Intra‐epithelial non‐canonical Activin A signaling safeguards prostate progenitor quiescence
2022; Springer Nature; Volume: 23; Issue: 5 Linguagem: Inglês
10.15252/embr.202154049
ISSN1469-3178
AutoresFrancesco Cambuli, Veronica Foletto, Alessandro Alaimo, Dario De Felice, Francesco Gandolfi, Maria Dilia Palumbieri, Michela Zaffagni, Sacha Genovesi, Marco Lorenzoni, Martina Celotti, Emiliana Bertossio, Giosuè Mazzero, Arianna Bertossi, Alessandra Bisio, Francesco Berardinelli, Antonio Antoccia, Marco Gaspari, Mattia Barbareschi, Michelangelo Fiorentino, Michael M. Shen, Massimo Loda, Alessandro Romanel, Andrea Lunardi,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle7 March 2022Open Access Source DataTransparent process Intra-epithelial non-canonical Activin A signaling safeguards prostate progenitor quiescence Francesco Cambuli Corresponding Author Francesco Cambuli [email protected] The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Department of Medicine, Genetics and Development, Urology, Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Veronica Foletto Veronica Foletto orcid.org/0000-0003-3115-7636 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Conceptualization, Data curation, Formal analysis, Validation, Visualization, Methodology, Writing - original draft Search for more papers by this author Alessandro Alaimo Alessandro Alaimo orcid.org/0000-0002-5888-6268 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Dario De Felice Dario De Felice orcid.org/0000-0001-8121-8260 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Francesco Gandolfi Francesco Gandolfi Laboratory of Bioinformatics and Computational Genomics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Maria Dilia Palumbieri Maria Dilia Palumbieri orcid.org/0000-0002-0462-844X The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Michela Zaffagni Michela Zaffagni orcid.org/0000-0002-2483-8727 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Sacha Genovesi Sacha Genovesi orcid.org/0000-0002-3568-0048 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology Search for more papers by this author Marco Lorenzoni Marco Lorenzoni orcid.org/0000-0002-3040-0633 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Martina Celotti Martina Celotti The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Emiliana Bertossio Emiliana Bertossio The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Giosuè Mazzero Giosuè Mazzero Santa Chiara Hospital-APSS, Trento, Trento, Italy Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Arianna Bertossi Arianna Bertossi The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Alessandra Bisio Alessandra Bisio orcid.org/0000-0002-3326-1923 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Resources, Investigation, Methodology Search for more papers by this author Francesco Berardinelli Francesco Berardinelli Department of Science, University of Roma Tre, Roma, Italy Laboratory of Neurodevelopment, Neurogenetics and Molecular Neurobiology Unit, IRCCS Santa Lucia Foundation, Roma, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Antonio Antoccia Antonio Antoccia Department of Science, University of Roma Tre, Roma, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Marco Gaspari Marco Gaspari Department of Experimental and Clinical Medicine, University of Catanzaro, Catanzaro, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Mattia Barbareschi Mattia Barbareschi orcid.org/0000-0003-0217-3223 Santa Chiara Hospital-APSS, Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Michelangelo Fiorentino Michelangelo Fiorentino orcid.org/0000-0002-1749-150X Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Michael M Shen Michael M Shen orcid.org/0000-0002-4042-1657 Department of Medicine, Genetics and Development, Urology, Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA Contribution: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - review & editing Search for more papers by this author Massimo Loda Massimo Loda orcid.org/0000-0001-9674-8379 Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, USA Contribution: Data curation, Formal analysis, Methodology, Writing - review & editing Search for more papers by this author Alessandro Romanel Alessandro Romanel orcid.org/0000-0003-4855-8620 Laboratory of Bioinformatics and Computational Genomics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Andrea Lunardi Corresponding Author Andrea Lunardi [email protected] orcid.org/0000-0001-6218-2565 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Francesco Cambuli Corresponding Author Francesco Cambuli [email protected] The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Department of Medicine, Genetics and Development, Urology, Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Veronica Foletto Veronica Foletto orcid.org/0000-0003-3115-7636 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Conceptualization, Data curation, Formal analysis, Validation, Visualization, Methodology, Writing - original draft Search for more papers by this author Alessandro Alaimo Alessandro Alaimo