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Single-cell transcriptomics identifies a distinct luminal progenitor cell type in distal prostate invagination tips

Nature Genetics volume 52, pages 908–918 (2020)Cite this article

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Abstract

The identification of prostate stem/progenitor cells and characterization of the prostate epithelial cell lineage hierarchy are critical for understanding prostate cancer initiation. Here, we characterized 35,129 cells from mouse prostates, and identified a unique luminal cell type (termed type C luminal cell (Luminal-C)) marked by Tacstd2, Ck4 and Psca expression. Luminal-C cells located at the distal prostate invagination tips (termed Dist-Luminal-C) exhibited greater capacity for organoid formation in vitro and prostate epithelial duct regeneration in vivo. Lineage tracing of Luminal-C cells indicated that Dist-Luminal-C cells reconstituted distal prostate luminal lineages through self-renewal and differentiation. Deletion of Pten in Dist-Luminal-C cells resulted in prostatic intraepithelial neoplasia. We further characterized 11,374 human prostate cells and confirmed the existence of h-Luminal-C cells. Our study provides insights into the prostate lineage hierarchy, identifies Dist-Luminal-C cells as the luminal progenitor cell population in invagination tips and suggests one of the potential cellular origins of prostate cancer.

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Fig. 1: Single-cell transcriptomic survey of mouse prostate cells.
Fig. 2: Localization and characterization of Luminal-C cells.
Fig. 3: Lineage tracing of Ck4-expressing Dist-Luminal-C cells.
Fig. 4: Lineage tracing of Ck4-expressing Prox-Luminal-C cells.
Fig. 5: Prostate hyperplasia and PIN derived from the Luminal-C cell population.
Fig. 6: Luminal-C cells in the normal human prostate.
Fig. 7: Schematic diagram of the self-renewal of Dist-Luminal-C cells and their role as prostate cancer-initiating cells.

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Data availability

All requests for raw and analyzed data and materials should be directed to the corresponding authors. Data and materials that can be shared will be released via a material transfer agreement. All raw sequencing data created in this study have been uploaded to the National Omics Data Encyclopedia (NODE; https://www.biosino.org/node/index) with the accession number OEP000825.

Code availability

The custom code used is available at GitHub (https://github.com/LinLi-0909/Single-cell-analysis).

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Acknowledgements

We acknowledge J. Huang (Duke University) and A. M. De Marzo (Johns Hopkins University) for histopathological data consultation, B. Wu and G. Chen for animal husbandry and W. Bian for technical help at the SIBCB Core Facility. We thank the Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, CAS for technical support. This study was supported by grants from the National Key Research and Development Program of China (grant no. 2017YFA0505500), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant nos. XDA16020905 and XDB19000000), the National Natural Science Foundation of China (grant nos. 81830054, 81772723, 61403363 and 11901272), the State Key Project for Infectious Diseases (grant no. 2018ZX10302207-004-002), the Shanghai Municipal Science and Technology Major Project (grant no. 2017SHZDZX01) and the US National Cancer Institute (grant nos. R01CA208100, R01CA193837, P50CA092629 and P30CA008748).

Author information

Author notes

  1. These authors contributed equally: Wangxin Guo, Lin Li.

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China

    Wangxin Guo, Lin Li, Juan He, Zhuang Liu, Ming Han, Fei Li, Xinyi Xia, Xiaoyu Zhang, Yunguang Li, Rebiguli Aji, Hao Dai, Hui Wei, Chunfeng Li, Luonan Chen & Dong Gao

  2. University of Chinese Academy of Sciences, Beijing, China

    Wangxin Guo, Lin Li, Juan He, Zhuang Liu, Ming Han, Fei Li, Xinyi Xia, Xiaoyu Zhang, Yunguang Li, Rebiguli Aji, Hao Dai & Dong Gao

  3. Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, China

    Yao Zhu & Yu Wei

  4. Department of Oncology, Shanghai Medical College, Shanghai, China

    Yao Zhu & Yu Wei

  5. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Yu Chen

  6. Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Yu Chen

  7. Department of Medicine, Weill Cornell Medical College and New York-Presbyterian Hospital, New York, NY, USA

    Yu Chen

  8. Department of Cell and Developmental Biology, Weill Cornell Medical College and New York-Presbyterian Hospital, New York, NY, USA

    Yu Chen

  9. Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China

    Luonan Chen

  10. Key Laboratory of Systems Biology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, China

    Luonan Chen

  11. Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China

    Dong Gao

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  1. Wangxin Guo
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Contributions

D.G. conceived and designed the experimental approach. W.G. performed most experiments. L.L., H.D. and L.C. contributed to the computational and statistical analyses. J.H., Z.L., M.H., X.X., X.Z., F.L., Y.G.L., R.A., Y.Z., Y.W., H.W. and C.L. helped with the experiments and provided technical support. D.G., L.C. and Y.C. prepared the manuscript as the senior authors.

