Author response: A single-cell atlas of the mouse and human prostate reveals heterogeneity and conservation of epithelial progenitors (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Introduction Significant anatomical differences between the mouse and human prostate have long hindered analyses of mouse models of prostate diseases. The mouse prostate can be separated into anterior (AP), dorsal (DP), lateral (LP), and ventral (VP) lobes; the mouse dorsal and lateral lobes are often combined as the dorsolateral prostate (DLP) (Cunha et al., 1987; Shappell et al., 2004; Shen and Abate-Shen, 2010). In contrast, the human prostate lacks defined lobes, and instead is divided into different histological zones (central, transition, and peripheral); the peripheral zone represents the predominant site of prostate adenocarcinoma, whereas benign prostatic hyperplasia (BPH) occurs in the transition zone (Cunha et al., 2018; Ittmann, 2018; Shappell et al., 2004). Moreover, unlike the mouse, the human prostate has distinct ductal and acinar regions. Although microarray gene expression profiling has suggested that the DLP is most similar to the human peripheral zone (Berquin et al., 2005), there is no consensus on the relationship between mouse lobes and human zones (Ittmann, 2018; Ittmann et al., 2013; Shappell et al., 2004). The adult prostate epithelium is comprised of luminal, basal, and rare neuroendocrine cells (Shen and Abate-Shen, 2010; Toivanen and Shen, 2017), and cellular heterogeneity has been suggested within the luminal (Barros-Silva et al., 2018; Chua et al., 2014; Karthaus et al., 2020; Karthaus et al., 2014; Kwon et al., 2016; Liu et al., 2016) and basal compartments (Goldstein et al., 2008; Lawson et al., 2007; Wang et al., 2020). Lineage-tracing analyses have shown that the hormonally intact adult prostate epithelium is maintained by unipotent progenitors within the basal and luminal epithelial compartments (Choi et al., 2012; Lu et al., 2013; Wang et al., 2013). However, following tissue dissociation, both basal and luminal cells can act as bipotent progenitors in organoid or tissue reconstitution assays (Chua et al., 2014; Karthaus et al., 2014). The progenitor properties of basal cells may reflect their ability to generate luminal progeny during tissue repair after wounding or inflammation (Kwon et al., 2014; Toivanen et al., 2016), but the role of presumptive luminal progenitors has been less clear. In particular, several studies have suggested increased progenitor potential in the proximal region of the prostate (nearest to the urethra) (Burger et al., 2005; Goto et al., 2006; Tsujimura et al., 2002), particularly for proximal luminal cells (Guo et al., 2020; Karthaus et al., 2020; Kwon et al., 2016; Wei et al., 2019; Zhang et al., 2018). However, the nature and distribution of these epithelial populations have been poorly characterized. Results Distinct luminal epithelial populations in the mouse prostate To examine cellular heterogeneity, we performed single-cell RNA-sequencing of whole prostates from adult wild-type mice at 10 weeks of age. We microdissected the full proximal-distal extent of each prostate lobe down to its junction with the urethral epithelium (Figure 1—figure supplement 1A–C). We noted that the anterior (AP), dorsal (DP), and lateral (LP) lobes joined the urethra in close proximity on the dorsal side, whereas the ventral lobe (VP) had a distinct junction ventrally. As previously described (Cunha et al., 1987; Shappell et al., 2004; Shen and Abate-Shen, 2010), each lobe has a characteristic morphology, pattern of ductal branching, and histological appearance (Figure 1—figure supplement 1D–F). Single-cell populations were obtained using a multi-step tissue dissociation protocol with successive enzymatic digestion, trituration, and filtering steps. Droplet-based single-cell RNA-sequencing was performed on the 10x Genomics Chromium platform (see Materials and methods for details). For analysis of the resulting data, we performed batch effect correction if needed for aggregation of datasets (Stuart et al., 2019), followed by application of Randomly, an algorithm that uses random matrix theory to reduce noise in single-cell datasets (Aparicio et al., 2020). Using the universality property of random matrix theory on eigenvalues and eigenvectors of sparse matrices, Randomly discriminates biological signals from noise and sparsity-induced confounding signals, which typically comprise approximately 98% of the data, based on a survey of published single-cell datasets (Aparicio et al., 2020). The Randomly algorithm is based on the three-fold structure of a single-cell dataset: a random matrix (95% or more), a sparsity-induced (fake) signal, and a biological signal. The algorithm uses the universality properties of random matrix theory for both eigenvalues and eigenvectors to detect the biological signal. After de-noising of single-cell data, we performed clustering using the Leiden algorithm as implemented in Wolf et al., 2018, with selection of the number of clusters based on the mean silhouette score. Processing by Randomly followed by dimensional reduction for