Single-cell analysis of mouse and human prostate reveals novel fibroblasts with specialized distribution and microenvironment interactions

Abstract

Stromal-epithelial interactions are critical to the morphogenesis, differentiation, and homeostasis of the prostate, but the molecular identity and anatomy of discrete stromal cell types is poorly understood. Using single-cell RNA sequencing, we identified and validated the in situ localization of three smooth muscle subtypes (prostate smooth muscle, pericytes, and vascular smooth muscle) and two novel fibroblast subtypes in human prostate. Peri-epithelial fibroblasts (APOD+) wrap around epithelial structures, whereas interstitial fibroblasts (C7+) are interspersed in extracellular matrix. In contrast, the mouse displayed three fibroblast subtypes with distinct proximal-distal and lobe-specific distribution patterns. Statistical analysis of mouse and human fibroblasts showed transcriptional correlation between mouse prostate (C3+) and urethral (Lgr5+) fibroblasts and the human interstitial fibroblast subtype. Both urethral fibroblasts (Lgr5+) and ductal fibroblasts (Wnt2+) in the mouse contribute to a proximal Wnt/Tgfb signaling niche that is absent in human prostate. Instead, human peri-epithelial fibroblasts express secreted WNT inhibitors SFRPs and DKK1, which could serve as a buffer against stromal WNT ligands by creating a localized signaling niche around individual prostate glands. We also identified proximal-distal fibroblast density differences in human prostate that could amplify stromal signaling around proximal prostate ducts. In human benign prostatic hyperplasia, fibroblast subtypes upregulate critical immunoregulatory pathways and show distinct distributions in stromal and glandular phenotypes. A detailed taxonomy of leukocytes in benign prostatic hyperplasia reveals an influx of myeloid dendritic cells, T cells and B cells, resembling a mucosal inflammatory disorder. A receptor-ligand interaction analysis of all cell types revealed a central role for fibroblasts in growth factor, morphogen, and chemokine signaling to endothelia, epithelia, and leukocytes. These data are foundational to the development of new therapeutic targets in benign prostatic hyperplasia. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: benign prostatic hyperplasia; fibroblast; prostate; prostatic urethra; single-cell RNA sequencing; smooth muscle; stroma.

Figure 1.. Identification and validation of fibromuscular stroma subtypes in the human prostate.

(A) Clustering of major cell lineages in normal human prostate samples (n=3 prostate donors). (B) Subsetted fibromuscular stroma from normal human prostate samples (n=3 prostate donors). (C) Dot plot of selected differentially expressed marker genes across each fibromuscular stromal subtype. (D) Section through prostate tissue labeled with fluorescent antibodies to the prostate smooth muscle marker DES (in green), vascular smooth muscle marker MCAM (in red) and basal epithelial marker KRT5 (in white). (E) Section through prostate tissue labeled with fluorescent antibodies to the prostate smooth muscle marker DES (in green), Pericyte marker THY1 (in red) and basal epithelial marker KRT5 (in white). DAPI (in blue) labeled nuclei. (F) Section through prostate tissue from organ donor labeled with probes to the peri-epithelial fibroblast marker APOD (in brown). Magnified images of distinct anatomical locations from (F) corresponding to (G) Urethra, (H) Transition zone, (I) Central zone and (J) Peripheral zone. (K) Section through prostate tissue from organ donor labeled with probes to the interstitial fibroblast marker C7 (in brown). Magnified images of distinct anatomical locations from (K) corresponding to (L) Urethra, (M) Transition zone, (N) Central zone and (O) Peripheral zone. Black arrowheads indicate individual fibroblasts. Grey scale bar represents 2000 microns. Black scale bar represents 100 microns. See also supplementary material, Figure S1.

Figure 2.. Identification and validation of fibromuscular stroma subtypes in the mouse prostate and prostatic urethra.