Corresponding authors

Correspondence to Yu Chen, Luonan Chen or Dong Gao.

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Extended data

Extended Data Fig. 1 Decoding of the identity of cell types within the main clusters.

a, T-SNE maps show the expression levels of epithelial cell marker Epcam and known luminal cell marker genes (Ck8, Ck18, Ar, EYFP) across 11 clusters. Red circles indicate all the luminal cell clusters. b-k, T-SNE maps show the expression levels of marker genes across 11 clusters. Red circles indicate the Luminal-A cluster (n = 2,773 cells) (b), Luminal-B cluster (n = 2,233 cells) (c), Luminal-C cluster (n = 232 cells) (d), Neuroendocrine cell cluster (n = 4 cells) (e), Basal cluster (n = 747 cells) (f), Seminal Vesicle cluster (n = 406 cells) (g), Stromal cluster (n = 1,208 cells) (h), Macrophage cluster (n = 660 cells) (i), Lymphocyte cluster (n = 114 cells) (j), Endothelial cluster (n = 168 cells) (k). t-SNE map shows cells that are colored by the log-scale normalized read count of marker genes.

Extended Data Fig. 2 Lobe specific distribution of luminal clusters.

a, Visualization of clustering of 13,632 single cells from freshly dissociated AP, VP and DLP of WT mice at age of 10 weeks, based on the expression of known marker genes by t-SNE. b, Visualization the lobe distribution patterns of different cell clusters (n = 13,632 cells). c, Bar graph shows the percentage of the three luminal cell lineages in AP, VP and DLP. d-i, T-SNE maps show the expression levels of marker genes across 7 clusters. Black circle indicate the Luminal clusters (n = 9,295 cells) (d), Luminal-A cluster (n = 4,874 cells) (e), Luminal-B cluster (n = 4,059 cells) (f), Luminal-C cluster (n = 362 cells) (g), Basal cluster (n = 836 cells) (h), Stromal cluster (n = 2,864 cells) (i). T-SNE map shows cells that are colored by the log-scale normalized read count of marker genes.

Extended Data Fig. 3 Location of Luminal-A, Luminal-B and Luminal-C cells in different prostate lobes.

a,b, Immunofluorescence of Hoxb13 (a) and Nkx3.1 (b) in WT mouse anterior prostate (AP) (red circles), dorsal-lateral prostate (DLP) (green circle), ventral prostate (VP) (purple circle), proximal prostate (yellow circle), and urethra (light blue circle). c, immunofluorescence of Tacstd2 in the whole prostate of T2Y mouse. White circles indicate all the proximal prostate; green circle indicate the urethra. d,e,f,g Co-immunofluorescence of Hoxb13, Nkx3.1 and Tacstd2 in AP (d), DLP (e), VP (f) and proximal prostate (g) of WT prostate. 3 independent mice were used for each experiment. Scale bars, 50 μm(d-g).

Extended Data Fig. 4 Transcriptomic analysis of Dist-Luminal-C cells, Dist-Luminal-C cells, Tacstd2-negative luminal cells and basal cells.

a,b, FACS plots, gating strategy of the prostate basal cells (a), distal (b, upper panel) and proximal (b, lower panel) prostate epithelial cells. c, Heatmap for analyzing the gene signatures of Dist-Luminal-C cells, Dist-Luminal-C cells, Tacstd-negative luminal cells and basal cells (log2(fold change) >=log2(1.5), p-value < =0.01,edgeR). Each row represents one gene, while each column represents one sample. (The expression values of the gene are row z-score log (FPKM + 1)). d, Heatmap shows the gene signatures of Dist-Luminal-C cells, Dist-Luminal-C cells, Tacstd-negative luminal cells. 6 independent mice were used for bulk RNA-seq assay.

Extended Data Fig. 5 Functional characterization of Luminal-C cells.

a, Co-immunofluorescence of Tacstd2 with Ki67 in proximal prostate and distal prostate of T2Y mouse. b, Percentage of Ki67+ cells in Tacstd2-negative luminal cells, Proximal-Luminal-C cells and Distal-Luminal-C cells. c,d, Co-immunofluorescence of Ck8, Trp63 and endogenous YFP in organoids which derived from Dist-Luminal-C (c) and Prox-Luminal-C cells (d) of T2Y mouse prostates. e, H&E and immunohistochemical staining of prostate duct from renal grafts of total Luminal-C cells express YFP, Trp63 and Ck8. f, Co-immunofluorescence of Tacstd2 and endogenous YFP in renal grafts which derived from total Luminal-C cells. g,h, Co-immunofluorescence of Hoxb13 (g), Nkx3.1 (h) and endogenous YFP in renal grafts which derived from total Luminal-C cells. 4 independent mice were used in proliferation assay (a,b). These experiments were administered three (e-h) and five (c, d) times with similar results. Scale bars, 50μm (a,c-h).

