Urethral luminal epithelia are castration-insensitive cells of the proximal prostate

Abstract

Background: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors.

Methods: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry.

Results: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor.

Conclusions: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.

Keywords: benign prostatic hyperplasia; castration; prostate stem cell; prostatic urethra; single-cell RNA sequencing.

FIGURE 1

Identification of epithelial cell types of the mouse prostate and prostatic urethra by scRNA-seq. A and B, Mouse prostate lobes (n = 4) were dissected away from the rhabdosphincter of the urethra (n = 3) and each anatomical region was processed into a single-cell suspension and barcoded separately for scRNA-seq. The data were aggregated and subclustered by epithelial lineage (see Figure S1), and separated into (A) prostate and (B) urethra. C, Statistical correlation of each epithelial cluster of the mouse prostate and urethra with human epithelial cell types. D, Dot plot of differentially expressed genes for each cluster. AP, anterior prostate; DLP, dorsolateral prostate; ED, ejaculatory duct; Lum, luminal; scRNA-seq, single-cell RNA sequencing; SV, seminal vesicle; Ur, urethra; VP, ventral prostate

FIGURE 2

Urethral luminal cells express prostate progenitor markers and extend into the proximal prostate. A, Transverse section through the mouse prostatic urethra labeled with antibodies to KRT4 (in white, labels urethral luminal cells), KRT5 (in green, labels basal cells) and NKX3.1 (in red, labels prostate secretory luminal cells). Magnified insets from (A) are shown in (A′) and (A″). B, Transverse section through a normal human prostate labeled with antibodies to KRT13 (in white, labels hillock urethral cells), KRT5 (in green, labels basal cells) and ACPP (in red, labels prostate secretory luminal cells). Magnified insets from (B) are shown in (B′) and (B″). DAPI staining is shown in blue. C, Dot plot of mouse prostate and urethral epithelial cell type-specific markers and progenitor cell markers. D, Correlation of mouse scRNA-seq clusters to the transcriptomic signature of LSC^med “luminal progenitors” from Sackmann Sala et al. AP, anterior prostate; DAPI, 4′,6-diamidino-2-phenylindole; DLP, dorsolateral prostate; ED, ejaculatory duct; Lum, luminal; scRNA-seq, single-cell RNA sequencing; SV, seminal vesicle; Ur, urethra; VP, ventral prostate

FIGURE 3

Trop2+ urethral luminal cells are enriched in the prostates of castrated mice. A, Dissection of mouse prostate lobes away from urethra, and into proximal and distal regions. B, Flow cytometry of CD26+ prostate luminal epithelia, PDPN+ basal epithelia, and Trop2+ urethral epithelia in the urethra, proximal prostate, and distal prostate from intact mice. Data are representative of n = 4 independent experiments. C, Flow cytometry of Trop2+ urethral luminal cells in prostate tissue from sham-castrated and castrated mice. D and E, Whole-mount lower urinary tract sections from sham-castrated and castrated mice labeled with antibodies to Trop2 (in red) and counterstained with hematoxylin to stain nuclei. Images are representative of n = 3 mice per group. AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate

FIGURE 4

Urethral epithelial identity is established early in prostate development. Mouse lower urinary tract sagittal sections labeled with antibodies to KRT4 (in red, labels urethral luminal cells) and KRT5 (in green, labels basal cells). Stages shown are (A) embryonic day 18.5 (budding), (B) postnatal day 9 (branching), and (C) adult mouse prostate. Human lower urinary tract sagittal sections labeled with antibodies to KRT13 (in white, labels hillock urethral cells), SCGB1A1 (in red, labels club urethral cells), and KRT5 (in green, labels basal cells). Stages shown are (D) 12-week gestation (budding), (E) 19-week gestation (branching), and (F) adult human prostate. DAPI staining is shown in blue. DAPI, 4′,6-diamidino-2-phenylindole

FIGURE 5

Urethral epithelia are enriched in human BPH and are resistant to 5ARI treatment. A, scRNA-seq of tissue from three patients with glandular BPH demonstrates an enrichment of club epithelia compared with the three young adult normal prostates. B, Quantification of PSCA+ urethral epithelia from normal prostate and BPH prostates by flow cytometry (n = 6 per group). P value was obtained from Student’s t test performed on the two independent groups. C, Fold enrichment of the top 15 KEGG pathways significantly upregulated in club urethral luminal cells from BPH vs normal prostate. D-F, Dual IHC for KRT13 (in red, labels hillock urethral cells) and SCGB1A1 (in brown, labels club urethral cells) on prostate sections from (D) normal adults (n = 5), (E) patients with glandular BPH (n = 10) and (F) 5ARI-treated BPH patients (n = 11). Nuclei were counterstained with hematoxylin. 5ARI, 5-α reductase inhibitor; BPH, benign prostatic hyperplasia; FACS, fluorescence-activated cell sorting; IHC, immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; PSCA, prostate stem cell antigen; scRNA-seq, single-cell RNA sequencing