Abstract
The prostate is an organ characterized by significant spatial heterogeneity. To better understand its intricate structure and cellular composition, we constructed a comprehensive single-cell atlas of the adult human prostate. Our high-resolution mapping effort identified 253,381 single cells and 34,876 nuclei sampled from 11 patients who underwent radical resection of bladder cancer, which were categorized into 126 unique subpopulations. This work revealed various new cell types in the human prostate and their specific spatial localization. Notably, we discovered four distinct acini, two of which were tightly associated with E-twenty-six transcription factor family (ETS)-fusion-negative prostate cancer. Through the integration of spatial, single-cell and bulk-seq analyses, we propose that two specific luminal cell types could serve as the common origins of prostate cancer. Additionally, our findings suggest that zone-specific fibroblasts may contribute to the observed heterogeneity among luminal cells. This atlas will serve as a valuable reference for studying prostate biology and diseases such as prostate cancer.
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Data availability
Processed data generated in this work have been deposited in the figshare database (https://doi.org/10.6084/m9.figshare.25965613). Raw sequencing data have been deposited at the Genome Sequence Archive—Human database under project PRJCA026045, but a Data Access Committee (DAC) approval is necessary due to policy restrictions (following Detailed Rules for the Implementation of the Regulations on the Administration of Human Genetic Resources, https://ngdc.cncb.ac.cn/gsa-human/policy). Every researcher could submit an application on the website, and it would commonly take several weeks (4 weeks on average) for the database administrator and DAC to review. Ensemble (v.91; https://ftp.ensembl.org/pub/release-91/) was used as a reference to map single-cell and spatial sequencing data to the human genome.
The level-3 GTEx expression matrix was downloaded from the GTEx data portal (https://gtexportal.org/). Prostate-derived samples were selected for the cross-validation of single-cell sequencing data. RNA-seq data of CPGEA was downloaded from the website (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA001124)29 and is available from the corresponding author. TCGA-PRAD datasets were downloaded from UCSC XENA (https://xena.ucsc.edu/public). Because the TCGA-PRAD dataset contains samples from two different labs and a strong batch effect was observed in the previous study, only 333 samples from the phase one work were used in this paper. Clinical information, ESTIMATE tumor purity and androgen receptor scores were obtained from the supplementary table of a previous publication33. The bulk RNA-seq cohort of CRPC samples was obtained from cBioPortal (http://www.cbioportal.org/, SU2C/PCF Dream Team (ref. 35)). Only 208 samples of the SU2C cohort with RNA-seq data were analyzed in this paper. Single-cell sequencing data of human prostate tissues sampled from younger and older individuals were obtained from the GEO database, and deposited as GSE117403 (ref. 9) and GSE181294 (ref. 33), respectively.
Code availability
All existing software packages used in the study are cited in the relevant sections of the Methods. Code used in this work has been deposited at GitHub (https://github.com/AndersonHu85/normal_prostate)68.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (8212502 and 82330091 to S.R., 82373330 to K.C., 32100631 to F.L., and 82073082 and 82311530050 to G.-H.W.), Shanghai Shenkang Hospital Development Center (SHDC12022117 and SHDC2022CRT005 to S.R.), Shanghai Municipal Education Commission (2023ZKZD46 to S.R.), China Postdoctoral Science Foundation (2023T160061 to F.L.), Macao Young Scholars Program (AM2023024 to F.L.), Capital’s Funds for Health Improvement and Research (2024-4-40215 to F.L.) and the Young Elite Scientists Sponsorship Program by China Association for Science and Technology (YESS20210056 to F.L.). The authors thank NovelBio Bio-Pharm Technology for the support of the scRNA-seq experiment and bioinformatics analysis with their NovelBrain Cloud Analysis Platform (www.novelbrain.com) and the computation resource supported by the Medical Science Data Center at Shanghai Medical College of Fudan University. We also thank Powerful Biology and Wuhan Pigeonbio Technology for their help with IHC staining, mFISH and Multicolor IF staining.