Extended Data Fig. 6 Single-cell transcriptomic survey for prostate cells of intact and regressed mice (castrated for 7 days and castrated for 28 days).

a, Visualization of clustering of 17,305 single cells (points; n = 6 mice), based on the expression of known marker genes by t-SNE (left panel). Cell numbers and percentages of assigned cell types are summarized in the right panel. Luminal-A, type A luminal cell; Luminal-B, type B luminal cell; Luminal-C, type C luminal cell; Basal, basal cell; Stromal, stromal cell; Macro, macrophage; SV, seminal vesicle epithelial cell. b, T-SNE map shows the cell distribution from intact mice (day 0; n = 8,545 cells), regressed mouse (day 7; n = 5,734 cells) and (day 28; n = 3,006 cells). c, Bar graph shows the percentage of Luminal-A, Luminal-B and Luminal-C cells in total cells of intact mice (day 0), regressed mouse (day 7) and (day 28). d-j, T-SNE maps show the expression levels of marker genes across 8 clusters. Black circles indicate all luminal cluster (n = 13,136 cells) (d), basal cluster (n = 1,090 cells) (e), the Luminal-A cluster (n = 7,532 cells) (f), Luminal-B cluster (n = 4,903 cells) (g), Luminal-C cluster (n = 701 cells) (h), stromal cluster (n = 1,415 cells) (i), macrophage cluster (n = 727 cells) (j). t-SNE map shows cells that are colored by the log-scale normalized read count of marker genes. k, Mean Expression in target cells (log2[TP10K], color) and fraction of expressing cells (dot size) of key luminal markers of Luminal-A, Luminal-B and Luminal-C (rows) in cells from each luminal sub-clusters of intact mice (day 0), regressed mouse (day 7) and (day 28) (columns).

Extended Data Fig. 7 Lineage tracing of Psca-expressing Dist-Luminal-C cells.

a, Schematic of targeting strategy to generate the PaY (PscaCreERT2/+; Rosa26EYFP/+) mouse is used to label Psca-expression cells by EYFP expression at 8-week male mice. b, Time-line for Luminal-C cell labeling and prostate regression-regeneration in PaY mouse prostate. c, Co-immunofluorescence of Ck5, Ck8 and Trp63 with endogenous YFP in intact PaY mouse prostates 2-weeks after tamoxifen injection. d, Percentage of YFP + cell in Ck5- cells, Ck5+ cells, Trp63- cells, Trp63+ cells, Ck8- cells and Ck8+ cells of 10-week PaY mouse prostate. e, Co-immunofluorescence of Tacstd2 with endogenous YFP in intact PaY mouse prostate distal regions 2-weeks after tamoxifen injection. f, Co-immunofluorescence of Ck5, Ck8 and Trp63 with endogenous YFP in regenerated prostate after one cycle of regression-regeneration. g, Co-immunofluorescence of Ck5, Ck8 and Trp63 with endogenous YFP in regenerated prostate after three cycles of regression-regeneration. h, Percentage of YFP-positive cell clone in intact, regenerated prostate and regenerated prostate after one or three cycles of regression-regeneration. 8 independent mice were used for each experiment. Scale bars, 50 μm (c, e, f, g). Data show mean ± standard deviation and two-way ANOVA (h). ****P < 0.0001 (h).

Extended Data Fig. 8 Dist-Luminal-C cells are favored to generate prostate cancer.