visualization using t-SNE (t-distributed Stochastic Neighbor Embedding) or UMAP (Uniform Manifold Approximation and Projection) plots facilitated the identification of cell populations with distinct transcriptional signatures (Figure 1—figure supplement 2). Additional description of computational methods is provided in Materials and methods. We identified distinct luminal, basal, and neuroendocrine populations that were annotated based on the expression of marker genes, as visualized in an aggregated dataset composed of 5288 cells from two whole prostates (tSNE plot shown in Figure 1A,D; UMAP plot shown in Figure 1—figure supplement 3A). Notably, we could identify five different luminal epithelial populations, a single basal population, rare neuroendocrine cells, and a small population of epithelial cells that expresses both basal and luminal markers. We could also identify distinct stromal and immune components, corresponding to two different stromal subsets (Kwon et al., 2019), as well as immune cells (macrophages, T cells, B cells); some datasets also contained small populations of contaminating vas deferens and seminal vesicle cells. Figure 1 with 5 supplements see all Download asset Open asset Single-cell analysis identifies prostate luminal epithelial heterogeneity. (A) t-distributed stochastic neighbor embedding (tSNE) plot of 5288 cells from an aggregated dataset of two normal mouse prostates, processed by Randomly and clustered using the Leiden algorithm. (B) tSNE representation of each prostate lobe (AP: 2735 cells; DP: 1781 cells; LP: 2044 cells; VP: 1581 cells). (C) Schematic model of prostate lobes with the urethral rhabdosphincter partially removed, with the distribution of luminal epithelial populations indicated. (D) Dot plot of gene expression levels in each epithelial population for selected marker genes. (E) Ridge plots of marker genes showing expression in each population. (F) Hematoxylin-eosin (H and E) and immunofluorescence (IF) images of selected markers in serial sections; the periurethral/proximal region shown is from the AP and DP. Arrow in VP distal indicates distal cell with Ppp1r1b expression. Scale bars indicate 50 µm. To assess whether some of these epithelial populations might be lobe-specific, we next performed single-cell RNA-seq analyses of individual lobes (tSNE plots shown in Figure 1B; UMAP plots shown in Figure 1—figure supplement 3B). We found that four of the luminal populations identified in the aggregated dataset were highly lobe-specific; hence, we named these populations LumA (AP-specific), LumD (DP-specific), LumL (LP-specific), and LumV (VP-specific). The remaining luminal population was observed in the datasets for all four lobes and was highly enriched in the proximal portion of each lobe; thus, we termed this population LumP (proximal) (Figure 1C). Spatial localization and morphology of epithelial populations To examine the lobe-specificity and spatial distribution of these luminal populations, we identified candidate markers based on gene expression patterns in our single-cell datasets (marker genes of interest and/or used in this study are shown in the dot plot of Figure 1D; a larger set of differentially expressed genes are shown in Figure 1—figure supplement 4). We focused our analyses on candidate markers with commercially available antibodies that displayed low background staining in control sections, and selected antibodies for further study that were specific for the cell populations predicted by single-cell analyses (Figure 1D–F; Figure 1—figure supplement 5). For example, we found that the LumA and LumD marker Tgm4 was highly expressed by luminal cells in the distal region of the AP and DP, and that the LumL marker Msmb marked distal luminal cells in the LP. In contrast, the LumP marker Ppp1r1b was highly enriched in luminal cells that were primarily found in the proximal regions of all four lobes (Figure 1D–F). However, more general luminal markers such as androgen receptor (Ar) were expressed in all luminal populations (Figure 1—figure supplement 5). Next, we investigated the spatial localization of these luminal populations along the proximal-distal axis in each lobe. We found that the LumP-containing proximal region extended from inside the rhabdosphincter to the first major ductal branch point in the AP, DP, and VP, but not the LP, whereas the bulk of the lobes corresponded to distal regions (Figure 1C). In the AP and DP, we found a discrete boundary in the medial region between the proximal LumP population and distal LumA or LumD populations, respectively (Figure 2A,C; Figure 2—figure supplement 1A). In contrast, the LP had a population between the proximal and distal regions that expressed low levels of LumL markers (Figure 2A; Figure 2—figure supplement 1A). Histological analyses revealed that distal luminal cells of each lobe had a tall columnar appearance consistent with secretory function, whereas proximal LumP cells typically had a cuboidal morphology. Notably, this analysis also revealed heterogeneity in the distal region of each lobe, with rare clusters of 1–10 LumP cells observed in the distal AP, DP, and LP, but more