Adora1 promotes colon cancer immune evasion via Irf1-PD-L1 signal axis

Immunotherapy targeting immune checkpoints such as programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) has emerged as a novel treatment option for various cancers, including colon cancer. However, immune evasion mechanisms can limit the efficacy of cancer immunotherapy. Understanding the regulatory mechanisms of PD-1/PD-L1 expression is therefore critical to enhancing immunotherapeutic outcomes. A previous study demonstrated that the adenosine A1 receptor (Adora1) regulates PD-L1 expression and tumor immune evasion in human melanoma; however, its role in colon cancer and associated immune escape remains poorly defined. To investigate this, we downregulated Adora1 expression in CT26 colon cancer cells using lentiviral transduction of Adora1-targeting shRNA. We assessed Adora1 and PD-L1 expression levels and evaluated cell proliferation in CT26 cells. <i>In vivo</i>, we inoculated CT26 cells into mice and monitored tumor growth, immune cell phenotypes, and T cell exhaustion within the tumors. Additionally, we evaluated T cell exhaustion <i>in vitro</i> by co-culturing T cells with CT26 cells. While Adora1 knockdown did not impact CT26 cell viability or proliferation <i>in vitro</i>, it significantly suppressed tumor growth <i>in vivo</i> (P<0.0001). Furthermore, Adora1 downregulation reduced T cell exhaustion (all P=0.0025) by decreasing PD-L1 expression in CT26 cells. Knockdown of Adora1 did not alter Atf3 expression but led to reduced Irf1 expression (P=0.0268), which contributed to the downregulation of PD-L1. Overall, these findings suggest that Adora1 downregulation inhibits immune escape in colon cancer by suppressing PD-L1 expression.