Abstract 5205: ECM-free patient-derived organoids preserve diverse prostate cancer lineages and uncover in vitro-enriched cell types

Background: Patient-derived organoids (PDOs) offer new opportunities to model various cancers. However, their application in prostate cancer (PCa) has been hampered by poor success rates and overgrowth of cell types which are not representative of the patient samples. Here, we aimed at modulating culture conditions and investigating in vitro-associated cellular heterogeneity to identify refined PCa PDO culture conditions. Methods: A cohort of 164 diverse samples was obtained from 162 PCa patients undergoing radical prostatectomy, prostatic biopsies, transurethral resection of the prostate or the bladder or metastasis biopsy/resection. Out of this cohort, PDOs were established in various extracellular matrices (ECM: Matrigel, Collagen I, Collagen IV, Laminin-I, or no ECM) and medium compositions. PDOs were characterized using IF, IHC, and FISH. Single-cell RNA sequencing (scRNA-seq) was performed on 11 tumor and organoid samples, followed by unsupervised clustering, trajectory inferring, cell type annotation, and CNV analysis. Published spatial and bulk transcriptomic data were re-analyzed to validate specific markers. A prostate PDO scRNA-seq atlas was generated by integrating three published PDO datasets (Huang 2023, Song 2022, McCray 2019) together with our newly-generated data. Results: Out of 5 ECM conditions, an ECM-free culture system increased the take-rate of PDOs with luminal-like (CK5-, CK8+, AR+) and PCa features (AMACR+, PTEN-), while standard Matrigel-based culture conditions yielded basal-like benign organoids (CK5+, AR-, AMACR-). Cellular composition of organoids generated in ECM-free conditions differed significantly from those generated in Matrigel, with minimal overlap observed in their transcriptomic profiles. ECM-free PDOs comprised cell populations associated with known PCa signatures, exhibited transcriptomic resemblance with their respective parental tumors, and maintained patient-specific epithelial populations. Furthermore, we defined organoid cell type signatures and identified markers discriminating between several types of benign cells (basal, hillock, club, transitioning) versus tumor cell populations ex vivo and in situ. Finally, our integrative single-cell atlas highlighted that Matrigel-based organoids derived from primary PCa are essentially composed of benign epithelial cells, irrespective of the dataset or the malignant nature of the tissue of origin. In contrast, ECM-free conditions preserved heterogenous tumor-like cell populations with higher inferred copy-number variations and were enriched in intermediate club-like cell populations. Conclusions: ECM-free PCa PDOs exhibit improved fidelity and in vitro-enriched cell populations, while aberrant benign lineage programs are associated with Matrigel culture. Our work contributes to significantly enhancing the potential of PDOs in PCa research. Citation Format: Robin Dolgos, Romuald Parmentier, Jing Wang, Raphaëlle Servant, Arnoud J. Templeton, Tobias Zellweger, Alastair Lamb, Kirsten Mertz, Svetozar Subotic, Tatjana Vlajnic, Helge Seifert, Ashkan Mortezavi, Cyrill A. Rentsch, Lukas Bubendorf, Clementine Le Magnen. ECM-free patient-derived organoids preserve diverse prostate cancer lineages and uncover in vitro-enriched cell types [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 5205.