frequent LumP clusters in the distal VP (Figure 2B). Figure 2 with 1 supplement see all Download asset Open asset Luminal epithelial populations display spatial and morphological heterogeneity. (A) H and E and IF of serial sections from the DP and LP, showing expression of proximal (Ppp1r1b) and distal (Tgm4, Msmb) markers; note apparent differences in the boundary regions of the two lobes. (B) Detection of distally localized LumP cells (arrows) in all four lobes; these are most abundant in the VP. (C) Scanning electron micrographs of the boundary region of the AP; central low-power image is flanked by high-power images of boxed regions. Red arrow, mitochondria; black arrow, membrane interdigitation; blue arrow, Golgi apparatus; green arrow, rough endoplasmic reticulum. (D) Identification of the periurethral region. Cells in the periurethral region generally express Ly6d, Ck7, Aqp3, and Ppp1r1b; notably, Cldn10-expressing LumP cells decrease approaching the periurethral region (E) Lineage-marking in Nkx3.1Cre/+; R26R-YFP mice (n = 3) shows widespread YFP expression in the periurethral, proximal, and distal AP; small patches remain unrecombined and lack YFP (arrows). Scale bars in (A,B,D,E) indicate 50 µm; scale bars in (C) indicate 2 µm. To clarify the phenotypic differences between proximal and distal luminal populations, we performed scanning electron microscopy of an 8-week-old anterior lobe (Figure 2C; Figure 2—figure supplement 1B). LumA cells displayed dense regions of rough endoplasmic reticulum throughout the cytoplasm, many free ribosomes, and abundant secretory vesicles on the apical surface, typical of secretory cells. In contrast, LumP cells displayed areas of high mitochondrial density, complex membrane interdigitation, and no vesicles. At the proximal-distal boundary, we observed an abrupt transition between cellular morphologies that took place within 1–2 cell diameters. These ultrastructural differences indicate that the LumA and LumP populations represent distinct cell types, rather than cell states. Next, we investigated the remaining epithelial population, which shares basal and luminal features in our single-cell RNA-seq analysis (Figure 1A). We found that this small population was enriched in the most proximal region of all four lobes, residing inside the rhabdosphincter and adjacent to the urethral junction (Figure 2D; Figure 1—figure supplement 1C); hence, we termed this novel population PrU (periurethral). Although this PrU population co-expresses some markers with LumP (Figure 1D), it also expresses several urothelial markers such as Ly6d and Aqp3 (Figure 2D). The proximity of the PrU and LumP populations to the urethra and their co-expression of multiple markers led us to investigate their developmental origin. Consequently, we examined the expression of Nkx3-1, whose mRNA expression marks epithelial cells in ductal derivatives of the developing urogenital sinus, such as the prostate, but not the urothelium (Bhatia-Gaur et al., 1999); similarly, the Nkx3-1Cre driver also marks early prostate bud cells but not the urogenital sinus during development (Thomsen et al., 2008; Zhang et al., 2008). In the adult prostate, Nkx3-1 is expressed by all four distal luminal populations (LumA, LumD, LumL, LumV), but is not expressed by LumP or PrU (Figure 1—figure supplement 5). However, we found the Nkx3-1Cre driver lineage-marks most of the cells in all of these populations, including LumP and PrU, but not the urethra (Figure 2E). These data indicate that the LumP and PrU populations are derived from Nkx3-1 expressing prostate epithelial cells and are distinct from the urothelium. Functional analysis of epithelial populations We used an approach based on optimal transport theory to ascertain the relationships of these prostate epithelial populations (see Materials and methods). We calculated the similarity among cell populations in mouse using the Wasserstein-1 distance as a measure for phenotypic distance among cell populations, based on clusters in the latent space previously obtained by the Randomly algorithm. The pair-wise comparisons between populations (Figure 3A) can be captured by a neighbor-joining tree (Figure 3B), in which lower Wasserstein distance indicates greater similarity. We found that the distal populations grouped together, with the LumA and LumD populations being most closely related, followed by LumL and LumV. These distal populations were next most closely related to LumP, which in turn was most similar to PrU, followed by basal cells, suggesting a lineage relationship between LumP and distal luminal populations. Figure 3 with 2 supplements see all Download asset Open asset Functional analysis of epithelial populations in organoid and tissue reconstitution assays. (A) Heatmap visualization of the Wasserstein distances between epithelial populations with hierarchical clustering. (B) Tree visualization of Wasserstein distances. (C) Flow sorting of distinct epithelial populations from the AP lobe. (D) Organoids grown from sorted epithelial cells in two distinct culture conditions. ENR conditions: sorted cells from UBC-GFP mice plated at 1000 cells/well, imaged