Patient-derived organoids (PDOs) are becoming increasingly important in prostate cancer (PCa) translational research. However, direct proof-of-concept studies demonstrating their ability to model disease evolution, identify relevant biomarkers, and accurately predict treatment response in PCa patients, remain scarce. Here, we report the establishment of serially transplantable xenografts series derived from two advanced PCa PDO lines, which can be further re-cultured as organoids. Newly-generated model series maintain key phenotypic, genomic, and functional characteristics of the original patient tumors, and emulate relevant molecular subtypes of advanced PCa. Single-cell RNA sequencing (scRNA-seq) analysis uncovers transcriptomic differences between xenograft and organoid models, as well as signaling pathways which are largely preserved and can be targeted ex vivo. Functional drug profiles correlate with molecular and clinical attributes, as exemplified by response to androgen receptor (AR) pathway inhibitors and glucocorticoid-mediated AR signaling activation. Longitudinal scRNA-seq analysis of perturbed PDOs identifies a rare PROX1+/ALDH1A1+ cell population, which pre-exist in the treatment-naïve setting and is significantly enriched upon androgen deprivation. Notably, this cell population is similarly enriched in post-treatment samples of the original patient and is associated with aggressive AR-negative PCa molecular subtypes, suggesting a potential link with PCa progression. Our study provides proof-of-concept evidence that organoids can mirror PCa patient-specific drug sensitivity profiles and molecular paths of disease progression, uncovering pertinent biomarkers. Ultimately, our organoid-xenograft model series provide a modular and scalable platform that can readily be used for mechanistic and translational studies. ### Competing Interest Statement The authors have declared no competing interest. Krebsliga Beider Basel, https://ror.org/05v5ag345, KLbB-5329-03-2021 Swiss National Science Foundation, https://ror.org/00yjd3n13, 320030_205086 University Hospital of Basel, Department of Surgery, PMC Platform

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. By exploiting a cohort of 164 PCa patient samples and tuning several culture parameters, we show that an extracellular matrix-free (ECM)-free culture system increases the take-rate of PDOs with luminal-like and PCa features. Single-cell RNA sequencing (scRNA-seq) reveals that ECM-free PDOs comprise cell populations associated with known PCa signatures and exhibit transcriptomic resemblance with their respective parental tumors. In addition, we define organoid-associated cell type signatures and identify markers discriminating tumors versus benign cells ex vivo and in situ. Furthermore, we generate the first prostate PDO single-cell atlas integrating previously-published scRNA-seq datasets and our newly-generated data. We show that Matrigel-based organoid cultures derived from primary PCa are essentially composed of benign-like epithelial cells, irrespective of the dataset or the malignant nature of the tissue of origin. In contrast, ECM-free conditions maintain heterogenous patient-specific luminal tumor cell populations and enrich in intermediate cell types. Ultimately, our work will significantly enhance the potential of PDOs in basic and translational PCa research. ### Competing Interest Statement The authors have declared no competing interest.

The application of patient-derived organoids (PDOs) in prostate cancer (PCa) research has been hampered by poor take rates and benign overgrowth. We highlight the limitations of existing culture conditions and identify extracellular matrix composition as a determinant of organoid outcome. Single-cell RNA sequencing reveals that Matrigel-free PDOs exhibit cellular heterogeneity, preserve patient-specific PCa cells with active androgen receptor signaling, and enrich in intermediate cells. In contrast, Matrigel fails to maintain primary PCa cells and produces in vitro basal-like features divergent from patient samples. Furthermore, we redefine cell-type signatures, identify biomarkers discriminating tumor versus other cell types, and show that expression of laminin-binding integrins is a hallmark of Matrigel-derived organoids. Finally, integrating previously published datasets with our data, we generate a prostate PDO single-cell atlas (PPScA), which captures a spectrum of cellular identities while revealing pathways altered in vitro. Our study provides methodological improvements for short-term culture and cellular biology insights.

