Abstract B011: Adenosine signaling and immune modulation in the tumor microenvironment: insights from lung cancer murine model of radiation therapy
Authors: Saini Krishan K. , Sarti Samanta , Rahiman Chelsea L. , Ragaishis Brian E. , McCann Patrick J. , Bansal Shruti , Cham Jason , An Julia , Martin Rosa , Chen Ariel , Spina Catherine S. 
Published: January 2025

Introduction: Radiation therapy (RT) induces adenosine signaling by releasing ATP in the tumor microenvironment (TME), promoting immune suppression and tumor progression. This study investigates how RT impacts adenosine signaling and immune cell infiltration in a preclinical model of non-small cell lung cancer (NSCLC). Using immunophenotyping of tumor-infiltrating immune cells in LLC1 murine model, we aim to explore the potential of targeting the adenosine pathway in combination with RT to enhance antitumor immunity and clinical outcomes. Methods: LLC1 tumor cells (5 x 105) were implanted subcutaneously in the flanks of C57BL/6 mice. After 12 days, mice received RT (4 Gy x 3 or 8 Gy x 3). Tumor volumes were measured thrice a week and harvested 5- and 10-days post-RT. We performed immunophenotyping of tumor tissue to assess changes in immune (CD45+) and CD45- compartments. The expression of adenosine signaling pathway members was quantified in distinct immune cell populations. Results: RT caused significant inhibition in tumor growth in a dose- and time-dependent manner. 5-days post-RT, CD73 (P&lt; 0.01) and CD39 expressions (P&lt; 0.01) were significantly upregulated in the immune compartment, while A2BR (P&lt; 0.01) and A2AR (P&lt; 0.05) were decreased in the non-immune compartment. RT decreased T cell populations, including CD4+ and CD8+ T cells. Further, CD4+ T cells downregulate adenosine signaling markers, particularly CD73 (P&lt; 0.05) and ENPP1 (P&lt; 0.05), while CD8+ T cells decreased in CD73 expression (P&lt; 0.01). Myeloid subtype abundance was unchanged, but A2AR, A2BR, CD39, and CD73 expressions increased in the M2-like macrophage compartment (F4-80+MHCII-). 10 days post-RT, all adenosine signaling pathway markers significantly increased in the immune compartment and significantly decreased in the non-immune compartment. T cells (TCRb+) and macrophages (F4-80+) increased in abundance, including immuno-suppressive Arg+ macrophages (P&lt; 0.05). Macrophage population that expressed the adenosine signaling markers increased by 10-15% (P&lt; 0.05). While the proportion of CD4+ T cells and Tregs (CD4+FoxP3+) that expressed CD39 and CD73 also increased (P&lt; 0.05). Conclusions: Our study revealed that RT promotes an immunosuppressive TME, in part through adenosine signaling. 5 days post-RT, we observed lymphocyte depletion and upregulation of the adenosine signaling pathway in M2-like macrophages. 10 days post-RT, adenosine signaling significantly increased across the immune compartment and decreased in the non-immune compartment, suggesting that the increase in adenosine signaling post-RT is immunologically mediated and largely driven by the myeloid compartment. These data lead to the hypothesis that inhibition of the adenosine signaling pathway may partially mitigate RT-induced immune suppression and improve RT efficacy. Ongoing work includes quantification of adenosine by LC-MS, extension to a preclinical SCC model of NSCLC, and evaluating adenosine pathway inhibition in combination with RT as a therapeutic strategy to improve antitumor immune responses.

Citation Format: Krishan K. Saini, Samanta Sarti, Chelsea L. Rahiman, Brian E. Ragaishis, Patrick J. McCann, Shruti Bansal, Jason Cham, Julia An, Rosa Martin, Ariel Chen, Catherine S. Spina. Adenosine signaling and immune modulation in the tumor microenvironment: insights from lung cancer murine model of radiation therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B011.