Article | Published:

Single luminal epithelial progenitors can generate prostate organoids in culture

Nature Cell Biology volume 16, pages 951961 (2014) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Primary cell cultures as models of prostate cancer development. Endocr. Relat. Cancer 12, 19–47 (2005).

  2. 2.

    & Molecular genetics of prostate cancer: new prospects for old challenges. Genes. Dev. 24, 1967–2000 (2010).

  3. 3.

    & p63 in prostate biology and pathology. J. Cell. Biochem. 103, 1354–1368 (2008).

  4. 4.

    Diagnosis of adenocarcinoma in prostate needle biopsy tissue. J. Clin. Pathol. 60, 35–42 (2007).

  5. 5.

    , , , & Isolation and functional characterization of murine prostate stem cells. Proc. Natl Acad. Sci. USA 104, 181–186 (2007).

  6. 6.

    , , , & Self-renewal and multilineage differentiation in vitro from murine prostate stem cells. Stem Cells 25, 2760–2769 (2007).

  7. 7.

    , & Anchorage-independent culture maintains prostate stem cells. Dev. Biol. 312, 396–406 (2007).

  8. 8.

    et al. Human prostate sphere-forming cells represent a subset of basal epithelial cells capable of glandular regeneration in vivo. Prostate 70, 491–501 (2010).

  9. 9.

    , , , & Isolation, cultivation and characterization of adult murine prostate stem cells. Nat. Protoc. 5, 702–713 (2010).

  10. 10.

    , & Isolation and characterization of human prostate stem/progenitor cells. Methods Mol. Biol. 879, 315–326 (2012).

  11. 11.

    et al. Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics. Proc. Natl Acad. Sci. USA 105, 20882–20887 (2008).

  12. 12.

    et al. Basal epithelial stem cells are efficient targets for prostate cancer initiation. Proc. Natl Acad. Sci. USA 107, 2610–2615 (2010).

  13. 13.

    et al. Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity. Nat. Cell Biol. 15, 274–283 (2013).

  14. 14.

    & Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

  15. 15.

    et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).

  16. 16.

    et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

  17. 17.

    et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell. Nat. Med. 18, 618–623 (2012).

  18. 18.

    et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

  19. 19.

    et al. Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc. Natl Acad. Sci. USA 108, 6235–6240 (2011).

  20. 20.

    et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701–706 (2009).

  21. 21.

    et al. Lgr5(+ ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).

  22. 22.

    et al. In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).

  23. 23.

    et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721 (2013).

  24. 24.

    et al. Artificial three-dimensional niches deconstruct pancreas development in vitro. Development 140, 4452–4462 (2013).

  25. 25.

    et al. A luminal epithelial stem cell that is a cell of origin for prostate cancer. Nature 461, 495–500 (2009).

  26. 26.

    et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148, 1015–1028 (2012).

  27. 27.

    et al. Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture. Cell Growth Differ. 12, 631–640 (2001).

  28. 28.

    , , , & Stromal-epithelial cell interactions and androgen receptor-coregulator recruitment is altered in the tissue microenvironment of prostate cancer. Cancer Res. 67, 511–519 (2007).

  29. 29.

    et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am. J. Pathol. 180, 599–607 (2012).

  30. 30.

    et al. ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency. PLoS ONE 6, e18271 (2011).

  31. 31.

    et al. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc. Natl Acad. Sci. USA 107, 8129–8134 (2010).

  32. 32.

    et al. Forkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation. Development 132, 3431–3443 (2005).

  33. 33.

    et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).

  34. 34.

    et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189–193 (2011).

  35. 35.

    et al. Multipotent and unipotent progenitors contribute to prostate postnatal development. Nat. Cell Biol. 14, 1131–1138 (2012).

  36. 36.

    et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

  37. 37.

    , , , & Adult murine prostate basal and luminal cells are self-sustained lineages that can both serve as targets for prostate cancer initiation. Cancer Cell 21, 253–265 (2012).