(A) Clustering of subsetted fibromuscular stroma from adult mouse prostates (n=4 mice) and prostatic urethras (n=4 mice). (B) Subsetted fibromuscular stroma from adult mice displaying contribution from prostate and prostatic urethra samples. (C) Dot plot of selected differentially expressed marker genes across each fibromuscular stromal subtype. (D) Pearson correlation of human (genes converted to mouse orthologs) and mouse fibromuscular stromal subtypes. Correlation coefficients are globally scaled between 1 and −1. (E) Quantitative Set Analysis for Gene Expression (QuSAGE) performed on mouse fibroblast subtypes. Selected significantly upregulated gene sets (false discovery rate < 0.05) displayed with log2 fold-change values. (F) Sagittal section through adult mouse lower urinary tract labeled with probes to the prostate fibroblast marker C3. (G–J) Magnified regions corresponding to distinct anatomical locations in (F). (K) Sagittal section through adult mouse lower urinary tract labeled with probes to the urethral fibroblast marker Lgr5. (L–O) Magnified regions corresponding to distinct anatomical locations in (K). (P) Sagittal section through adult mouse lower urinary tract labeled with probes to the ductal fibroblast marker Wnt2. (Q–T) Magnified regions corresponding to distinct anatomical locations in (P). Black arrowheads indicate individual fibroblasts. Grey scale bar represents 1000 microns. Black scale bar represents 50 microns. See also supplementary material, Figure S2.

Figure 3.. Fibroblast density and distribution in the prostate.

(A) Illustration depicting stromal subtypes in the human prostate. (B) Illustration depicting distribution of mouse stromal subtypes across the prostate lobes and prostatic urethra. (C) Whole mount section through a young normal prostate labeled with antibodies to Desmin (in green, labels prostate smooth muscle), Decorin (in red, labels fibroblasts) and Keratin 5 (in white, labels basal epithelial cells). DAPI (in blue) labeled nuclei. Magnified insets from (C) correspond to the proximal region (C’) and the distal region (C”) of the prostate. (D) Red channel corresponding to Decorin immunostaining isolated from image (C) to visualize fibroblast density differences. (E) Fibroblast to smooth muscle ratio in the proximal and distal region from four donor prostates. See also supplementary material, Figure S3, S4.

Figure 4.. Fibroblasts in human benign prostatic hyperplasia (BPH).

(A) Subsetted fibromuscular stroma from normal human prostate samples (n=3 prostate donors) and BPH samples (n=6 BPH patients). (B) Fibromuscular stromal composition of glandular (n=6) and stromal samples (n=6) from six BPH patients. (C) Dot plot of selected differentially regulated genes (padj < 0.05) in BPH versus normal. (D) Section through BPH tissue labeled with probes to the peri-epithelial fibroblast marker APOD (in brown). Magnified images of distinct histologic phenotypes from (D) corresponding to (E) glandular nodule and (F) stromal nodule. Black arrowheads indicate APOD+ fibroblasts in the peri-epithelial compartment. (G) Section through BPH tissue labeled with probes to the interstitial fibroblast marker C7 (in brown). Magnified images of distinct histologic phenotypes from (G) corresponding to (H) glandular nodule and (I) stromal nodule. Grey scale bar represents 2000 microns. Black scale bar represents 50 microns. Black dotted lines outline stromal nodules. (J) Quantitative Set Analysis for Gene Expression (QuSAGE) performed for each fibroblast subtype comparing donor (normal) and BPH (diseased). Significant gene sets (false discovery rate < 0.05) were subsetted to obtain unique and shared gene sets upregulated by the fibroblast subtypes in BPH. See also supplementary material, Figure S5.

Figure 5.. Human fibroblast interactions with endothelial and epithelial cells.

(A) Heatmap of predicted interactions from CellPhoneDB. (B) Dot plot of predicted interactions between fibroblast subtypes and endothelial cells. (C) Dot plot of predicted interactions from fibroblasts to epithelial cells. (D) Dot plot of predicted interactions from epithelial cells to fibroblasts. (E) Illustration depicting signals from fibroblasts to epithelial and endothelial cells. Up and down arrows indicate genes upregulated or downregulated respectively in the fibroblast subtype in BPH.

Figure 6.. Immune subtypes and interactions with fibroblasts in BPH.

(A) Subsetted leukocytes from normal human prostate samples (n=3 prostate donors) and BPH samples (n=6 BPH patients) with major immune lineages identified. (B) Immune composition of glandular (n=6) and stromal samples (n=6) from six BPH patients. (C) Dot plot of predicted interactions from fibroblasts to immune cells. (D) Immune subtypes in the prostate identified using Travaglini et al [21]. (E) Illustration depicting signals from fibroblasts to immune cells. Up arrows indicate genes upregulated in the fibroblast subtype in BPH. See also supplementary material, Figures S6, S7.