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D.G., K.C. and S.R. conceptualized and designed the project. L.Y., W.C., W.X., Y.C., Y.H., L.L., F.L. and J.Z. performed experiments or data collection. L.Y., J.H., S.C. and G.Z. performed computational, multi-omic and statistical analyses. J.Z., Y.W. and J.W. performed data interpretation and biological analysis. J.H., G.D. and J.J. wrote the original draft of the manuscript. K.C., S.R., Z.W., G.W., H.H. and F.L. did the writing, reviewing and editing of the manuscript.
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Extended data
Extended Data Fig. 1 Identification of subpopulations of ductal luminal (dLum) and basal cells in human prostate.
a, UMAP for expression of known markers of prostate epithelial lineages in the scRNA-seq (top) and snRNA-seq (bottom). b, UMAP of dLum cells in scRNA-seq data colored by given cell identities, related to Fig. 2a. c, Heatmap of AUROC scores showing similarity between epithelial subsets identified by scRNA-seq (y axis) and snRNA-seq (x axis). Higher AUROC score represents higher similarity. d, Overview of the sampling strategy of the prostate of HP05. e, H&E staining of a slice adjacent to the urethra (left). Spatial transcriptomic slice colored by inferred cell proportions (right). f, Overview of the sampling strategy of the prostate of patient 2. g, Spatial transcriptomic slice, H3_5, of patient 2 colored by inferred cell proportions.
Extended Data Fig. 2 Spatial localization of dLum cell subsets.
a, Multicolor IF staining of a slice surrounding the urethra, related to Fig. 2d. Experiment has been repeated three times. b, IHC staining of selected markers on glandular adjacent to the urethra (top) and in the distal acini (bottom). Experiment has been repeated three times. c, Overview of the sampling strategy of the prostate of patient 2. d, H&E staining (left) and spatial transcriptomic slice, V1_5 of patient 2, colored by inferred cell proportions (right). e, IHC staining of LTF on a coronal plane of human prostate. Experiment has been repeated three times. f, UMAP of epithelial cells in scRNA-seq data colored by gene expression levels. g, Heatmap showing cell fractions of each subset of epithelial cells in scRNA-seq data (left). Cell fractions were transformed into row z score. Bar plot of cell fraction in each sample was shown on the right. h, Spatial transcriptomic slice, V1_5 of patient 2, colored by expression level of CNMD (left) and KLK5 (right), respectively. i, Pie plot showing the proportion of spots detected d6_dLum-CNMD signature in glandular ducts and other epithelial spots, respectively. P value was calculated by two-side Fisher’s exact test. j, Bar plot showing enriched pathway in d7_dLum-SPIB. T values were calculated by comparing GSVA scores per cell in d7_dLum-SPIB to all other dLum cells. k, Violin plot of expression of MHC molecules in dLum cell subsets.
Extended Data Fig. 3 Identification of basal cell lineages in human prostate.
a, A 2D projection of 3D UMAP of snRNA-seq data, related to Fig. 2a. The arrow showed the potential differentiation direction from club to basal cells. b,c, Pseudoheatmap showing gradual shift in expression of transcription factors (TFs) (b) and motifs activities (c) in snRNA-seq data from club cells to basal cells in two directions. d, Diffusion map with superimposed RNA velocity analysis of the selected dLum and basal cell subsets, showing two differentiation directions from d1_dLum-Club to basal cells. DC, diffusion component. e, Diffusion map with superimposed PAGA-velocity analysis of the selected dLum and basal cell subsets, related to Extended Data Fig. 3d. f, Spatial transcriptomic slice, H3_2 of patient 2, colored by inferred cell proportions. g, Overview of the sample strategy of the prostate of patient 2. h,i, Spatial transcriptomic slice, V2_1 (h) and V2_5 (i) of patient 2, colored by inferred cell proportions. j, UMAP of epithelial cells in scRNA-seq data colored by expression level of KIT in each cell. KIT is expressed in FOXI1+ basal cell lineage. k, Multicolor IF staining of a slice shows the presence of KIT+CK5+ basal cells in the human prostate, related to Fig. 2m. Experiment has been repeated three times.
