Understanding the cellular constituents of the prostate is essential for identifying the cell of origin for prostate adenocarcinoma. Here we describe a comprehensive single-cell atlas of the adult mouse prostate epithelium, which displays extensive heterogeneity. We observe distal lobe-specific luminal epithelial populations (LumA, LumD, LumL, and LumV), a proximally-enriched luminal population (LumP) that is not lobe-specific, and a periurethral population (PrU) that shares both basal and luminal features. Functional analyses suggest that LumP and PrU cells have multipotent progenitor activity in organoid formation and tissue reconstitution assays. Furthermore, we show that mouse distal and proximal luminal cells are most similar to human acinar and ductal populations, that a PrU-like population is conserved between species, and that the mouse lateral prostate is most similar to the human peripheral zone. Our findings elucidate new prostate epithelial progenitors, and help resolve long-standing questions about anatomical relationships between the mouse and human prostate.

Embryonic (ES) and trophoblast (TS) stem cells reflect the first, irrevocable cell fate decision in development that is reinforced by distinct epigenetic lineage barriers. Nonetheless, ES cells can seemingly acquire TS-like characteristics upon manipulation of lineage-determining transcription factors or activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Here we have interrogated the progression of reprogramming in ES cell models with regulatable Oct4 and Cdx2 transgenes or conditional Erk1/2 activation. Although trans-differentiation into TS-like cells is initiated, lineage conversion remains incomplete in all models, underpinned by the failure to demethylate a small group of TS cell genes. Forced expression of these non-reprogrammed genes improves trans-differentiation efficiency, but still fails to confer a stable TS cell phenotype. Thus, even ES cells in ground-state pluripotency cannot fully overcome the boundaries that separate the first cell lineages but retain an epigenetic memory of their ES cell origin.

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A single-cell atlas of the mouse and human prostate reveals heterogeneity and conservation of 3 epithelial progenitors 4 5 6 Summary 24 Understanding the cellular constituents of the prostate is essential for identifying the 25 cell of origin for benign prostatic hyperplasia and prostate adenocarcinoma. Here we 26 describe a comprehensive single-cell atlas of the adult mouse prostate epithelium, which 27 demonstrates extensive heterogeneity. We observe distinct lobe-specific luminal epithelial 28 populations (LumA, LumD, LumL, and LumV) in the distal region of the four prostate lobes, 29 a proximally-enriched luminal population (LumP) that is not lobe-specific, as well as a 30 periurethral population (PrU) that shares both basal and luminal features. Functional 31 analyses suggest that LumP and PrU cells have multipotent progenitor activity in organoid 32 formation and tissue reconstitution assays. Furthermore, we show that mouse distal and 33 proximal luminal cells are most similar to human acinar and ductal populations, that a PrU-34 like population is conserved between species, and that the mouse lateral prostate is most 35 similar to the human peripheral zone. Our findings elucidate new prostate epithelial 36 progenitors, and help resolve long-standing questions about the anatomical relationships 37 between the mouse and human prostate. 38 39 40 3

Therapy resistance is a major roadblock in oncology. Exacerbation of molecular dysfunctions typical of cancer cells have proven effective in twisting oncogenic mechanisms to lethal conditions, thus offering new therapeutic avenues for cancer treatment. Here, we demonstrate that selective agonists of Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), a cation channel characteristic of the prostate epithelium frequently overexpressed in advanced stage III/IV prostate cancers (PCa), sensitize therapy refractory models of PCa to radio, chemo or hormonal treatment. Overall, our study demonstrates that pharmacological-induced Ca2+ cytotoxicity is an actionable strategy to sensitize cancer cells to standard therapies.

The imminent release of tissue atlases combining multichannel microscopy with single-cell sequencing and other omics data from normal and diseased specimens creates an urgent need for data and metadata standards to guide data deposition, curation and release. We describe a Minimum Information about Highly Multiplexed Tissue Imaging (MITI) standard that applies best practices developed for genomics and for other microscopy data to highly multiplexed tissue images and traditional histology.

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-b 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.

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