orcid.org/0000-0002-5888-6268 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Dario De Felice Dario De Felice orcid.org/0000-0001-8121-8260 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Francesco Gandolfi Francesco Gandolfi Laboratory of Bioinformatics and Computational Genomics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Maria Dilia Palumbieri Maria Dilia Palumbieri orcid.org/0000-0002-0462-844X The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Michela Zaffagni Michela Zaffagni orcid.org/0000-0002-2483-8727 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Sacha Genovesi Sacha Genovesi orcid.org/0000-0002-3568-0048 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology Search for more papers by this author Marco Lorenzoni Marco Lorenzoni orcid.org/0000-0002-3040-0633 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Martina Celotti Martina Celotti The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Emiliana Bertossio Emiliana Bertossio The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Giosuè Mazzero Giosuè Mazzero Santa Chiara Hospital-APSS, Trento, Trento, Italy Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Arianna Bertossi Arianna Bertossi The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Alessandra Bisio Alessandra Bisio orcid.org/0000-0002-3326-1923 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Resources, Investigation, Methodology Search for more papers by this author Francesco Berardinelli Francesco Berardinelli Department of Science, University of Roma Tre, Roma, Italy Laboratory of Neurodevelopment, Neurogenetics and Molecular Neurobiology Unit, IRCCS Santa Lucia Foundation, Roma, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Antonio Antoccia Antonio Antoccia Department of Science, University of Roma Tre, Roma, Italy Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Marco Gaspari Marco Gaspari Department of Experimental and Clinical Medicine, University of Catanzaro, Catanzaro, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Mattia Barbareschi Mattia Barbareschi orcid.org/0000-0003-0217-3223 Santa Chiara Hospital-APSS, Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Michelangelo Fiorentino Michelangelo Fiorentino orcid.org/0000-0002-1749-150X Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Michael M Shen Michael M Shen orcid.org/0000-0002-4042-1657 Department of Medicine, Genetics and Development, Urology, Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA Contribution: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - review & editing Search for more papers by this author Massimo Loda Massimo Loda orcid.org/0000-0001-9674-8379 Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, USA Contribution: Data curation, Formal analysis, Methodology, Writing - review & editing Search for more papers by this author Alessandro Romanel Alessandro Romanel orcid.org/0000-0003-4855-8620 Laboratory of Bioinformatics and Computational Genomics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Andrea Lunardi Corresponding Author Andrea Lunardi [email protected] orcid.org/0000-0001-6218-2565 The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Francesco Cambuli *,1,2,10,†, Veronica Foletto1,†, Alessandro Alaimo1, Dario De Felice1, Francesco Gandolfi3, Maria Dilia Palumbieri1, Michela Zaffagni1, Sacha Genovesi1, Marco Lorenzoni1, Martina Celotti1, Emiliana Bertossio1, Giosuè Mazzero4, Arianna Bertossi1, Alessandra Bisio1, Francesco Berardinelli5,6, Antonio Antoccia5, Marco Gaspari7, Mattia Barbareschi4, Michelangelo Fiorentino8, Michael M Shen2, Massimo Loda9, Alessandro Romanel3 and Andrea Lunardi *,1 1The Armenise-Harvard Laboratory of Cancer Biology & Genetics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy 2Department of Medicine, Genetics and Development, Urology, Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA 3Laboratory of Bioinformatics and Computational Genomics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy 4Santa Chiara Hospital-APSS, Trento, Trento, Italy 5Department of Science, University of Roma Tre, Roma, Italy 6Laboratory of Neurodevelopment, Neurogenetics and Molecular Neurobiology Unit, IRCCS Santa Lucia Foundation, Roma, Italy 7Department of Experimental and Clinical Medicine, University of Catanzaro, Catanzaro, Italy 8Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 9Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, USA 10Present address: Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA † These authors contributed equally to this work as first authors *Corresponding author. Tel: +1 646 888 3997; E-mail: [email protected] *Corresponding author. Tel: +39 0461 285288; E-mail: [email protected] EMBO Reports (2022)23:e54049https://doi.org/10.15252/embr.202154049 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-β ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra-epithelial non-canonical Activin A signaling inhibits cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non-canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors. Synopsis Non-canonical Activin A signalling in the prostate inhibits cell proliferation in a Smad-independent manner and safeguards epithelial quiescence. This represents a key molecular mechanism controlling prostate quiescence, with implications for benign prostate hyperplasia and tumorigenesis. Activin A signalling inhibits prostate progenitor proliferation through a non-canonical pathway mediated by Tak1/p38. Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Reduced Activin A signalling is associated with DNA replication stress and aneuploidy in 3D organoids. Dysregulation of the pathway leads to dysplastic growth in an orthotopic transplantation mouse model. Introduction The healthy prostate is a relatively quiescent tissue during adulthood (De Marzo et al, 1998; Toivanen & Shen, 2017). In contrast, the overgrowth of the prostatic epithelium is one of the most common conditions experienced by aging men, being linked with urinary dysfunction and tumorigenesis (Ørsted & Bojesen, 2013). The molecular mechanisms causing exit from quiescence are poorly understood. Chronic inflammation—potentially induced by infection (e.g., prostatitis) (Shinohara et al, 2013; Kwon et al, 2014; Simons et al, 2015), chemical damage (e.g., urine reflux) (Kirby et al, 1982), physical trauma (e.g., corpora amylacea) (DuPre et al, 2018), dietary carcinogens (Nakai et al, 2007), obesity (Kwon et al, 2016a), hormonal imbalance (e.g., low systemic androgen levels) (Zhang et al, 2016; Wang et al, 2017), and aging (Crowell et al, 2019)—has been implicated in DNA damage, oxidative stress, and atrophy, leading to a proliferative response (Sfanos et al, 2018; de Bono et al, 2020). Considering the high frequency of these events, it would be logical to hypothesize specialized mechanisms to safeguard epithelial quiescence, but they have been rarely investigated. It has long been known that transforming growth factor β (TGF-β) signaling inhibits the proliferation of a large variety of epithelial cell types (Tucker et al, 1984; Moses et al, 2016), including those of the prostate (McKeehan & Adams, 1988). SMAD factors are the canonical intracellular mediators of this signaling, but additional non-canonical pathways can also be triggered by TGF-β receptors (Massagué, 2012; Derynck & Budi, 2019). In gastrointestinal (GI) carcinomas (e.g., pancreas, colon), the canonical pathway is frequently mutated (Bailey et al, 2016; Yaeger et al, 2018). However, outside of the GI tract, TGF-β/SMAD components are rarely inactivated in tumors, leaving unexplained the nature of the intracellular signaling responsible for the cytostatic effect of TGF-β (David & Massagué, 2018; Gerstung et al, 2020). Enhanced Tgf-β signaling has been linked with the presence of quiescent epithelial progenitors in the proximal/periurethral region of the mouse prostate (Salm et al, 2005; Wei et al, 2019). Recent single-cell studies have confirmed the enrichment of a variety of epithelial progenitors—basal, luminal proximal (LumP), and periurethral (PrU) cells—in this anatomical district, though also present at low frequency in the distal compartment (Henry et al, 2018; Crowley et al, 2020; Guo et al, 2020; Joseph et al, 2020; Karthaus et al, 2020; Mevel et al, 2020). Such cells are known to be particularly quiescent during homeostasis (Pignon et al, 2015; Kwon et al, 2020), but also to exhibit extensive regenerative potential in ex vivo assays (Kwon et al, 2016b; Crowley et al, 2020). Thus, the TGF-β-induced cytostatic response in epithelial progenitors may be relevant for the control of quiescence, but the complexity of this pathway, the lack of interpretable genetic alterations in patients, and the heterogeneous cellular composition of the prostate have so far hampered mechanistic investigations. Here, we reasoned that prostate organoid models (Chua et al, 2014; Karthaus et al, 2014)—in combination with orthotopic transplantation approaches—may provide a biologically relevant, and experimentally amenable, system for addressing this question. Results Mouse prostate organoid cultures enable the continuous expansion of epithelial progenitors in a near-physiological manner Initially, we set out to assess whether mouse prostate organoids are a representative and informative model for the study of the prostate epithelium, in light of recent discoveries on prostate cellular heterogeneity and dynamics (Barros-Silva et al, 2018; Henry et al, 2018; Crowley et al, 2020; Guo et al, 2020; Karthaus et al, 2020; Kwon et al, 2020; Mevel et al, 2020). Considering our interest in signaling, we focused on a culture method in defined media conditions. This protocol relies on a mix of growth factors and inhibitors, including Egf, Noggin, R-spondin 1, the Tgf-β receptors inhibitor A83-01, and dihydrotestosterone (ENRAD) (Karthaus et al, 2014; Drost et al, 2016). We generated a biobank of mouse prostate organoids, starting from bulk populations of cells from distinct prostate lobes and mouse strains (Fig 1A, Appendix Fig S1A–E). In line with previous studies (Chua et al, 2014; Karthaus et al, 2014; Drost et al, 2016), we found that, upon tissue dissociation, only a small fraction of cells (approx. 