A Way to Visualize CAR-T Cells Has Been Found

CAR-T therapy is one of the newest and most promising directions in the treatment of oncological diseases. The method involves 'manually training' the patient's immune cells to fight cancer. They are extracted from the blood, modified, and endowed with special receptors that help them recognize malignant cells and destroy them. The genetically modified lymphocytes are then reintroduced into the patient's bloodstream, where they begin to actively multiply and attack the enemy. This approach allows for personalized treatment, creating a unique medication for each patient, and consequently achieving the best results without side effects.

This type of immunotherapy has already helped increase the effectiveness of treatment for certain forms of cancer, but it remains unknown where CAR T-cells go after entering the patient's body. How do doctors know that they have successfully reached their target and continue to fight tumours weeks, months, or even years after administration?

In a recent study, specialists discovered a new way to track such cells in the patient's body. Using genetic engineering methods, they created CAR T-cells with molecular markers, allowing them to be observed using positron emission tomography (PET) in animal models of cancer. They reported the results of their work in the latest issue of the scientific journal Molecular Therapy.

“Currently, the only way to know if gene or cell therapy is working is through regular biopsies of tumours or blood samples, which provide only a very rough estimate of the effectiveness of the treatment being conducted,” says one of the authors of the study, Associate Professor of Radiology Mark Sellmeyer. “With our technology, doctors will be able to literally see the number and location of CAR T-cells that have persisted in the body over time, which is an indicator of the duration and potential effectiveness of treatment. Visualizing therapeutic cells will also allow scientists to more easily test and refine treatment methods for various diseases.”

PET allows for the acquisition of colorful three-dimensional images of the human body using radioactive indicators. These indicators are small molecules similar to glucose that can accumulate in tumours or bind to specific proteins, indicating the presence of a particular disease. For example, when a radiopharmaceutical called fluorodeoxyglucose is introduced into the body, malignant cells begin to absorb its molecules much more actively than the surrounding cells. Cameras take pictures of these tumour foci and transmit them to a computer, allowing doctors to see the localization of the disease.

However, in the case of cell therapy, the therapeutic cells look like ordinary immune cells of the body, which does not allow PET to detect them. To solve this problem, researchers equipped CAR T-cells with a specific indicator, which was an enzyme produced by the bacterium E. coli – dihydrofolate reductase (eDHFR).

To visualize the difference between ordinary and therapeutic cells, the team created a radioactive label derived from the antibiotic trimethoprim, which has a high affinity for the bacterial enzyme and a low affinity for human cells. “This difference led us to believe that during visualization we could achieve high contrast, or a high 'signal-to-noise' ratio, for CAR T-cells expressing the bacterial enzyme,” says Sellmeyer.

During the study, CAR T-cells were labeled with the bacterial protein eDHFR (which was named a reporter gene for PET) and returned to mouse models of cancer. After the rodents were administered trimethoprim, the therapeutic cells began to glow, allowing scientists to track their movement in real-time using PET-CT scanning. And since these labels were genetically encoded, once CAR T-cells multiplied, the new cells also became carriers of the same visualization marker.

PET-CT scanning of mouse models of the disease showed that after seven days, the therapeutic cells accumulated in the spleen, and after 13 days began to accumulate in antigen-containing tumours. According to the scientists, these results indicate that CAR T-cells may have early and late 'dislocation sites', and that there is still much to learn about their quantity and exact location in the human body.

Researchers were also surprised to find that the radioactive indicator they used has an extremely high sensitivity for detecting CAR T-cells within tumours: 11,000 cells per cubic millimeter. “It would be great if we could see 10 million cells, but knowing that therapeutic cells are still present in the body, even in a quantity of just 10,000, is already good,” says Sellmeyer. “The level of quantitative visualization that we

“I hope that in the future the effectiveness of many types of gene or cell therapy, including CAR T, can be assessed by tracking the number and location of therapeutic cells in the body,” notes Mark Sellmeyer.