Androgen deprivation therapy (ADT) is the mainstay for treatment of advanced castration-sensitive prostate cancer (CSPC); yet most patients eventually relapse and progress to lethal castration-resistant prostate cancer (CRPC). Here we aimed at generating single-cell transcriptomic profiles from advanced CSPC samples and ADT-treated patient-derived organoids (PDOs) to dissect cellular dynamics underlying response to androgen deprivation at single-cell resolution. Nine radical prostatectomy or metastasis resection specimens obtained from treatment-naïve high-grade CSPC patients were collected and processed into single-cell suspensions. Single-cell RNA sequencing (scRNA-seq) was carried out on six specimens meeting necessary quality criteria. In parallel, four PDO lines were established, characterized using whole exome sequencing, IHC and immunofluorescence, and cultured in androgen-deficient conditions to mimic ADT. Viability assay confirmed treatment efficacy before scRNA-seq was performed with a lipid-based multiplexing strategy (MULTI-seq). We generated an atlas encompassing the transcriptomic profiles of more than 20 000 cells originating from rare tissue material. While highlighting patient-specific tumor features and integrating cells from the microenvironment, our single-cell atlas also revealed various epithelial subtypes. This included a subset of intermediate cells, referred to as club/hillock cells, which were previously proposed to play a role in tumor progression. Interestingly, these cells were maintained in PDOs derived from non-metastatic tissues, whereas PDOs derived from metastatic ones were exclusively composed of tumor-like cells. Treated organoids exhibited diverse viability responses and pathway alterations, including changes in the Androgen Response Signature, reflecting their sensitivity to ADT and underscoring their value as clinically relevant in vitro models. Simultaneously, scRNA-seq revealed transcriptional shifts within specific subpopulations of treated PDOs, highlighting significant changes in additional pathways that provide insights into the mechanisms driving the onset of castration resistance. Ongoing analyses aim to determine whether these populations pre-exist in the tumor or if this state is gradually acquired during treatment. PDOs effectively mirror original prostate tumor characteristics and represent functional castration-sensitive models. By leveraging the generated scRNA-seq atlases and transcriptomic shift analyses, we anticipate identifying castration-tolerant cell populations potentially contributing to tumor heterogeneity and relapse. Romuald Parmentier, Julien Roux, Robin Dolgos, Raphaëlle Servant, Kirsten Mertz, Subotic Svetozar, Lukas Bubendorf, Helge Seifert, Cyrill A. Rentsch, Clémentine Le Magnen. Investigating prostate cancer cellular heterogeneity and treatment response at singe-cell level [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 1244.

Introduction: An important clinical need remains to improve survival of men with advanced metastatic prostate cancer (PCa). A better understanding of the mechanisms underlying PCa progression and treatment resistance is a prerequisite to address this challenge, which is currently limited by the scarcity of experimental models. Here, we aimed at establishing and characterizing new patient-derived organoids xenograft (PDOX) models of advanced PCa. Materials & methods: We used two patient-derived organoid lines (PDOs), previously generated in our laboratory. P20-11 PDOs were derived from a lung metastasis obtained from a patient with hormone-naïve PCa. P20-23 PDOs were derived from a transurethral prostate resection obtained from a patient with metastatic castration-resistant PCa, previously treated with goserelin, docetaxel, and enzalutamide. PDOX were generated by subcutaneous injection of PDOs in NOD scid gamma (NSG) male mice. Organoids were derived from the PDOX tumors (PDOX-O). Matched patients’ tumor, PDOs, PDOX and PDOX-O samples were characterized using immunohistochemistry (IHC), immunofluorescence (IF), and whole exome sequencing (WES). Organoids were treated with different concentrations of drugs and cell viability was measured using CellTiter-Glo 3D, after a 5-day long treatment. Results: P20-11 PDOs developed tumors in 2 out of 6 mice, 10 months after injection. IHC analysis highlighted a loss of PTEN expression, overexpression of ERG and P53, as well as strong AR and NKX3.1 expression in the tumor, PDOs, PDOX and PDOX-O samples. WES analysis uncovered mutations in CTNNB1, PTEN and TP53 in all samples, and approximately 87% of shared non-synonymous mutations between the PDOX and the original patients’ tumor. Similar to the original PDOs, P20-11 PDOX-O displayed androgen sensitivity in vitro. P20-23 PDOs formed tumors in 3 out of 3 mice 8 months following injection. IHC and IF analyses highlighted a strong expression of CK8, PSMA, AR and NKX3.1, as well as a loss of PTEN expression in the tumor, PDOs, PDOX and PDOX-O samples. WES identified a pathogenic mutation in the PCa-associated gene ZMYM3 in all samples, associated with loss of protein expression. The activating AR point mutation L702H, previously linked to AR signaling inhibitors resistance, was detected in the tumor and in 2 out of 3 PDOX and their derived organoids. Overall, an average of 80% of non-synonymous mutations were shared between the patients’ tumor and PDOX. Finally, P20-23 PDOX-O did not respond to docetaxel or enzalutamide but exhibited sensitivity to the PI3K/AKT inhibitor ipatasertib. Conclusion: We have successfully generated two novel PDOX models, which highly resemble the original patients’ tumor and can be further cultured as organoids. These models are representative of relevant clinical and molecular subtypes of advanced PCa, providing further opportunities for translational studies. Citation Format: Raphaëlle Servant, Zoi Diamantopoulou, Michele Garioni, Luca Roma, Tatjana Vlajnic, Arnoud J. Templeton, Heike Pueschel, Salvatore Piscuoglio, Nicola Aceto, Helge Seifert, Cyrill A. Rentsch, Bubendorf Lukas, Clémentine Le Magnen. Establishment and characterization of two novel patient-derived organoid xenograft models of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3085.

