs, the researchers coined the term tumor-homing GPRs, or thGPRs.
The thGPRs identified are known to recognize and migrate toward specific types of phospholipids, fatty acids and derivatives of cholesterol, which are generated by cancer cells in their headlong dash to proliferate. The study's analyses of patient data indicate that these metabolites recruit tumor-friendly immune cells, creating an environment that supports tumor growth and resists drug treatment. But they can also serve as a smoking gun, indicating a tumor is nearby.
"It's been known for decades that cancer cells are metabolically unique in many ways," Jerby said. "Clearly there are certain metabolic features that either directly aid tumor growth or are a byproduct of uncontrolled cell proliferation. These features are routinely exploited for cancer diagnostic scans such as PET imaging, which pinpoints areas in the body with high metabolic activity."
Chemoattracting metabolites stimulate responding cells to migrate toward higher concentrations of the target to reach the signaling cells — a kind of "follow the yellow brick road" approach in which the path widens and becomes easier to follow as the destination is neared. The researchers exploited this feature to equip NK or killer T cells to track down and infiltrate tumors based on their telltale metabolite trails.
Examining this therapeutic approach the researchers focused on one of the thGPRs, GPR183, in breast cancer. GPR183 is a receptor of oxidized forms of cholesterol called GPR183. Engineering NK or T cells to express GPR183 on their surfaces markedly enhanced the ability of the cells to migrate toward cancer cells in laboratory dishes or in mice. Expressing GPR183 on the surface of NK cells, CAR-NK cells, CAR-T cells and other types of tumor reactive T cells led to significantly better tumor control and survival of laboratory mice with breast cancer tumors.
"We saw a more than doubling in the number of complete responses in the animals," Jerby said. "T cells engineered to express GPR183 on their surfaces were far better at completely eradicating highly aggressive breast tumors. The tumors did not come back, and the mice went back to being healthy."
Jerby and her lab members are now investigating whether thGPRs can be modified to recognize other tumor metabolites that are not normally chemoattracting as navigation cues, or to have immune cells interpret tumor metabolites as "on switches" to become killing machines only in the tumor. They are also moving toward testing the GPR183-engineered cells in clinical trials and testing the other thGPRs for their therapeutic potential.
"To the best of our knowledge, no one has tried to use cancer metabolism, a hallmark of drug resistance and aggressive tumor growth, to attract cancer-killing immune cells to the tumor," Jerby said. "But our study uncovered the potential of this approach, and the results are quite promising."
Jerby is a member of the Stanford Cancer Institute , Bio-X and a Chan Zuckerberg Biohub investigator.
The study was funded by the National Institutes of Health (grant U01HG012069), the Chan Zuckerberg Biohub, Under One Umbrella and the Stanford Cancer Institute, Stanford's Discovery Innovation Funds, the Ovarian Cancer Research Alliance, the Burroughs Wellcome Fund, the National Research Foundation of Korea, the Ovarian Cancer Research Alliance, the Department of Defense, and Alba Tull Molecular Therapeutics Award for Innovative Medicines.
Stanford University has filed a patent based on the research in which Jerby and Kim are named as inventors.