1%) was capable to generate organoids in culture and that organoid-forming efficiency increased over passages, suggesting enrichment for epithelial progenitors (Fig 1B). To gain greater insights, we longitudinally tracked organoid formation—from single cells to fully formed organoids—and we observed a progressive expansion of cells expressing the progenitor epithelial surface antigen Sca-1 (encoded by Ly6a; Fig 1C) (Kwon et al, 2016b; Crowley et al, 2020). Thereafter, the level of Sca-1 appeared to be stable over a long culture period (e.g., 10 weeks). To extend our observations, we performed transcriptomic analyses on three organoid lines derived from distinct mouse prostate lobes (Fig 1D and E). Consistently with enrichment for epithelial progenitors, organoids expressed high levels of genes specific for the proximal and periurethral compartments (e.g., Psca, Tacstd2, and Ly6d), as well as basal (e.g., Krt5, Krt14, Trp63) and luminal marker genes (e.g., Krt8, Ar, Foxa1). In contrast, distal luminal markers were lowly expressed (e.g., Nkx3.1, Pbsn, Sbp). Histological (H&E) and immunofluorescent (IF) analyses confirmed that prostate organoids, for the most part, are made up of a bilayer of cuboidal cells, displaying progenitor marker proteins (e.g., Krt7, Ppp1r1b), and resembling the cyto-architecture of the periurethral/proximal compartment (Figs 1F–H and EV1A). As expected for periurethral/proximal cells—which are known to be castration resistant—mouse prostate organoids were reversibly dependent on androgen for lumen formation (Fig EV1B–D), but minimally for their survival (Fig EV1E). Figure 1. Mouse prostate organoids are highly enriched in epithelial progenitor cells Schematic diagram describing organoid culture derivation (AP, Anterior Prostate; DLP, Dorso-Lateral Prostate; VP, Ventral Prostate; SV, Seminal Vesicle; DHT, dihydrotestosterone). Mouse prostate organoid forming efficiency. Efficiency at derivation (left; n = 6 biological replicates, data points are shown with crossing line representing mean value). Efficiency at passage 1-3 (right; n ≥ 3 biological replicates per organoid line/passage; data are presented as mean). Representative longitudinal flow cytometry analysis of dissociated organoid cells (Bas, Basal; LumP, Luminal Proximal; PrU, Periurethral; Lum Distal, Luminal Distal). End-point RT–PCR analysis for selected marker genes (n = 3 biological replicates). Bulk-RNAseq analysis (n = 3 biological replicates; individual data points are shown with bar graphs representing mean value). Representative hematoxylin-eosin (H&E) staining of mouse prostate tissue and organoid sections (scale bars = 50 μm). Representative immunofluorescence (IF) analysis for selected markers in mouse prostate tissue and organoid sections (scale bars = 50 μm). IF staining for selected markers in mouse prostate tissue and organoid sections (scale bars = 50 μm). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Mouse prostate organoids are dependent on androgen signaling for lumen formation—but not for survival, related to Fig 1 Immunofluorescence analysis for selected markers in mouse prostate tissue and organoid sections. Scale bars = 50 µm. Stereoscopic images of mouse prostate organoids experiencing transient DHT removal (androgen cycling). Lumen formation necessitates androgen signaling. Scale bar = 1 mm. Western blot analysis for androgen receptor (Ar) expression in mouse prostate organoids experiencing androgen cycling, and control cell lines expressing (LNCaP) or not expressing (PC3) the receptor. qRT–PCR mRNA expression analysis for selected androgen-responsive genes in the presence or absence of DHT. Data points are shown with crossing line representing mean value (n = 3 biological replicates; Student's t-test, two-tailed, P-value *< 0.05, **< 0.01, ***< 0.001). Western blot analysis for apoptotic markers (Cleaved Caspase 3, Full-length, and Cleaved Parp) expression in mouse prostate organoids cultured with or without DHT (n = 2 biological replicates). Source data are available online for this figure. Download figure Download PowerPoint The epithelium of the prostate is characterized by a slow cellular turnover. In contrast, prostate organoids appeared to proliferate indefinitely (Fig EV2A and B)—while retaining low levels of genomic instability (Fig EV2C and D)—raising the question of how culture conditions enable persistent cycling in a near-physiological manner. Either an excess of stimulatory cues in culture, or a lack of inhibitory ones—or both—may explain the shift from homeostatic quiescence in vivo to unrestrained mitotic activity in vitro. Using a "n -1 approach" for assessing the requirement for growth factors and inhibitors in culture (Fig EV3A), we found that prostate organoids are strictly dependent on exogenous Egf (Fig EV3B–D), as well as Tgf-β receptors inhibition by A83-01. Given the limited understanding of prostate quiescence, we focused on the requirement for the Tgf-β receptors inhibitor A83-01 for the continuous expansion
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