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

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.

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

(n = 8,545 cells). 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 (Psca^CreERT2/+; Rosa26^EYFP/+) mouse is used to label Psca-expression cells by EYFP expression at 8-week male mice. b, Timeline 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 (Hoxb13^high; Tacstd2⁻; pAkt⁺) and Dist-Luminal-C (Tacstd2⁺; Hoxb13^low; pAkt⁺) cells with Pten deletion in T2P mouse prostate 7 days after tamoxifen treatment. c, Co-immunofluorescence of Luminal-B (Nkx3.1^high; Tacstd2⁻; pAkt⁺) and Dist-Luminal-C (Tacstd2⁺; Nkx3.1^low; pAkt⁺) cells with Pten deletion in T2P mouse prostate 7 days after tamoxifen treatment. d, Co-immunofluorescence of Luminal-A (Hoxb13^high; Tacstd2⁻; pAkt⁺) and Dist-Luminal-C (Tacstd2⁺; Hoxb13^low; pAkt⁺) cells with Pten deletion in T2P mouse prostate 14 days after tamoxifen treatment. e, Co-immunofluorescence of Luminal-B (Nkx3.1^high; Tacstd2⁻; pAkt⁺) and Dist-Luminal-C (Tacstd2⁺; Nkx3.1^low; pAkt⁺) cells with Pten deletion in T2P mouse prostate 14 days after tamoxifen treatment. f, Percentage of Luminal-A (Hoxb13^high; Tacstd2⁻; pAkt⁺), Luminal-B (Nkx3.1^high; Tacstd2⁻; pAkt⁺) and Dist-Luminal-C (Tacstd2⁺; Nkx3.1^low; 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).

Figure 1 |. Single-cell transcriptomic survey of mouse prostate cells.

a, Visualization of the clustering of 8,545 single cells (points; n  =  4 mice) based on the expression of known marker genes by t-SNE (left panel). The numbers and percentages of the 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; Neuro, neuroendocrine cell; SV, seminal vesicle epithelial cell; Stromal, stromal cell; Macro-A, macrophage-A; Macro-B, macrophage-B; Lymph, lymphocyte; Endo, endothelial cell. b, Violin plots showing the expression levels of representative marker genes across the main clusters (n = 8,545 cells). c, Violin plots showing the expression levels of representative marker genes across the three luminal cell clusters. The y axis shows the log-scale normalized read count (n = 5,238 cells). d, Top 40 significantly enriched (Methods; P value < 0.01; −log₁₀ (P value)) gene ontology terms in the gene signature for the Luminal-C subtype (n = 232 cells).

Figure 2 |. Localization and characterization of Luminal-C cells.

a,b, Immunofluorescence staining for YFP and Tacstd2 shows that Luminal-C cells are in the distal prostate invagination tip (arrow) (a) and proximal prostate region (b) of T2Y mice. c, Violin plot comparing the Markov-chain entropy (MCE) of Tacstd2-negative luminal cells (n = 3 samples), Dist-Luminal-C cells (n = 3 samples) and Prox-Luminal-C cells (n = 3 samples). The P value is from the one-sided t test. d, Micrographs of organoids formed by Tacstd2-negative luminal cells, Prox-Luminal-C cells and Dist-Luminal-C cells isolated from the T2Y mouse prostate and cultured in Matrigel with mouse prostate organoid medium (scale bars, 500 μm). e,f, Organoid formation efficiency (n = 6, n = 11, n = 12) (e) and organoid size (n = 109, n = 891, n = 1,029) (f) of Tacstd2-negative luminal cells, Pro-Luminal-C cells and Dis-Luminal-C cells calculated after one week of culture. The data are presented as the mean ± standard deviation of five replicates, and 3 mice were used for each experiment. g, Photographs of renal capsule regeneration with 50 cells (I), 200 cells (II), 1,000 cells (III) and 10,000 cells (IV) at 2.5 months after transplantation. The red arrowheads show Luminal-C cell grafts, and the blue arrowheads show Tacstd2⁻ luminal cell grafts. Bioluminescence intensity is shown in photons per second per centimeter squared per steradian (p s⁻¹ cm⁻² sr⁻¹) h, Box plot of the graft efficiency of Tacstd2-negative luminal cells and total Luminal-C cells. Six mice were used for each experiment, and each experiment was performed three times. Scale bars, 50 μm (a,b). Data were analyzed by unpaired two-tailed Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (e,f).

Figure 3 |. Lineage tracing of Ck4-expressing Dist-Luminal-C cells.