at day 10. Hepatocyte Media (HM) conditions: sorted cells from wild type C57BL/6 mice plated at 2000-5000 cells/well and imaged on day 12-13. (E) Organoid formation efficiency plots. Maximum p-values for each pair-wise comparison are indicated. (F) Grafting efficiency in tissue reconstitution assays (average p-value shown). LumP, PrU, and basal are significantly more efficient at generating grafts from smaller number of cells relative to distal luminal populations. (G) H&E and IF of sections from fully-differentiated renal grafts; positive staining corresponds to results found in ≥3 independent grafts. Scale bars indicate 50 µm. To investigate the functional properties of each epithelial population, we developed a flow sorting strategy to isolate these populations (Figure 3C; Figure 3—figure supplement 1A); we performed re-sorting experiments to assess the purity of the isolated populations (Figure 3—figure supplement 1B). We performed organoid formation assays with isolated cell populations using the defined ENR-based medium (Drost et al., 2016; Karthaus et al., 2014) as well as hepatocyte medium (HM), which has a more complex composition including serum (Chua et al., 2014). Despite differences in overall efficiency between media conditions, we consistently found that the PrU and basal populations were most efficient at forming organoids, followed by LumP, whereas the efficiency of distal LumA, LumD, LumL, and LumV was significantly lower (Figure 3D,E). Next, we assessed the progenitor potential of isolated epithelial populations using in vivo tissue reconstitution assays. These assays involve recombination of dissociated epithelial cells with rat embryonic urogenital mesenchyme followed by renal grafting, and have been extensively utilized for analysis of progenitor properties in the prostate (Lawson et al., 2007; Wang et al., 2013; Xin et al., 2003). We observed significant variation in the frequency of graft formation depending on the number and type of input epithelial cells (Figure 3F). Based on histological and immunostaining analyses, we found that each epithelial population typically gave rise to cells of the same type, but their ability to generate cells of other populations varied considerably (Figure 3G; Figure 3—figure supplement 2A). Notably, grafts using distal luminal cells required relatively large numbers of input cells to grow (approximately 30,000 cells) (Figure 3F), and were mostly composed of the same population (i.e. LumA cells generated LumA cells) together with a normal or reduced percentage of basal cells; additionally, these grafts could contain small patches of cells expressing LumP markers on their periphery (Figure 3G; Figure 3—figure supplement 2A). Interestingly, LumV cells had the lowest grafting efficiency and generally formed small ductal structures that lacked basal cells. In contrast, LumP, PrU, and basal cells could produce grafts with significantly lower input cell numbers (approximately 1,000 cells), which contained LumP cells together with multiple distal luminal populations as well as a normal ratio of basal cells. We excluded contribution of host cells and rat urogenital epithelium to grafts using control tissue reconstitution assays with GFP-expressing donor epithelial cells (Figure 3—figure supplement 2B). Taken together, these results suggest a spectrum of progenitor potential among the different epithelial populations. Both PrU and basal cells possessed high progenitor activity in these assays, with LumP cells also displaying enhanced activity. In contrast, the distal luminal populations are much less efficient in these assays. These findings are consistent with the inferred relationships between these populations based on molecular (Figure 3A,B) as well as histological and ultrastructural analyses (Figure 2A,C). Comparison of human and mouse prostate epithelial populations To examine the conservation of epithelial populations between the mouse and human prostate, we performed single-cell RNA-seq analyses of tissue samples from human prostatectomies (Figure 4—source data 1). We identified two distinct luminal populations as well as a PrU-like population (tSNE plots shown in Figure 4A–C; UMAP plots shown in Figure 4—figure supplement 1), and examined their spatial distribution by immunostaining of benign human prostate tissue (Figure 4—source data 1). Using specific markers, we found that one luminal population (Lum Ductal, expressing KRT7 and RARRES1) was primarily localized to ducts, whereas the second population was predominantly acinar (Lum Acinar, expressing MSMB and MME), although some intermixing could be observed within ducts (Figure 4G). As in the mouse, the PrU-like population expressed both basal and luminal genes; similarly, some PrU-like markers were shared with Lum Ductal. Figure 4 with 1 supplement see all Download asset Open asset Heterogeneity and conservation of luminal populations in the human prostate. (A–C) tSNE plot of scRNA-seq data (A, 1600 cells; B, 2303 cells; C, 2825 cells) from three independent human prostatectomy samples. (D–F) Heatmap visualization of Wasserstein distances between the human and mouse prostate