Background Natural killer (NK) cells are part of innate immunity with high cytolytic activity against tumor cells, along with the ability to secrete cytokines and chemokines. However, solid tumors have been largely resistant to treatment, owing to unfavorable interactions between immune cells and tumors driven by an immunosuppressive tumor microenvironment (TME). Among tumor-driving metabolites, we have found that adenosine (ADO), has the ability to alter the phenotypic profile of NK cells in solid tumors and enhance their cytotoxicity via inhibiting LAG-3 expression. At the same time, targeting these in the TME of solid tumors with CAR-engineered NK cells enables tumor control and the identification of drivers of resistance to CAR-NK cell killing.

Methods NK cells from lung cancer patients were isolated from blood samples. To study the effect and role of oncometabolites, we treated NK cells with various concentrations of ADO for 24 hours before either removing or leaving them in the medium. NK cell phenotyping, including LAG-3 expression, was measured using flow cytometry. Subsequently, we generated dual-specific CAR-NK cells and identified drivers of resistance to CAR-NK-mediated killing via a genome-based CRISPR screen involving patient-derived GBM.

Using the same conditions as above, NK cells from both groups were then co-cultured with wild type and CD73 knockout (KO) lung carcinoma cell line A549 (A549 WT and A549 CD73KO).

Results Our results showed that NK cell phenotype is altered in response to ADO, including increasing cytotoxicity and aberrant expression of LAG-3, which was downregulated upon ADO treatment, and could be partially recovered upon removal of ADO from the culture medium.

Finally, genome-based CRISPR screening of dual-specific CAR-NK cells co-targeting CD73 identified metabolic drivers of GBM response to NK attack and could represent new targets for adoptive cell therapy with NK cells.

Conclusions Our data showed that metabolites affect NK cell killing capacity. Interestingly, we found that ADO heterogeneously modulates the killing ability of NK cells , suggesting that context- and receptor-specific activation – such as the continued presence of cytokines and metabolites – drives NK cell responses to metabolic factors in the TME. We corroborated these findings with the phenotypic characterization of NK cells isolated from tumors of lung cancer patients and revealed a dysfunctional state linked with an inability of NK cells to support proliferative or cytolytic functions. This dysfunction could be repaired by targeting new transcriptional drivers of GBM resistance to NK cell attack.