Single luminal epithelial progenitors can generate prostate organoids in culture

Abstract

The intrinsic ability to exhibit self-organizing morphogenetic properties in ex vivo culture may represent a general property of tissue stem cells. Here we show that single luminal stem/progenitor cells can generate prostate organoids in a three-dimensional culture system in the absence of stroma. Organoids generated from CARNs (castration-resistant Nkx3.1-expressing cells) or normal prostate epithelia exhibit tissue architecture containing luminal and basal cells, undergo long-term expansion in culture and exhibit functional androgen receptor signalling. Lineage-tracing demonstrates that luminal cells are favoured for organoid formation and generate basal cells in culture. Furthermore, tumour organoids can initiate from CARNs after oncogenic transformation and from mouse models of prostate cancer, and can facilitate analyses of drug response. Finally, we provide evidence supporting the feasibility of organoid studies of human prostate tissue. Our studies underscore the progenitor properties of luminal cells, and identify in vitro approaches for studying prostate biology.

Figures

Figure 1

Generation of prostate epithelial organoids from lineage-marked CARNs. (a) Time course of lineage-marking of CARNs in Nkx3.1CreERT2/+; R26R-YFP/+ mice. (b) Isolation of YFP-positive lineage-marked CARNs by flow cytometry. (c,d) Bright-field (c) and epifluorescent (d) views of CARN-derived organoids that are filled or hollow (arrow). (e,f) Hematoxylin-eosin (H&E) staining of CARN organoids at low-power (e) showing range of phenotypes, and high-power (f). (g) Uniform YFP expression with Ki67 immunostaining (arrows). (h) The basal marker CK5 is expressed on the exterior (arrowheads), while the luminal marker CK8 is expressed internally. (i,j) Strong nuclear expression of AR (arrows, i) and Foxa1 (j). (k–m) Renal grafts generated by tissue recombination of CARN-derived organoids with rat embryonic urogenital mesenchyme (k) display normal stratification of basal (arrowheads, l) and luminal cells (l), and uniform YFP and nuclear AR immunostaining (m); note that the slightly atypical histology in (k) likely reflects the heterozygous phenotype of Nkx3.1 mutants, . (n) Efficiency of organoid formation by lineage-marked CARNs (YFP-positive cells from tamoxifen-induced and castrated Nkx3.1CreERT2/+; R26R-YFP/+ mice; n=4 experiments) and non-CARNs (YFP-negative cells from the same mice; n=3 experiments). Source data are provided in Supplementary Table 1. Error bars represent one standard deviation; the difference between CARNs and non-CARNs is statistically significant (p = 0.002, two-tailed Student’s t-test). (o) Generation of organoids from single CARNs. Time course of paired images shown under bright-field (top) and epifluorescent (bottom) illumination shows organoid growth from isolated single CARN. Scale bars in o correspond to 25 microns, in f–j,l,m to 50 microns, and in c–e,k to 100 microns.

Figure 2

Growth and androgen-responsiveness of prostate organoids from normal prostate epithelium. (a) Flow-sorting strategy to eliminate EpCAM−E-cadherin− cells from dissociated prostate tissue for organoid culture. (b) Low-power view of organoids at 20 days after plating, showing heterogeneity of phenotype. (c) Higher-power view showing hollow and filled budding organoid (arrow). (d,e) H&E staining of sections from a hollow organoid (d) and a multi-layered organoid (e). (f) Many proliferating cells are detectable by Ki67 immunostaining (arrows). (g) Organoids have an outer layer that expresses the basal marker CK5 (arrowheads). (h) Outer cells express the basal marker p63 (arrowheads), while interior cells are positive for the luminal marker CK18. (i) Nuclear immunostaining of AR (arrows) in organoids cultured in standard conditions with DHT. (j) Nuclear immunostaining for Foxa1 (arrows). (k) Tissue recombination of normal organoids with rat embryonic urogenital mesenchyme followed by renal grafting results in reconstitution of prostate tissue. (l,m) Organoids at passage 4 were passaged as single-cell suspensions and plated in the presence of DHT (l) or absence of DHT (m). (n,o) Strong nuclear AR immunostaining in the presence of DHT (n) and weak cytoplasmic AR immunostaining in the absence of DHT (o). (p) qPCR analysis of expression of AR downstream genes in organoids cultured in the presence or absence of DHT. Results are from a single experiment representative of 2 independent experiments. All assays were performed using three technical replicates and normalized to GAPDH expression; Scale bars in e–j,n,o correspond to 50 microns, and in b–d,k,l,m to 100 microns.