populations for each dataset. (G) H and E and IF images of serial sections from human prostate, showing regions of the prostatic utricle, central, transition, and peripheral zones. Arrows indicate regions of ductal morphology. Scale bars indicate 50 µm. Figure 4—source data 1 Human prostate samples and corresponding clinical data. https://cdn.elifesciences.org/articles/59465/elife-59465-fig4-data1-v2.docx Download To this we the Wasserstein distances for each pair-wise comparison between epithelial and major populations (Figure analysis that a PrU-like population is conserved between Furthermore, the human Lum cells are most closely related to mouse LumP, whereas the Lum cells are most closely related to LumL followed by LumV. Notably, these relationships were observed in each dataset. Discussion We have generated a comprehensive cellular atlas of the prostate epithelium and have defined and functional properties of each epithelial population. Our analyses have revealed spatial and functional heterogeneity primarily in the luminal epithelial including distinct cell populations along the proximal-distal axis as well as lobe-specific Notably, we have shown marked differences in progenitor potential between cell which to distinct cell rather than cell In tissue reconstitution assays, the ability of LumP and PrU cells to generate luminal distal and basal cells that both populations have properties of multipotent In to the luminal basal cells relatively suggesting that previously basal heterogeneity (Goldstein et al., 2008; Lawson et al., 2007; Wang et al., may be more Notably, the PrU population is not in as it is comprised of cells with both basal and luminal features. Our findings also a for other of epithelial heterogeneity. single-cell RNA-seq analysis of the mouse anterior prostate identified three distinct luminal populations et al., to to our LumA and to we also identified a population expressing genes in both mouse and human but have annotated this as deferens based on marker expression and on findings et al., based on single-cell analysis of the whole mouse prostate has described three luminal populations, in which to to our to our LumA and LumD, and to our notably, this analysis used to progenitor properties of distal (LumP) cells (Guo et al., 2020). Based on patterns of gene we suggest that the LumP population corresponds to the previously luminal cells (Kwon et al., 2016) as well as cells et al., 2019), which have been described as proximal progenitor populations with distal cells, and may also be for the enhanced serial grafting efficiency of proximal prostate et al., In the human prostate, the progenitor activity of LumP and/or PrU-like cells may have been observed by lineage using mitochondrial et al., In the LumP and PrU populations may some with the and cells described in a cellular atlas of the mouse et al., and in the human prostate et al., 2018; et al., and in benign human prostate et al., 2019), but the relationship of these populations is LumP and PrU cells display multipotent progenitor activity in both organoid formation and tissue reconstitution assays, their spatial distribution may reflect the ability of the prostate to repair from a proximal to distal in response to extensive tissue as well as from distal progenitors in response to more localized Our findings suggest that the novel PrU population in may a role in prostate tissue repair and/or consistent with the identification of cells as a progenitor (Barros-Silva et al., 2018). Notably, the ability of Nkx3-1Cre to both PrU and LumP, but not the that these cell populations are distinct from the the molecular between the proximal prostate and the urethra noted in a et al., 2020). our results that lineage relationships among the derived from the urogenital sinus et al., are of for the of in the urogenital Our findings help resolve a long-standing about the relationship of the mouse and human we that mouse proximal and distal regions are most related to human ductal and acinar and that the mouse LP is most similar to the human peripheral zone. if studies have assessed in the LP, as it is small and combined with the DP as the dorsolateral prostate our analyses show that the DP significantly from the LP at the anatomical and molecular Consequently, we suggest that a of in mouse models may a similarity to human prostate than previously the of prostate epithelial heterogeneity has significant for the cell of origin for prostate adenocarcinoma. studies have suggested that luminal cells as well as basal cells can as the cell of origin for prostate et al., 2013; Wang et al., 2014; 2019), differences in human prostate et al., be on this Notably, studies suggest that both distal and proximal luminal cells can as cells of origin for prostate For example, prostate following of by the which is specific for distal luminal cells et al., as well as by the which is specific for LumP cells (Guo et al., 2020). further analyses of epithelial heterogeneity and progenitor potential to into prostate and Materials and methods type or or background to background to background to background to background to background to background to background