Patient‐derived organoids (PDOs) represent promising preclinical models in various tumor types. In the context of prostate cancer (PCa), however, their establishment has been hampered by poor success rates, which impedes their broad use for translational research applications. Along with the necessity to improve culture conditions, there is a need to identify factors influencing outcomes and to determine how to assess success versus failure in organoid generation. In the present study, we report our unbiased efforts to generate PDOs from a cohort of 81 PCa specimens with diverse pathological and clinical features. We comprehensively analyzed histological features of each enrolled sample (Gleason score, tumor content, proliferation index) and correlated them with organoid growth patterns. We identified improved culture conditions favoring the generation of PCa organoids, yet no specific intrinsic tumor feature was broadly associated with sustained organoid growth. In addition, we performed phenotypic and molecular characterization of tumor–organoid pairs using immunohistochemistry, immunofluorescence, fluorescence in situ hybridization, and targeted sequencing. Morphological and immunohistochemical profiles of whole organoids altogether provided a fast readout to identify the most promising ones. Notably, primary samples were associated with an initial take‐rate of 83% (n = 60/72) in culture, with maintenance of cancer cells displaying common PCa alterations, such as PTEN loss and ERG overexpression. These cancer organoids were, however, progressively overgrown by organoids with a benign‐like phenotype. Finally, out of nine metastasis samples, we generated a novel organoid model derived from a hormone‐naïve lung metastasis, which displays alterations in the PI3K/Akt and Wnt/β‐catenin pathways and responds to androgen deprivation. Taken together, our comprehensive study explores determinants of outcome and highlights the opportunities and challenges associated with the establishment of stable tumor organoid lines derived from PCa patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Department of Pathology, Institute of Medical Genetics and Pathology, and Department of Urology, University Hospital Basel, Basel, Switzerland; Department of Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland; Department of Urology, University Hospital Basel, Basel, Switzerland; Division of Urology, St. Clara Hospital, Basel, Switzerland; Division of Medical Oncology, St. Claraspital, Basel, and Faculty of Medicine, University of Basel, Basel, Switzerland; Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, and Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland; Department for BioMedical Research, University of Bern, Bern, Switzerland; Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland; Department of Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, and Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland; 1Department for BioMedical Research, University of Bern, Bern, Switzerland and Bern Center for Precision Medicine, University of Bern and Inselspital, Bern, Switzerland

