Background Natural Killer (NK) cells can eliminate cancer cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC).1–3 NK cell-based treatments have had therapeutic success for hematological malignancies but strategies to treat solid tumors have been limited due to immunosuppression within the tumor microenvironment (TME).4–6 An important and understudied aspect of NK cell immunosuppression is the low oxygen (hypoxia) environment created by proliferating tumor cells. We used the novel AVATAR incubator system to model oxygen levels of three key tissues that NK cells inhabit in vivo: the peripheral blood (12% O2), the bone marrow (5% O2 ) and the TME (1% O2).
Methods NK cells were incubated in the AVATAR incubators for 24 hours, 72 hours and 7 days. We conducted a mass cytometry (CyTOF) analysis to assess phenotype, flow cytometry-based assays to assess proliferation and an IncuCyte machine and immunofluorescent imaging to measure cytotoxicity of NK cells incubated at different oxygen conditions. We evaluated NK cell metabolism using Seahorse assays, gene expression using RNA-Seq and are in the process of evaluating epigenetic regulation using ATAC-Seq.
Results NK cells from the 1% O2 condition express fewer activating receptors (CD16, NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions (figure 1). NK cells in the 1% O2 condition also have decreased aggregation of perforin and granzyme granules at the immune synapse. This translates to reduced natural cytotoxicity and ADCC responses against tumor targets (figure 2). We also observe a sharp decrease in proliferation in the NK cells at 1% O2 (figure 3). This is partly due to an increase in CISH gene expression that makes the cells less responsive to cytokine stimulation.7 The RNA-Seq analysis revealed that NK cell metabolism closely resembles cancer cell metabolism under hypoxic conditions, specifically an increased expression of genes related to glycolysis, amino acid synthesis and central carbon metabolism. This change in metabolism was confirmed using Seahorse assays. We also observed changes in genes related to epigenetic regulation specifically, increases in histone demethylases and decreases in DNA methyltransferases (figure 4).
Conclusions These results indicate that NK cells who enter the solid TME are fundamentally different than those in the bone marrow or the blood stream. Overall, the insights gained from these experiments can help overcome hypoxia induced immune suppression in the tumor microenvironment and improve NK cell-based immunotherapy for solid tumors.
Acknowledgements We thank XCell biosciences for providing us with the AVATAR incubators used for these experiments
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