Figure 3

Lineage-tracing shows that luminal cells are favored for generation of prostate organoids. (a) Strategy for lineage-marking of basal and luminal epithelial cells for organoid culture. (b) Isolation of YFP-positive luminal cells from CK8-CreERT2; R26R-YFP (CK8-trace) mice by flow cytometry. (c) CK5-trace organoid. (d) Many cells within a CK5-trace organoid are CK5-positive, including internal cells (arrowheads). (e) CK8-trace organoid. (f) CK18-trace organoid. (g) Efficiency of organoid formation from YFP-positive CK5-trace (n=4 experiments), CK8-trace (n=3 experiments), and CK18-trace (n=2 experiments) epithelial cells. The differences in efficiency between CK5-trace and CK8-trace (p=0.001) and between CK5-trace and CK18-trace (p=0.0009) are statistically significant (**) using a two-tailed Student’s t-test; error bars correspond to one standard deviation. Source data are provided in Supplementary Table 1. (h,i) Expression of the basal marker CK5 (arrowheads) in a CK8-trace organoid, shown with (h) and without (i) YFP overlay. (j,k) Expression of the basal marker p63 (arrowheads) in a CK8-trace organoid, shown with (j) and without (k) YFP overlay. (l,m) Expression of the luminal marker CK18 in a CK8-trace organoid, shown with (l) and without (m) YFP overlay. (n) Organoid generated from mixing of red CK18-trace cells and green CK5-trace cells shows green cells on the exterior, consistent with the localization of basal cells. (o) Serial passaging of CK18-trace organoids at passage 3. (p,q) CK18-trace organoids at passage 9 cultured in the presence (p) and absence (q) of DHT. (r,s) AR immunostaining is nuclear in CK18-trace organoids in the presence of DHT (r), but is weakly cytoplasmic in the absence of DHT (s). Scale bars in c–f,h–n correspond to 50 microns, and in o–s to 100 microns.

Figure 4

Tumor organoids can be generated from single transformed CARNs. (a) Time course for generation of transformed CARNs in Nkx3.1CreERT2/+; Ptenflox/flox; KrasLSL/+; R26R-YFP/+ (NPK) mice. (b) NPK-CARN organoid shows extensive budding. (c) H&E staining of NPK-CARN organoids. (d–f) NPK-CARN organoids display Ki67 immunostaining (arrows, d), membrane localization of phospho-Akt (e), and patchy expression of phospho-Erk (arrows, f). (g,h) Luminal phenotype of NPK-CARN organoids, with limited expression of p63 (arrowheads, g) or CK5 (h). (i) Nuclear expression of Foxa1 (arrows). (j,k) AR expression is nuclear in the presence of DHT (arrows, j), but is cytoplasmic in the absence of DHT (k). (l–p) Renal grafts generated by recombination of NPK-CARN organoids with rat embryonic urogenital mesenchyme display high-grade PIN/carcinoma histological phenotypes (l), abundant Ki67 immunostaining (arrows, m), membrane-localized pAkt (n), patchy pErk (arrows, o), and nuclear AR (arrows, p). (q) Generation of organoids from single NPK CARNs. Time course of paired images shown under bright-field (top) and epifluorescent (bottom) illumination shows organoid growth from isolated single NPK CARN. Scale bars in q correspond to 25 microns, in d–h,m–p to 50 microns, and in b,c,i–l to 100 microns.

Figure 5

Modeling tumor phenotypes in organoid culture. (a–j) Formation of organoids from mouse models of prostate cancer, shown in bright-field (a–e) and H&E stained sections (f–j). (a,f) Organoids generated from TRAMP mice at 22 weeks of age. (b,g) Organoids from Nkx3.1CreERT2/+; Ptenflox/flox; p53flox/flox (NPP53) mice induced with tamoxifen at two months of age and assayed at 10 months. (c,h) Organoids from Nkx3.1− − null mutant mice at 14 months of age. (d,i) Organoids from Hi-Myc transgenic mice at 9 months. (e,j) Organoids from Nkx3.1+/−; Pten+/− mice at 10 months. (k) Organoid formation efficiency from the indicated mouse models (data are from 10 technical replicates). (l–r) Induction of tumor phenotypes in culture by tamoxifen treatment of organoids derived from CK8-CreERT2; Ptenflox/flox; KrasLSL-G12D/+; R26R-CAG-YFP mice. (l) Time course of induction experiment. (m–p) Immunostaining for YFP and pAkt in control untreated organoids (m,o) or 4-hydroxy-tamoxifen (4OHT) treated organoids (n,p) at passage 1 (m,n) and at passage 4 (o,p). (q,r) H&E staining of control (q) and 4OHT-treated organoids (r) at passage 4. Scale bars in a–j correspond to 100 microns, and in m–r to 50 microns.

Figure 6

Modeling drug treatment response in organoid culture. (a) Efficiency of organoid formation using organoids from Nkx3.1CreERT2/+; Ptenflox/flox; R26R-YFP/+ (NP) mice. Passaged organoids were treated with the indicated compounds (n=3 samples analyzed per treatment condition); source data are provided in Supplementary Table 1. (b–g) Bright-field images of treated NP organoids. (h,i) H&E sections from control +DHT organoids (h) and enzalutamide + MK8669 treated organoids (i). (j,k) AR expression in control +DHT organoids (arrows, j) and enzalutamide treated organoids (k). (l,m) pAkt expression in control +DHT organoids (arrow, l) and enzalutamide + MK8669 treated organoids (m). Scale bars in h–m correspond to 50 microns, and in b–g correspond to 100 microns. Error bars represent one standard deviation; ** p < 0.01.