New components of the intestinal stem cell niche are constantly being discovered. Recently, the subepithelial Foxl1+telocytes (TCFoxl1+) were found to play a key role in maintaining and regulating intestinal crypt homeostasis. TCFoxl1+ produce crucial niche components such as WNT2b, WNT5a and RSPO3 as well as the Bone Morphogenic Proteins (BMP) inhibitors Gremlin 1 and 2. Previous work in our laboratory showed that epithelial BMP signaling is involved in terminal differentiation and maturation of cells from the secretory lineage. Our recent work showed that the loss of mesenchymal BMP signaling leads to the development of a pathological microenvironment inducing colonic and gastric polyposis in older mice, drawing attention to the impact of a pathogenic microenvironment in polyposis.By genetic means, we propose to address how BMP signaling in TCFoxl1+ modulates the intestinal stem cell niche. Using the Cre/loxP system, we generated mice with a deletion of BmpR1a in all gastrointestinal subepithelial TCFoxl1+. Histological analysis and terminal differentiation assessment were performed with cellular staining and immunofluorescence (H&E, Alcian Blue and Lysozyme). Niche components were analysed by RT‐qPCR from crypt epithelium or total ileal RNA extracts. H&E staining on 9 months‐old BmpR1aΔFoxl1+ and control mice ileum demonstrated perturbed architecture in the BmpR1aΔFoxl1+ such as fused and enlarged villi compared to control mice. Alcian Blue staining showed an increase of goblet cells in the mutant mice. Electron microscopy showed that Paneth cells from mutant mice have larger vesicles than controls. The RT‐qPCR analysis showed a decrease in WNT signaling targets in the crypts epithelium cells. Indeed, LGR5 is decreased by 50% and mTERT by 34% in the mutant mice, whereas the stem cell marker BMI1 is not modulated. Maturity of Paneth cells is impaired in the mutant mice as shown by a 41% decrease in SOX9 expression. This lack in Paneth cell maturity is also reflected by a 60% decrease in WNT3 expression in BmpR1aΔFoxl1+ mice, an important stem cell niche factor. Secretory cell determination transcription factor ATOH1 is decreased by 35% in the mutant mice. Analysis from total ileal extract revealed a decrease of WNT2b, a key niche factor from the mesenchymal cells.Thus, our results show that altering Bmp signaling in subepithelial TCFoxl1+ modulates crucial niche factors influencing intestinal stem cells homeostasis and their subsequent determination.Support or Funding InformationCanadian Institutes of Health Research‐Institute of Nutrition, Metabolism and Diabetes

Studies of colorectal cancer (CRC) traditionally focused on the role of epithelial genetic mutations and signaling pathway dysregulation with an underappreciation of faulty signaling impact from the stromal microenvironment in this context.1 The colonic stroma consists of professional and nonprofessional cells with fibroblasts, myofibroblasts, and telocytes found in close contact with the epithelia.2,3 These latter cells are responsible for the contribution of the microenvironment surrounding the epithelium.

Intestinal epithelial cells form a protective barrier in limiting gut luminal content potentially harmful to the host. Upon gut epithelium injury, several signals instruct epithelial cells to undergo a rapid healing process. Defects in this process induce inflammatory responses and can further evolve into chronic gut inflammatory diseases. We previously identified the transcription factor CUX1 as crucial for protecting against experimental colitis in mice. However, the precise molecular mechanisms by which CUX1 intervenes during this biological process are unknown. Our aim was to evaluate CUX1 biological and functional roles during intestinal epithelial cell wound healing. RNAi knockdown of CUX1 in intestinal epithelial cells revealed a crucial role for this regulator in migratory response following wounding assays. Gene expression profiling identified several gene transcripts modulated in absence of CUX1 during wound healing for which a significant number was associated with cell motility and cytoskeleton function. Chromatin immunoprecipitation assays identified the guanine nucleotide exchange factor Vav2 gene as a direct target for CUX1. Coincidently, reduction of VAV2 in absence of CUX1 was associated with a significant decrease of RAC1 activity in response to epithelial wounding. Our results identify a novel pathway by which CUX1 regulates normal intestinal epithelial cell restitution.