Abstract PR-005: Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment
Authors: Bansal Shruti , Chaimowitz Mathew , Ager Casey R. , Obradovic Aleksandar A. , Drake Charles G. , Spina Catherine S. 
Published: April 2021

Abstract
Introduction/Purpose: Radiation therapy drives pro-inflammatory changes to the tumor microenvironment (TME). We have shown that this may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation therapy induces immunosuppressive myeloid cells in the TME in an adenosine-dependent fashion. Methods: Using the 4T1 orthotopic breast cancer mouse model, we tested the immunophenotypic changes to the TME by spectral flow cytometry and bulk RNASeq of tumor infiltrating leukocytes (CD45+, TIL) 72-hours after tumor-directed radiation therapy (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). We cultured human peripheral blood mononuclear cells (PBMCs) 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. Results: Radiation increases the abundance of CD45+CD11b+ myeloid cells (77.4% vs. 84.4%) and decreased CD45+TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME, compared to untreated controls. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a significant reduction 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 an induction of granulocytic and monocytic myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). Radiation caused a decrease in the tumor-associated macrophage population (TAM: 50% vs 31.1%) in the setting of a phenotypic shift to from pro-inflammatory M1-like to immunosuppressive M2-like TAM phenotype: M2-like TAM/M1-like TAM ratio 2.0 vs 13.3. Radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92) by TILs, but not CD73 that metabolizes AMP to adenosine. The addition of NECA to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) at all NECA concentrations tested and a dose-dependent decrease in GM-CSF, IL-12p40 and IL-23. There was a significant reduction of IL-1 β, IL-8, IL-12p70 and MCP-1 only at the highest dose (10uM) tested. IL-6 was increased at the intermediate dose (1uM) of NECA with an overall reduction with high-dose NECA. High-dose NECA alone significantly increased both IFN- β and TGF- β. Conclusion: Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs, M-MDSCs and a shifting the TAM population towards an immunosuppressive phenotype. Radiation increases the expression of adenosine receptors on TILs and treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu suggesting a mechanistic link between radiation and adenosine signaling.
Citation Format: Shruti Bansal, Mathew Chaimowitz, Casey R. Ager, Aleksandar A. Obradovic, Charles G. Drake, Catherine S. Spina. Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-005.