Abstract 1150: Fluorescent dCFPyL (GUL-Cy5) for detecting circulating tumor and immune cells containing cancer extracellular vesicles in early metastatic prostate cancer prognostication

Abstract Introduction: Circulating tumor cell (CTCs) liquid biopsies for metastatic prostate cancer are in need of clinically relevant biomarkers. We propose using the GUL probe (GUL-Cy5), which is already used in PSMA PET imaging. Additionally, we synthesized an isotype for GUL-Cy5, called tBu-Cy5, to quantitate non-specific binding. To validate GUL-Cy5, we used a commercial antibody specific for PSMA. These reagents were utilized with imaging flow cytometry (imFC) to characterize the probes (GUL-Cy5 and anti-PSMA mAb) and their distribution on CTCs and in immune cells. Methods: Metastatic prostate cancer (mPCa) whole blood samples were processed using the density isolation medium Ficoll-Paque to isolate PBMCs and CTCs. The isolated samples were stained with DAPI for nucleus staining, CD45 as a marker for immune cells, EpCAM as a lead marker for CTCs, GUL-Cy5 for PSMA, and a commercial antibody specific for PSMA. Imaging software for visualizing probe binding patterns to define several subclasses of CTCs and immune cells containing cancer extracellular vesicles was used. Results: CTCs were successfully detected in mPCa patient samples and classified into various subclasses based on combinations of EpCAM, GUL, and PSMA biomarkers. Notably, events positive for desired markers and those specific to prostate markers were observed. The GUL signal pattern was primarily cytoplasmic, and similar to the signal from anti-PSMA mAbs. Surprisingly, a large proportion of CD45+ve events contained PSMA punctuate signal representing cancer EVs. Conclusion: GUL-Cy5 shows promise as a next-generation probe for PSMA in liquid biopsies. Imaging flow cytometry effectively detects and characterizes CTCs and immune cells that contain cancer EVs, allowing investigators to gain insights into their biology, providing a powerful tool for cancer research and future oncology applications. Citation Format: Omar Alawamry, Shuyang Feng, Minzhi Sheng, John Hayward, Kristen Cimolai, John F. Trant, Nesan Bandali, Bianca L. Tiglao, Urban Emmenegger, Stanley Liu, Hon S. Leong. Fluorescent dCFPyL (GUL-Cy5) for detecting circulating tumor and immune cells containing cancer extracellular vesicles in early metastatic prostate cancer prognostication [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 1150.