Extended Data Fig. 4 Identification of morphologically benign acinar with somatic copy number alterations (SCNAs).
a, UMAP of epithelial cells in snRNA-seq data colored by expression level of given marker genes of intraepithelial neoplasia (PIN) in ST slice sampled from the transition zone of HP09. b, Spatial transcriptomic slice of the transition zone of HP09 colored by the expression level of selected marker genes of the PIN in this slice. c, Heatmap showing CNA in each single nucleus identified as luminal cell sampled from the peripheral zone of HP09. d, Sampling strategy of HP12 (upper left) and H&E staining of this slice. Selected region in the dashed box was used to perform Visium analysis. e, Enlarged partition of slice sampled from the peripheral zone of HP12. f, Spatial transcriptomic slices of HP12 colored by CNV Leiden clusters. g, Heatmap grouped by CNA Leiden clusters of the peripheral zone slice of HP12. h, Spatial transcriptomic slice colored by expression level of Type 2 luminal signature in each spot. i, Enlarged partition of the slice expressing high level of Type 2 luminal signature. j, Sampling strategy of HP13. k, Spatial transcriptomic slices of HP13 colored by CNV Leiden clusters. l, Heatmap grouped by CNA Leiden clusters of the peripheral zone slice of HP13. m, Enlarged partition of the slice sampled from the peripheral zone of HP13 expressing high level of Type 2 luminal signature. The region in dashed line is an early prostate cancer (PCa) lesion. n, Spatial transcriptomic slices of HP13 colored by expression level of Type 2 luminal signature.
Extended Data Fig. 5 Identification of morphologically benign TGM4+ acinar containing SCNAs in human prostate.
a, Sampling strategy of patient 1. b, H&E staining of the slice V2_5 of patient 1. c, Spatial dot plot of V2_5 colored by Seurat clusters, each dot represents a sampling point of 1K-array slice. d, Spatial dot plot of V2_5 colored by TGM4+ luminal signature (top-left), Type 2 signature (top-right), expression level of ETV4 (bottom-left) and PCA3 (bottom-right). e, Heatmap of CNA per spot in cluster 2 and 4 of slice V2_5, related to c. f, Boxplot showing the Type 2, SFTPA2+ and Type 1 signature in each tumor sample of TCGA-PRAD grouped by molecular subtype. ERG, n = 152; ETV1, n = 28; ETV4, n = 14; others, n = 135. Adjusted P values were calculated by two-sided Tukey’s test. In boxplots, the central line represents the median value, box limits indicate the interquartile ranges and the whiskers extend to 1.5× interquartile range.
Extended Data Fig. 6 Type 2 and SFTPA2+ signature represent the feature of ETS− prostate cancer.
a, Correlation between ABSOLUE estimated tumor purity and signature score of dLum cells. Dots were colored by the molecular subtypes. Error bands represent 95% confidence intervals of the correlation curve. b, Heatmap showing expression levels of selected genes in CPGEA. Expression level of each gene was transformed into row z score before visualization. c, Overview of the sampling strategy of the prostate of patient 1. d, Spatial transcriptomic slices of patient 1 color by CNV Leiden clusters. e, Heatmap grouped by CNV Leiden clusters showing CNA in each spot. f, Spatial transcriptomic slice color by gene signatures calculated by AddModuleScore. g, Overview of the sampling strategy of the prostate of patient 1 (top-left) and spatial transcriptomic slices of patient 1 color by CNV Leiden clusters. h, Heatmap grouped by CNV Leiden clusters showing CNA in each spot. i, Spatial transcriptomic slice colored by gene signatures calculated by AddModuleScore (left and middle) and expression level of SFTPA2 (right) in each spot.
Extended Data Fig. 7 Identification of expression patterns of ERG+ prostate cancer.
a, Spatial transcriptomic slices colored by gene signatures calculated by AddModuleScore and expression level of ERG in each spot. b, Overview of the sampling strategy of the prostate of patient 2. c, H&E staining of the slices sampled from the site encircled in b. d, Spatial transcriptomic slices of patient 2 color by CNV Leiden clusters. e, Enlarged partition of slice H3_1 of patient 2. f, Heatmap grouped by CNV Leiden clusters showing CNA in each spot. g, Spatial transcriptomic slices colored by gene signatures calculated by AddModuleScore. h, Spatial transcriptomic slice colored by expression level of ERG in each spot.