  38. 38.

    et al. Conditionally ablated Pten in prostate basal cells promotes basal-to-luminal differentiation and causes invasive prostate cancer in mice. Am. J. Pathol. 182, 975–991 (2013).

  39. 39.

    et al. ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer. Proc. Natl Acad. Sci. USA 110, E3506–E3515 (2013).

  40. 40.

    et al. Roles for Nkx3.1 in prostate development and cancer. Genes Dev. 13, 966–977 (1999).

  41. 41.

    et al. Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis. Cancer Res. 62, 2999–3004 (2002).

  42. 42.

    et al. Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis. Proc. Natl Acad. Sci. USA 99, 2884–2889 (2002).

  43. 43.

    et al. Prostate cancer in a transgenic mouse. Proc. Natl Acad. Sci. USA 92, 3439–3443 (1995).

  44. 44.

    et al. A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential. Cancer Res. 61, 2239–2249 (2001).

  45. 45.

    et al. Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 4, 223–238 (2003).

  46. 46.

    et al. Cross-species analysis of genome-wide regulatory networks identifies a synergistic interaction between FOXM1 and CENPF that drives prostate cancer malignancy. Cancer Cell 25, 638–651 (2014).

  47. 47.

    et al. A molecular signature predictive of indolent prostate cancer. Sci. Transl. Med. 5, 202ra122 (2013).

  48. 48.

    et al. Dual targeting of the Akt/mTOR signaling pathway inhibits castration-resistant prostate cancer in a genetically engineered mouse model. Cancer Res. 72, 4483–4493 (2012).

  49. 49.

    et al. Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer Cell 19, 575–586 (2011).

  50. 50.

    et al. VCaP, a cell-based model system of human prostate cancer. In Vivo 15, 163–168 (2001).

  51. 51.

    Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell 12, 520–530 (2013).

  52. 52.

    , & Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture. Cancer Res. 44, 1998–2010 (1984).

  53. 53.

    et al. Differentiation of prostate epithelial cell cultures by matrigel/stromal cell glandular reconstruction. In Vitro Cell. Dev. Biol. Anim. 42, 273–280 (2006).

  54. 54.

    , & E-cadherin-mediated survival of androgen-receptor-expressing secretory prostate epithelial cells derived from a stratified in vitro differentiation model. J. Cell Sci. 123, 266–276 (2010).

  55. 55.

    , , & Hormonal, cellular, and molecular control of prostatic development. Dev. Biol. 253, 165–174 (2003).

  56. 56.

    Mesenchymal-epithelial interactions: past, present, and future. Differentiation 76, 578–586 (2008).

  57. 57.

    et al. Differentiated Troy + chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 155, 357–368 (2013).

  58. 58.

    et al. Regenerated luminal epithelial cells are derived from preexisting luminal epithelial cells in adult mouse prostate. Mol. Endocrinol. 25, 1849–1857 (2011).

  59. 59.

    et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods 9, 81–83 (2012).

  60. 60.

    et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

  61. 61.

    & Organoid cultures for the analysis of cancer phenotypes. Curr. Opin. Genet. Dev. 24, 68–73 (2014).

  62. 62.

    , & Drug discovery through stem cell-based organoid models. Adv. Drug Deliv. Rev. 69-70, 19–28 (2014).

  63. 63.

    , , , & Ex vivo culture of human prostate tissue and drug development. Nat. Rev. Urol. 10, 483–487 (2013).

  64. 64.

    et al. A preclinical xenograft model identifies castration-tolerant cancer-repopulating cells in localized prostate tumors. Sci. Transl. Med. 5, 187ra171 (2013).

  65. 65.

    et al. High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development. Cancer Res. 74, 1272–1283 (2014).

  66. 66.

    et al. Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis. J. Cell. Biol. 187, 91–100 (2009).

  67. 67.

    et al. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis 32, 148–149 (2002).

  68. 68.

    et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001).

