In recent years, great advances have been made in the field of immunotherapy to treat cancer. One of the most promising treatments involves engineering immune cells to express chimeric antigen receptors (CAR). These receptors are carefully designed to recognize antigens expressed on the surface of tumor cells. Once the target is recognized, the CAR-engineered immune cells can attack and kill the tumor cells. CAR T cells have been successfully used to treat certain blood cancers—three CAR T therapies for lymphoma and leukemia have gained US FDA approval. In these cases, T cells were taken from individual patients, grown and genetically-altered in the lab, then reintroduced into the same patient.
Despite the high promise of CAR T cells, there are many challenges to this approach. There is the risk of CAR T cells recognizing and attacking non-tumor cells, which could cause serious side effects. Also, the process of engineering the patient’s own T cells and reintroducing back to the patient is cost and labor-intensive. Another big challenge is that CAR T cells do not work well against non-blood solid tumors—abnormal masses of tissue that occur in bone, muscles or organs. This is because solid tumors reject T cells that try to enter.
Applying CAR to natural killer (NK) cells may be a way to get around these obstacles. NK cells differ from T cells in that they rely on other receptors to detect tumor cells, not just CAR. This means they can recognize tumor cells even if they alter their antigens to evade detection. Unlike CAR T therapy, NK cells don’t have to be taken from each individual patient. Instead, they can be obtained from healthy donors, developed into cell lines and introduced to patients with little risk of rejection. This significantly reduces the cost and side-effects of treatment.
Researchers are eager to test CAR NK therapy, but there is one problem. No suitable cell model exists for evaluating the efficacy and safety of CAR NK treatment on different patients. That is until now. In a recent study published in The EMBO Journal, researchers in Germany developed a new approach to assess CAR NK therapy efficacy on 3D colorectal organoids derived from patient tumors.
To determine whether the CAR NK cell line was effectively killing the colorectal tumor cells, they needed a quantitative readout for cytotoxicity during prolonged periods that would work on 3D organoids. A luciferase-based 3D reporter assay was their method of choice. They designed a luciferase/GFP-expressing transgene and introduced it into the organoids via lentiviral transduction. Luciferase expressed from the transgene allowed quantitative and simple measurement of cytotoxicity: higher luminescent signal detected means more live cells. This allowed them to determine what percentage of tumor cells have been killed by the CAR NK cell treatment using a simple readout from a luminometer. The stable expression of GFP within the transgene also allowed them to monitor cytotoxicity over time using live-cell imaging.
Using this new approach, the researchers proceeded to evaluate the efficacy of CAR NK therapy. They first engineered patient-derived colorectal organoids to express a common tumor-specific antigen, called EGFRvIII. The tumor organoids were co-cultured with healthy colon organoids to mimic in vivo tumors. The tumors were then treated with a CAR NK-92 cell line for 10 hours. They found that the CAR NK cells specifically attacked the tumor cells and not the healthy cells. They also tested a new CAR strategy targeting an endogenously expressed tumor antigen called FRIZZELED, and found that it could result in off-target effects against healthy colorectal cells.
This study is the first step toward assessing CAR NK immunotherapy using patient-derived 3D organoids. Compared to existing 2D cell models or animal models, the new approach allows better physiological analysis of CAR-mediated cytotoxicity in patient cells. The luciferase-based detection is also adaptable to high-throughput applications. In the future, this technology may help improve CAR-T strategies for colorectal cancer and other solid cancers.
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