Abstract 4033: Adenosine regulates radiation therapy-induced antitumor immunity
Authors: Wennerberg Erik , Mediero Aranzazu , Wilder Tuere , Formenti Silvia , Cronstein Bruce , Demaria Sandra 
Published: July 2016

Abstract
Radiation-induced immunogenic cell death (ICD) is a key mechanism whereby local radiation therapy (RT) can elicit anti-tumor immune responses and synergize with immune checkpoint inhibitors in enhancing tumor responses. ATP, which is an essential signal of ICD, activates tumor-resident dendritic cells (DCs) promoting their ability to cross-present tumor-derived antigens to T cells. Interestingly, while release of ATP by RT is dose-dependent (Golden et al., OncoImmunology 2014), a large RT dose of 20 Gy was not effective in inducing anti-tumor T cells and synergize with anti-CTLA-4 (Dewan et al., Clin Cancer Res 2009). Extracellular ATP is rapidly catabolized to adenosine (ADO) by ectonucleotidases CD39 and CD73, which are expressed on tumor cells and immune cells. ADO has immunosuppressive effects, inhibiting DC- and effector T cell-activation, while promoting regulatory T cells (Tregs). Here, we tested the hypothesis that conversion of ATP to ADO hinders generation of effective anti-tumor immunity by high dose RT.
Mice were inoculated s.c. with TSA breast cancer cells or MCA38 colon cancer cells on day 0 and assigned to treatment with: (1) control mAb; (2) anti-CD73 (TY/23); (3) RT (4) RT+TY/23. TY/23 (200 μg) was administered i.p. on day 11, 14, 17 and 20. RT was given locally as single 20 Gy dose on day 12. On day 18, some tumors were harvested for flow cytometry analysis of DC and T cells. Mice were monitored for tumor progression. HPLC was used to measure ADO levels in supernatants from 24 h co-cultures of bone marrow-derived DCs and irradiated TSA cells.In vitro, antibody blockade of CD73, the rate-limiting enzyme in the generation of ADO, reduced the levels of ADO in the supernatant and restored the activation of DCs cultured with irradiated TSA cells. In irradiated tumors, anti-CD73 reduced the percentage of Tregs within the tumor-infiltrating CD4+ T cell population (7.9±2.5% in RT+TY/23 vs 20±0.8% in RT, p&lt;0.01) while increasing CD8+ T cells (38.3±0.1% in RT+TY/23 vs 17.3±4% in RT, p&lt;0.05). Among intratumoral DCs, the CD8a+ DC subpopulation was increased after CD73-blockade (37.9±15.7% in TY/23+RT vs 11.3±4.9% in RT, p&lt;0.01). Importantly, in irradiated tumors, anti-CD73 enhanced expression of activation markers CD40 on CD8a+ DCs (MFI: 218±1 in RT+TY/23 vs 54±41 in RT, p&lt;0.05) and CD69 on CD8+ T cells (MFI: 513±126 in RT+TY/23 vs 148±59 in RT, p&lt;0.01). Furthermore, tumor-bearing mice treated with RT in combination with anti-CD73 had a significantly delayed tumor progression (p&lt;0.05) and prolonged survival (p&lt;0.01) compared to mice receiving RT alone. Anti-CD73 given alone had no effect on tumor growth.
Our data show that adenosinergic signaling regulates the ability of RT to induce anti-tumor immunity, affecting activation of both DCs and effector T cells. ADO blockade may represent a promising strategy to enhance the immunogenicity of irradiated tumors by improving the ability of RT to induce in situ tumor vaccination.
Citation Format: Erik Wennerberg, Aranzazu Mediero, Tuere Wilder, Silvia Formenti, Bruce Cronstein, Sandra Demaria. Adenosine regulates radiation therapy-induced antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4033.