  69. 69.

    et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl Acad. Sci. USA 106, 12771–12775 (2009).

  70. 70.

    , , & Aberrant expression of p63 in adenocarcinoma of the prostate: a radical prostatectomy study. Am. J. Surg. Pathol. 37, 1401–1406 (2013).

Download references

Acknowledgements

We thank M. Kruithof-de Julio, M. Hanoun and P. Frenette for initial discussions about organoid culture, C. Sawyers and C. Abate-Shen for providing pathway inhibitors, C. Liu and the HICCC Flow Cytometry Shared Resource for flow-sorting, D. Sun for assistance with specimen acquisition, the HICCC Molecular Pathology Shared Resource for organoid sectioning and H&E staining, F. Talos for helpful comments on the culture protocol, D-E Parfitt for assistance with cell-picking, and C. Abate-Shen and F. Talos for insightful discussions and comments on the manuscript. This work was supported by postdoctoral fellowships from the DOD Prostate Cancer Research Program (C.W.C., M.S. and R.T.), by a Residency Research Award from the Urology Care Foundation (L.J.B.), and by grants from the National Institutes of Health (M.M.S.).

Author information

Author notes

    • Chee Wai Chua
    • , Maho Shibata
    •  & Ming Lei

    These authors contributed equally to this work.

Affiliations

  1. Department of Medicine, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA

    • Chee Wai Chua
    • , Maho Shibata
    • , Ming Lei
    • , Roxanne Toivanen
    • , Sarah K. Bergren
    •  & Michael M. Shen
  2. Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA

    • Chee Wai Chua
    • , Maho Shibata
    • , Ming Lei
    • , Roxanne Toivanen
    • , Sarah K. Bergren
    •  & Michael M. Shen
  3. Department of Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA

    • Chee Wai Chua
    • , Maho Shibata
    • , Ming Lei
    • , Roxanne Toivanen
    • , Sarah K. Bergren
    •  & Michael M. Shen
  4. Department of Urology, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA

    • Chee Wai Chua
    • , Maho Shibata
    • , Ming Lei
    • , Roxanne Toivanen
    • , LaMont J. Barlow
    • , Sarah K. Bergren
    • , Ketan K. Badani
    • , James M. McKiernan
    • , Mitchell C. Benson
    •  & Michael M. Shen
  5. Department of Pathology and Cell Biology, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA

    • Hanina Hibshoosh

Authors

  1. Search for Chee Wai Chua in:

  2. Search for Maho Shibata in:

  3. Search for Ming Lei in:

  4. Search for Roxanne Toivanen in:

  5. Search for LaMont J. Barlow in:

  6. Search for Sarah K. Bergren in:

  7. Search for Ketan K. Badani in:

  8. Search for James M. McKiernan in:

  9. Search for Mitchell C. Benson in:

  10. Search for Hanina Hibshoosh in:

  11. Search for Michael M. Shen in:

Contributions

C.W.C. and M.L. developed the organoid culture protocol, C.W.C. and M.S. performed analyses of CARN-derived and normal prostate organoids, M.S. performed lineage-tracing studies, M.L. performed analyses of transformed CARN organoids and drug response, R.T. performed analyses of benign human prostate organoids, M.S. and C.W.C. analysed VCaP organoids, L.J.B. performed studies of tumour organoids from mouse models, and S.K.B. assisted with single-cell experiments and tissue grafts, K.K.B. and J.M.M. provided surgical specimens, and H.H. performed pathological analyses. C.W.C., M.S., M.L., R.T., L.J.B., M.C.B, H.H. and M.M.S. designed experiments, analysed data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Chee Wai Chua or Maho Shibata or Ming Lei or Michael M. Shen.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Information

  2. 2.

    Supplementary Table 2

    Supplementary Information

  3. 3.

    Supplementary Table 3

    Supplementary Information

  4. 4.

    Supplementary Table 4

    Supplementary Information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb3047

Further reading