Single-cell RNA sequencing of human prostate basal epithelial cells reveals zone-specific cellular populations and gene expression signatures.

Despite evidence of genetic signatures in normal tissue correlating with disease risk, prospectively identifying genetic drivers and cell types that underlie subsequent pathologies has historically been challenging. The human prostate is an ideal model to investigate this phenomenon because it is anatomically segregated into three glandular zones (central, peripheral, and transition) that develop differential pathologies: prostate cancer in the peripheral zone (PZ) and benign prostatic hyperplasia (BPH) in the transition zone (TZ), with the central zone (CZ) rarely developing disease. More specifically, prostatic basal cells have been implicated in differentiation and proliferation during prostate development and regeneration; however, the contribution of zonal variation and the critical role of basal cells in prostatic disease etiology are not well understood. Using single-cell RNA sequencing of primary prostate epithelial cultures, we elucidated organ-specific, zone-specific, and cluster-specific gene expression differences in basal cells isolated from human prostate and seminal vesicle (SV). Aggregated analysis identified ten distinct basal clusters by Uniform Manifold Approximation and Projection. Organ specificity compared gene expression in SV with the prostate. As expected, SV cells were distinct from prostate cells by clustering, gene expression, and pathway analysis. For prostate zone specificity, we identified two CZ-specific clusters, while the TZ and PZ populations clustered together. Despite these similarities, differential gene expression was identified between PZ and TZ samples that correlated with gene expression profiles in prostate cancer and BPH, respectively. Zone-specific profiles and cell type-specific markers were validated using immunostaining and bioinformatic analyses of publicly available RNA-seq datasets. Understanding the baseline differences at the organ, zonal, and cellular level provides important insight into the potential drivers of prostatic disease and guides the investigation of novel preventive or curative treatments. Importantly, this study identifies multiple prostate basal cell populations and cell type-specific gene signatures within prostate basal epithelial cells that have potential critical roles in driving prostatic diseases. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Sox2 Expression Marks Castration-Resistant Progenitor Cells in the Adult Murine Prostate.

Identification of defined epithelial cell populations with progenitor properties is critical for understanding prostatic development and disease. Here, we demonstrate that Sox2 expression is enriched in the epithelial cells of the proximal prostate adjacent to the urethra. We use lineage tracing of Sox2-positive cells during prostatic development, homeostasis, and regeneration to show that the Sox2 lineage is capable of self-renewal and contributes to prostatic regeneration. Persisting luminal cells express Sox2 after castration, highlighting a potential role for Sox2 in cell survival and castration-resistance. In addition to revealing a novel progenitor population in the prostate, these data implicate Sox2 as a regulatory factor of adult prostate epithelial stem cells. Stem Cells 2019;37:690-700.

Single-cell RNA-Seq identifies factors necessary for the regenerative phenotype of prostate luminal epithelial progenitors.

Benign prostate hyperplasia and prostate cancer are common diseases that involve the overgrowth of prostatic tissue. Although their pathologies and symptoms differ, both diseases show aberrant activation of prostate progenitor cell phenotypes in a tissue that should be relatively quiescent. This phenomenon prompts a need to better define the normal prostate progenitor cell phenotype and pursue the discovery of causal networks that could yield druggable targets to combat hyperplastic prostate diseases. We used single-cell (sc) RNA-Seq analysis to confirm the identity of a luminal progenitor cell population in both the hormonally intact and castrated mouse prostate. Using marker genes from our scRNA-Seq analysis, we identified factors necessary for the regeneration phenotype of prostate organoids derived from mice and humans in vitro. These data outline potential factors necessary for prostate regeneration and utilization of scRNA-Seq approaches for the identification of pharmacologic strategies targeting critical cell populations that drive prostate disease.

The role of the androgen receptor in prostate development and benign prostatic hyperplasia: A review.

Benign prostatic hyperplasia (BPH) is a benign enlargement of the prostate in which incidence increases linearly with age, beginning at about 50 years old. BPH is a significant source of morbidity in aging men by causing lower urinary tract symptoms and acute urinary retention. Unfortunately, the etiology of BPH incidence and progression is not clear. This review highlights the role of the androgen receptor (AR) in prostate development and the evidence for its involvement in BPH. The AR is essential for normal prostate development, and individuals with defective AR signaling, such as after castration, do not experience prostate enlargement with age. Furthermore, decreasing dihydrotestosterone availability through therapeutic targeting with 5α-reductase inhibitors diminishes AR activity and results in reduced prostate size and symptoms in some BPH patients. While there is some evidence that AR expression is elevated in certain cellular compartments, how exactly AR is involved in BPH progression has yet to be elucidated. It is possible that AR signaling within stromal cells alters intercellular signaling and a "reawakening" of the embryonic mesenchyme, loss of epithelial AR leads to changes in paracrine signaling interactions, and/or chronic inflammation aids in stromal or epithelial proliferation evident in BPH. Unfortunately, a subset of patients fails to respond to current medical approaches, forcing surgical treatment even though age or associated co-morbidities make surgery less attractive. Fundamentally, new therapeutic approaches to treat BPH are not currently forthcoming, so a more complete molecular understanding of BPH etiology is necessary to identify new treatment options.

Single-cell RNA-Seq analysis identifies a putative epithelial stem cell population in human primary prostate cells in monolayer and organoid culture conditions.

Human primary prostate epithelial (PrE) cells represent patient-derived in vitro models and are traditionally grown as a monolayer in two-dimensional culture. It has been recently demonstrated that expansion of primary cells into three-dimensional prostatic organoids better mimics prostate epithelial glands by recapitulating epithelial differentiation and cell polarity. Here, we sought to identify cell populations present in monolayer PrE cells and organoid culture, grown from the same patient, using single-cell RNA-sequencing. Single-cell RNA-sequencing is a powerful tool to analyze transcriptome profiles of thousands of individual cells simultaneously, creating an in-depth atlas of cell populations within a sample. Organoids consisted of six distinct cell clusters (populations) of intermediate differentiation compared to only three clusters in the monolayer prostate epithelial cells. Integrated analysis of the datasets allowed for direct comparison of the monolayer and organoid samples and identified 10 clusters, including a distinct putative prostate stem cell population that was high in Keratin 13 (KRT13), Lymphocyte Antigen 6D (LY6D), and Prostate Stem Cell Antigen (PSCA). Many of the genes within the clusters were validated through RT-qPCR and immunofluorescence in PrE samples from 5 additional patients. KRT13+ cells were observed in discrete areas of the parent tissue and organoids. Pathway analyses and lack of EdU incorporation corroborated a stem-like phenotype based on the gene expression and quiescent state of the KRT13+ cluster. Other clusters within the samples were similar to epithelial populations reported within patient prostate tissues. In summary, these data show that the epithelial stem population is preserved in PrE cultures, with organoids uniquely expanding intermediate cell types not present in monolayer culture.