a, H&E and Co-immunofluorescence of pAkt, Tacstd2 and Ck4 in T2P mouse prostate one month after tamoxifen treatment. Most of Pten-loss luminal cells gained the Luminal-C marker Tacstd2 and Ck4 expression. b, Co-immunofluorescence of Luminal-A (Hoxb13high; Tacstd2-; pAkt + ) and Dist-Luminal-C (Tacstd2 + ; Hoxb13low; pAkt + ) cells with Pten deletion in T2P mouse prostate 7 days after tamoxifen treatment. c, Co-immunofluorescence of Luminal-B (Nkx3.1high; Tacstd2-; pAkt + ) and Dist-Luminal-C (Tacstd2 + ; Nkx3.1low; pAkt + ) cells with Pten deletion in T2P mouse prostate 7 days after tamoxifen treatment. d, Co-immunofluorescence of Luminal-A (Hoxb13high; Tacstd2-; pAkt + ) and Dist-Luminal-C (Tacstd2 + ; Hoxb13low; pAkt + ) cells with Pten deletion in T2P mouse prostate 14 days after tamoxifen treatment. e, Co-immunofluorescence of Luminal-B (Nkx3.1high; Tacstd2-; pAkt + ) and Dist-Luminal-C (Tacstd2 + ; Nkx3.1low; pAkt + ) cells with Pten deletion in T2P mouse prostate 14 days after tamoxifen treatment. f, Percentage of Luminal-A (Hoxb13high; Tacstd2-; pAkt + ), Luminal-B (Nkx3.1high; Tacstd2-; pAkt + ) and Dist-Luminal-C (Tacstd2 + ; Nkx3.1low; pAkt + ) cell clone in T2P mouse prostates after 7 or 14 days of tamoxifen treatment. g, Percentage of cell types in T2P mouse prostates after 7 or 14 days of tamoxifen treatment. In (f) and (g) data were from at least three independent experiments, which involving five mice (**P < 0.01; ****P < 0.0001, 2way ANOVA for multiple comparisons and unpaired t test). Scale bars, 50μm (a), 25μm (b-e). These data were from three independent experiments, which involving five mice(a-g). Data show mean ± standard deviation and 2way ANOVA for multiple comparisons (f,g). Scale bars, 50μm (a), 25μm (b-e). **P < 0.01; ****P < 0.0001 (f,g).

Extended Data Fig. 9 Decoding of the identity of cell types within the main human prostate cell clusters.

a, T-SNE maps show the expression levels of epithelial cell marker Epcam across 7 clusters. Blue circles indicate all the epithelial cell clusters (n = 9,825 cells). b-h, T-SNE maps show the expression levels of marker genes across 7 clusters. Blue circles indicate the h-Basal cluster (n = 5,696 cells)(b), all luminal cell clusters (n = 4,129 cells)(c), h-Luminal-A/B cluster (n = 3,434 cells) (d), h-Stromal cluster (n = 387 cells) (e), h-Macrophage cluster (n = 439 cells) (f), h-Lymphocyte cluster (n = 106 cells) (g), and h-Endothelial cluster (n = 617 cells) (h). i, Visualization of clustering of prostate single luminal cells, based on the expression of known marker genes by t-SNE (n = 4,129 cells). Decoding of the identity of cell types within the main luminal cell clusters. j, T-SNE maps show the expression levels of h-Luminal-C cell markers CK4, PSCA, TACSTD2 and PIGR (n = 908 cells). k, T-SNE maps show the expression levels of mouse Luminal-A/B cell markers HOXB13 and NKX3.1 (n = 3,166 cells). l, T-SNE maps show the expression levels of h-Luminal-A cell markers MSMB, KLK3, KLK2 and NPY (n = 2,743 cells). m, T-SNE maps show the expression levels of Luminal-B cell markers SLC14A1, MEG3, FHL2 and KRT23 (n = 423 cells). Black circle indicate the indicated cell clusters. t-SNE maps showed cells that are colored by the log-scale normalized read count of marker genes.

Extended Data Fig. 10 The expression levels of h-Luminal-C markers in human prostate cancer.

a, TACSTD2 immunohistochemistry staining of human prostate cancer samples (left panel), the graph indicates the percentage of TACSTD2 expression level (low, medium, high) in human prostate cancer samples (right panel). b, KRT4 immunohistochemistry staining of human prostate cancer samples (left panel), the graph indicates percentage of KRT4 expression level (low, medium, high) in human prostate cancer samples (right panel). c, PSCA immunohistochemistry staining of human prostate cancer samples (left panel), the graph indicates percentage of PSCA expression level (low, medium, high) in human prostate cancer samples (left panel). d,e, The expression levels of h-Luminal-C markers (TACSTD2, KRT4, PSCA) were analyzed in human prostate tumors and normal prostates from TCGA (n = 547) (d) and GSE8218 (n = 136) (e) data set. f, Graphs indicate the relationship between expression levels of h-Luminal-C markers (TACSTD2, KRT4 and PSCA) and tumor stage (n = 547). Scale bars, 50μm (a-c). 13 prostate cancer samples are from Fudan University Shanghai Cancer Center, the others are from the Human Protein Atlas (https://www.proteinatlas.org/) (a-c). These experiments were administered three times with similar results (a-c). Data show mean ± standard deviation and unpaired two-tailed Student’s t-test (d-f).

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Guo, W., Li, L., He, J. et al. Single-cell transcriptomics identifies a distinct luminal progenitor cell type in distal prostate invagination tips. Nat Genet 52, 908–918 (2020). https://doi.org/10.1038/s41588-020-0642-1

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