a, Schematic of the targeting strategy to generate C4T (Ck4^CreERT2/+;Rosa26^td-Tomato/+) mice to label Ck4-expressing cells by tdTomato expression. b, Timeline of Luminal-C cell labeling and prostate regression-regeneration in the C4T mouse prostate. c, Co-immunofluorescence of Ck5, Ck8, Ck14 and Trp63 with endogenous tdTomato in the distal region of the intact C4T mouse 2 weeks after tamoxifen injection. d, Percentage of tdTomato⁺ cells among Ck5⁻ cells, Ck5⁺ cells (n = 1,491), Ck14⁻ cells, Ck14⁺ cells (n = 1,219), Trp63⁻ cells, Trp63⁺ cells (n = 1,685), Ck8⁻ cells and Ck8⁺ cells (n = 1,641) in the 10 week C4T mouse prostate. e, Co-immunofluorescence of Tacstd2 and Ck4 with endogenous tdTomato in the distal region of the intact C4T mouse prostate 2 weeks after tamoxifen injection. f, Immunostaining of Ck5, Ck8 and Trp63 with endogenous tdTomato in the regenerated prostate after one regression-regeneration cycle. g, Percentage of tdTomato-positive cells among Ck8-positive luminal cells in the distal region of the intact and regenerated C4T mouse prostate after one or three regression-regeneration cycles (n = 6 mice/group). h, Immunostaining of Ck5, Ck8 and Trp63 with endogenous tdTomato in the regenerated prostate after three regression-regeneration cycles. i,j, Percentage of tdTomato⁺ cell clones (i) and average colony size (j) in the intact and regenerated prostate after one or three regression-regeneration cycles. k-m, Luminal-C cells drive prostate luminal cell regeneration by a self-renewal mechanism (k). Quantification of tdTomato-labeled cell progeny showed that Tacstd2⁺/tdTomato⁺ Dist-Luminal C cells both self-renew to replenish Tacstd2⁺ Luminal-C cells and differentiate into Tacstd2⁻ daughter luminal cells (n = 6 mice/group) (l). The percentage of Tacstd2⁺/tdTomato⁺ Dist-Luminal C cells was sustained during the regression-regeneration period (n = 6 mice/group) (m). n, Immunostaining of Tacstd2, Nkx3.1 and Hoxb13 with endogenous tdTomato in the regenerated prostate after one regression-regeneration cycle. Six mice were used for each experiment. Data show mean ± standard deviation (g, j, l and m), two-way ANOVA (i) and unpaired two-tailed Student’s t test (g,j,l and m). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001, (d,g,i,j,l,m). Scale bars, 50 μm (c,e,f,h,n).

Figure 4 |. Lineage tracing of Ck4-expressing Prox-Luminal-C cells.

a, Immunofluorescence of Tacstd2 in the proximal region of the 10-week-old adult mouse prostate. b, Co-immunofluorescence of Ck5, Ck8 and Tacstd2 with endogenous tdTomato in the proximal region of the intact C4T mouse prostate 2 weeks after tamoxifen injection. c, Co-immunofluorescence of Tacstd2 with endogenous tdTomato in the proximal region of the intact C4T mouse prostate 2 weeks after tamoxifen injection. The yellow lines indicate the boundary of the proximal and distal regions. d, Prox-Luminal-C daughter cells will cross the boundary and generate Tacstd2-negative cells if they contribute to distal prostate regeneration. e, Immunostaining of Ck5, Ck8 and Trp63 with endogenous tdTomato in the regenerated prostate after one regression-regeneration cycle. f, Prox-Luminal-C cells did not cross the boundary and contribute to distal prostate regeneration. Immunostaining of Tacstd2 with endogenous tdTomato in the regenerated prostate after one regression-regeneration cycle. These experiments were performed three times with similar results (a-c,e,f). Scale bars, 50 μm (a-c,e,f).

Figure 5 |. Prostate hyperplasia and PIN derived from the Luminal-C cell population.

a, Targeting strategy to generate the C4TP (Psca^CreERT2/+;Rosa26^EYFP/+;Pten^flox/flox) mouse, label Ck4-expressing cells with tdTomato and perform lineage-specific deletion of the tumor suppressor Pten. b, Schematic of the Luminal-C labeling and Pten deletion strategy in C4TP mice. c,d, Images of H&E staining and pAkt(S473) immunohistochemistry of the C4TP distal prostate at 1 month (c) or 2 months (d) after tamoxifen injection: anterior lobe (left panel), ventral lobe (middle panel), and dorsal-lateral lobe (right panel). e,f Immunofluorescence staining of Tacstd2 and endogenous tdTomato shows that all Luminal-C lineage-marked daughter cells are Tacstd2-positive with Pten deletion. C4TP prostate distal region (left panel) and proximal region (right panel). g,h Images of H&E staining and pAkt(S473) immunohistochemistry of the distal region (g) and proximal region (the square shows the PIN lesion adjacent to the intermediate region) (h) of the C4TP mouse prostate 5 months after tamoxifen injection. Five mice were analyzed for each experiment (c-h). Scale bars, 50 μm (c-h).

Figure 6 |. Luminal-C cells in the normal human prostate.

a, Visualization of the clustering of 11,374 single human prostate cells based on the expression of known marker genes by t-SNE. h-Luminal-A/B, type A/B luminal cell; h-Luminal-C, type C luminal cell; h-Basal, basal cell; h-Stromal, stromal cell; h-Macro, macrophage; h-Endo, endothelial cell; h-Lymph, lymphocyte. b, T-SNE maps show the expression levels of h-Luminal-C marker genes (CK4, PSCA, TACSTD2 and PIGR) across 7 clusters (n = 11,374 cells). c, Immunofluorescence staining of TACSTD2, TP63 (upper panel) and CK8 (lower panel) showed that Luminal-C cells reside in normal human prostate invagination tips (red arrows). d, Immunofluorescence staining of CK4 (upper panel), PIGR (middle panel), PSCA (lower panel) and TP63 showed that Luminal-C cells reside in normal human prostate invagination tips (red arrows). These experiments were administered three times with similar results (c,d). Scale bars, 50 μm (c-d)

Figure 7 |. Schematic diagram of the self-renewal of Dist-Luminal-C cells and their role as prostate cancer-initiating cells.

Dist-Luminal-C cells are located in invagination tips of the mouse prostate. Luminal-C cells undergo self-renewal in prostate invagination tips during mouse prostate regression and regeneration. Dist-Luminal-C cells can serve as the cellular origin of prostate cancer.