Abstract 246: Innovative use of a fluorescent probe and 3D spheroids to study A3 adenosine receptor-targeted compounds in cancer cell lines

Abstract Introduction: The A3 adenosine receptor (A3AR) is a promising cancer therapy targeted due to its role in tumor progression, immune regulation, and apoptosis. Its selective expression in tumors like the liver and pancreas makes it ideal for therapies with minimal off-target effects. However, live-cell imaging techniques to study A3AR, including receptor expression, membrane localization, and interactions with AR modulators, are limited. Here, we present CELT-228, an A3AR-specific fluorescent probe, as a tool for visualization and functional analysis in live cells and 3D spheroid cultures. This method also enables competitive binding assays to identify and validate new A3AR agonists and antagonists. Material and methods: The reporter cell line ES-012-A, overexpressing A3AR, was treated with the CELT-228 (Celtarys, Spain) and imaged via spinning disc confocal microscopy (Yokogawa CV7000). Fluorescent signal localization and intensity were analyzed using Signal Image Artist (Revity). Competitive binding assays using CELT-228 were developed for High Content Screening (HCS) of newly synthesized nucleoside-based compounds and for studying A3AR interactions in cancer cell lines of diverse origins. Additionally, CELT-228 staining was applied to 3D spheroids from Hep-3B cells to investigate receptor localization under varying microenvironmental conditions. Results: In 2D and 3D models, CELT-228 predominantly localized to cell membranes of reporter and cancer cell lines. Competitive assays showed that new compounds bind to the orthosteric site, competing effectively with CELT-228. In 3D spheroids, staining revealed A3AR distribution across different regions, including cell membranes and intracellular compartments. A3AR expression patterns varied in hypoxic, nutrient-deprived, and proliferative regions, providing insights into microenvironmental influences on A3AR regulation. Conclusion: CELT-228 is a versatile tool for studying A3AR in 2D and 3D models, enabling receptor localization, competitive binding assays, and receptor mapping in spheroids. This approach holds great potential for A3AR-targeted drug discovery and advancing cancer therapies. Supported by the MEYS Czech Republic: CZ-OPENSCREEN (LM2023052), EATRIS-CZ (LM2023053), SALVAGE (811100711/37), National Institute for Cancer Research (Program EXCELES, ID Project No. LX22NPO5102) and IGA_LF_2024_038. Citation Format: Katerina Jecmenova, Alžběta Srovnalova, Marián Hajdúch, Petr Džubák. Innovative use of a fluorescent probe and 3D spheroids to study A3 adenosine receptor-targeted compounds in cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 246.