PURPOSE: Docetaxel resistance is a significant obstacle in the treatment of prostate cancer (PCa), resulting in unfavorable patient prognoses. Intratumoral heterogeneity, often associated with epithelial-to-mesenchymal transition (EMT), has previously emerged as a phenomenon that facilitates adaptation to various stimuli, thus promoting cancer cell diversity and eventually resistance to chemotherapy, including docetaxel. Hence, understanding intratumoral heterogeneity is essential for better patient prognosis and the development of personalized treatment strategies. METHODS: To address this, we employed a high-throughput single-cell flow cytometry approach to identify a specific surface fingerprint associated with docetaxel-resistance in PCa cells and complemented it with proteomic analysis of extracellular vesicles. We further validated selected antigens using docetaxel-resistant patient-derived xenografts in vivo and probed primary PCa specimens to interrogate of their surface fingerprint. RESULTS: Our approaches revealed a 6-molecule surface fingerprint linked to docetaxel resistance in primary PCa specimens. We observed consistent overexpression of CD95 (FAS/APO-1), and SSEA-4 surface antigens in both in vitro and in vivo docetaxel-resistant models, which was also observed in a cell subpopulation of primary PCa tumors exhibiting EMT features. Furthermore, CD95, along with the essential enzymes involved in SSEA-4 synthesis, ST3GAL1, and ST3GAL2, displayed a significant increase in patients with PCa undergoing docetaxel-based therapy, correlating with poor survival outcomes. CONCLUSION: In summary, we demonstrate that the identified 6-molecule surface fingerprint associated with docetaxel resistance pre-exists in a subpopulation of primary PCa tumors before docetaxel treatment. Thus, this fingerprint warrants further validation as a promising predictive tool for docetaxel resistance in PCa patients prior to therapy initiation.

Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

Department of Clinical and Molecular Pathology Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital Olomouc 779 00 Czech Republic

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 Brno 612 00 Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno 625 00 Czech Republic

Department of Molecular Oncology H Lee Moffitt Cancer Center and Research Institute FL 33612 Tampa USA

Department of Urology Erasmus MC Cancer Institute Erasmus University Medical Center Wytemaweg 80 Rotterdam 3015 CN The Netherlands

Department of Urology Experimental Urology Medical University of Innsbruck Anich Strasse 35 Innsbruck A 6020 Austria

Department of Urology University Hospital Olomouc Olomouc 779 00 Czech Republic

International Clinical Research Center St Anne's University Hospital in Brno Brno 602 00 Czech Republic

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