Extended Data Fig. 8 Identification of factors accounting for loss of type 2 and SFTPA2+ features in prostate cancer.
a, Spatial transcriptomic slices of patient 2 colored by CNV Leiden clusters. Loss of PTEN was detected in tumor region inside the red dashed line. b, Heatmap grouped by CNV Leiden clusters showing CNA in each spot. c, Spatial transcriptomic slices colored by gene signatures calculated by AddModuleScore and expression level of PTEN (middle) in each spot. d, Chromosomal ___location of SFTPA2 and PTEN on chr 10 of human. e, Boxplot showing the type 2 and type 1 signature in each ETS− sample of TCGA-PRAD grouped by Gleason score (top) and pathological T stage (bottom). Gleason 6: n = 30; 7: n = 67, ≥8: n = 38; pT stage T2: n = 56, T3: n = 75, T4: n = 2. Adjusted P values were calculated by two-sided Tukey’s test. The central line represents the median value, box limits indicate the interquartile ranges and the whiskers extend to 1.5× interquartile range. f, Heatmap showing expression levels of selected genes in a CRPC cohort. Expression level of each gene was transformed into row z score before visualization.
Extended Data Fig. 9 Spatial analysis identified zone-specific fibroblast subgroups.
a, Sapling strategy (bottom) and H&E staining (top) of the slice H2_4 of patient 2. b, Spatial transcriptomic slice H2_4 of patient 2 colored by inferred cell fractions and expression level of CHGB in each spot. c, Enlarged partition of H2_4 encircled in a. d, Spatial transcriptomic slices sampled from the peripheral zone of HP05 colored by inferred cell fractions. e, H&E staining of the slice H2_5 of patient 2. f, Spatial transcriptomic slices, H2_5, of patient 2 colored by inferred cell fractions.
Extended Data Fig. 10 Identification of a special structure in the stroma of human prostate.
a, Heatmap showing Pearson’s correlation coefficient between cell proportions of selected cell types in total single cells in scRNA-seq data. b, Heatmap showing Pearson’s correlation coefficient between cell signature intensity and marker genes of IM and RTM in GTEx prostate dataset. Signature scores of specific cell subpopulations were calculated by the ssGSEA algorithm. c, Sapling strategy of the slices V2_3 and V2_4 of patient 2. d, Spatial transcriptomic slices, V2_3, of patient 2 colored by signature scores calculated by AddModuleScore (top), expression level of CHGB and inferred cell fractions in each spot. H&E staining image of the type 1 fibroblast-enriched region was enlarged to visualize the histological feature of the structure (top-left). e, Spatial transcriptomic slices, V2_4, of patient 2 colored by signature scores calculated by AddModuleScore (top), expression level of CHGB (bottom-left) and inferred cell fractions in each spot (bottom-middle and bottom-right). f, Multicolor IF staining of the special structure in human prostate, related to Fig. 6k. Experiment has been repeated three times. g, UMAP of fibroblasts in scRNA-seq data colored by expression level of CHGB in fibroblast from the central zone (left), transition zone (middle) and peripheral zone (right) of human prostate.
Supplementary information
Supplementary Information
Supplementary Note and Supplementary Figs. 1–11.
Supplementary Tables 1–5
Supplementary Table 1: Clinical characteristics of prostate donors. Supplementary Table 2: Sequencing strategy of each sample. Supplementary Table 3: Top markers of each major cell types and cell subpopulations. Supplementary Table 4: Relationship between epithelial cells subgroups identified in this work and already known subpopulations. Supplementary Table 5: Gene list used to score visium slice and bulk RNA-seq data.
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Hu, J., Liu, F., Zhang, J. et al. Spatially resolved transcriptomic analysis of the adult human prostate. Nat Genet 57, 922–933 (2025). https://doi.org/10.1038/s41588-025-02139-9
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DOI: https://doi.org/10.1038/s41588-025-02139-9
