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).

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Abstract 675 Figure 1

Oxygen concentration alters NK cell phenotype300,000 enriched NK cells were incubated for 24 hrs, 72 hrs and 7 days at noted incubator conditions with 1 ng/ml IL-15. At the end of the incubation, cells were barcoded and stained with a custom panel for CyTOF evaluation. Data is show here for CD16 (A), NKG2D (B), Perforin (C) and Granzyme B (D). N=3 (data concatenated).

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Abstract 675 Figure 2

Oxygen concentration effects NK cell cytotoxicity300,000 enriched NK cells were plated per well in 96-well round-bottom plates with 1 ng/ml IL-15. Plates were inserted in noted incubator conditions for 24 hours, 72 hours or 7 days. At the end of the incubation period NK cells were counted and plated at a 2:1 E:T ratio with fluorescently labeled K562 targets or fluorescently labeled labeled Raji targets + Rituximab (10 ug/ml) and cells were imaged every 30 minutes. Data is shown here for K562 targets (A) and raji targets (B) at the 7 day timepoint. Representative of six separate experiments.

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Abstract 675 Figure 3

Oxygen concentration impacts NK cell proliferation300,000 PMBCs were CellTrace labeled and plated per well in 96-well round-bottom plates with noted treatments. The NK cells were incubated under noted incubator conditions for 7 days. At the end of 7 days, LiveDead dye was used to assess viability (A), while proliferation was assessed by evaluating CellTrace dye dilution on gated (CD56+CD3-) NK cells (B). (N=6)

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Abstract 675 Figure 4

RNA-Seq reveals changes in gene expressionAn RNA-Seq analysis was performed on enriched NK cells incubated in noted oxygen and pressure concentrations for 24 hours, 72 hours or 7 days. A heat map of epigenetic regulation genes (A) and glycolysis genes (B) are shown for the day 7 timepoint. (N=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

Trial Registration N/A

Ethics Approval N/A

Consent N/A

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