NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its autoxidation and generation of H2O2, which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H2O2 in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H2O2 in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H2O2-scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo. The addition of AUR to L-ASC-treated cells triggers the accumulation of H2O2 in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings.

Immunotherapy revolutionized cancer treatment in the last decade. Both NK cells and T cells are key components of host immunity against malignant cells that are being extensively investigated in the field of cancer immunotherapy. While approaches have been developed to improve the antitumor activity of NK and T cells, the tumor microenvironment remains an obstacle to effective NK and T-cell–based therapies. Here, we demonstrated that cancer-conditioned medium suppresses the antitumor activity of NK cells. Ammonia, a by-product of cancer cell metabolism, accumulated in cancer-conditioned medium and in the tumor microenvironment, and impaired the cytotoxicity of NK cells as well as the efficacy of antibody-based and chimeric antigen receptor (CAR) NK and CAR T-cell–based therapies in vitro. Ammonia induced NK and T-cell dysfunction by decreasing the amount of mature perforin in secretory lysosomes, which was dependent on its lysosomotropic features and ability to increase pH in acidic compartments. These findings demonstrate that, in addition to its previously described role in promoting tumor growth as a source of nitrogen for tumor biomass, ammonia promotes tumor immune escape by inhibiting both NK and CAR T-cell cytotoxicity. Significance: Ammonia is elevated in the tumor microenvironment and functions as an immunoinhibitory metabolite in cancer by reducing perforin levels, inhibiting NK and T-cell–mediated immunity and limiting the efficacy of immunotherapies.

Supplementary Fig. 13 shows the effects of ammonia on T cells.

Ammonia affects NK cell functions and decreases the level of perforin. A, The surface levels of NK cell activation receptors (NKG2D, NKp30, NKp44, NKp46) after incubation with different concentrations of NH4Cl were assessed after 4 hours using flow cytometry. Mean fluorescence intensity (MFI) was normalized to control (medium without exogenously added ammonium). B, Formation of conjugates of NK and target cells in different concentrations of NH4Cl (n = 3). NK cells were stained with CellTrace Deep Red (CTDR), and target cells (K562 cells) were stained with CFSE. Cells were mixed and incubated with different concentrations of ammonia at 37°C for 30 minutes. After this time, 4% PFA was added, followed by a 10-minute incubation and wash. The percentage of conjugates defined as CTDR+CFSE+ double-positive events was determined using flow cytometry. The graph shows a representative experiment from one donor. C, Degranulation of NK cells in response to stimulation with target cells (K562) at different concentrations of NH4Cl (n = 4). The percentage of degranulating NK cells (CD107a+) was assessed after 4 hours using flow cytometry. D, The level of cytokines expressed in NK cells in response to stimulation with target cells (K562) at different concentrations of NH4Cl (n = 6). The percentage of TNFα-positive and IFNγ-positive NK cells was assessed after 4 hours using flow cytometry. E, Detachment of NK cells from target cells in different concentrations of NH4Cl (n = 3). NK cells were stained with CTDR, and target cells (K562 cells) were stained with CFSE. Cells were mixed and incubated in different concentrations of ammonia. After 0 to 45 minutes, 4% PFA was added, followed by a 10-minute incubation and wash. The percentage of conjugates, defined as CTDR+CFSE+ double-positive events, was determined using flow cytometry. F, The level of granzyme B in NK cells incubated in different concentrations of NH4Cl (n = 4). The level of granzyme B in CD107a− and CD107a+ NK cells was assessed by intracellular staining using flow cytometry. G, The level of granzyme B in NK cells incubated with ammonia determined by Western blot methods using an anti–granzyme B antibody (M3304B06 clone; n = 2). β-Actin is presented as a loading control. Representative blot from one donor is shown. H, The level of granzyme H in NK cells incubated with ammonia determined by Western blot methods using an anti–granzyme H antibody (E3H7W clone; n = 2). β-Actin is presented as a loading control. Representative blot from one donor is shown. I, The level of perforin in NK cells incubated in different concentrations of NH4Cl (n = 4). The level of total perforin in CD107a− and CD107a+ NK cells was assessed by intracellular staining (B-D48 antibody) using flow cytometry. P values were calculated using two-way ANOVA with Tukey post hoc test. Data show means ± SEM. n values are the numbers of biological replicates in in vitro experiments.

Supplementary Fig. 2 shows that low molecular fraction of conditioned medium suppresses cytotoxicity of NK cells