Radiation, Adenosine Signaling, and Immunosuppressive Changes to the Tumor Microenvironment

Abstract

Tumor irradiation causes pro-inflammatory changes in the tumor microenvironment (TME). We have shown that these changes may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation activates adenosine signaling and induces immunosuppressive myeloid cells in the TME.Using the 4T1 orthotopic breast cancer mouse model, we tested immunophenotypic changes to the TME by spectral flow cytometry and by bulk RNASeq of tumor infiltrating leukocytes (CD45+, TILs) 72-hours after tumor irradiation (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro with 0.1, 1, and 10uM of adenosine receptor agonist, 5'-N-ethylcarboxamindoadenosine (NECA), in the presence of CD3/CD28 beads. Cytokine quantification of cell-free supernatant was performed 48-hours later using the Meso Scale Discovery platform.Radiation increases the abundance of CD11b+ myeloid cells (77.4% vs. 84.4%) and decreases TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a proportional reduction in Tregs (1.4% vs 0.63%), with no change in abundance of CD8+ T-cells (0.53% vs 0.43%). The increase in myeloid cells was predominantly influenced by granulocytic- and monocytic-myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). By bulk RNASeq of CD45+ TILs, radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92), but not CD73 - an ecto-nucleotidase that metabolizes AMP to adenosine. By flow cytometry, radiation increased expression of both CD73 and A2AR in the myeloid population, with CD73 expression significantly increased in the monocytic compartment (Ly6C+, 63% vs 70%) and A2AR expression increased in the granulocytic compartment (Ly6G, 89% vs 97%). The addition of adenosine analog, NECA, to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) across doses, an increase in IL-6 and MCP-1/CCL2 at the intermediate dose tested, and increased IFN-β and TGF- β while decreasing IL-1 β, IL-8 only at the highest dose.Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs and M-MDSCs and activating adenosine signaling. Radiation increases the expression of adenosine receptors on CD45+ TILs at the transcriptomic level and cell surface expression of CD73 on monocytes and A2AR on granulocytes. Treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu. These data suggest a mechanistic link between radiation, adenosine signaling and the myeloid compartment.