Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell– and patient-derived tumor organoids

Abstract

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Induction of polarized organoids from human pluripotent stem cells.
Figure 2: Organoids express markers associated with pancreatic progenitor cells.
Figure 3: Differentiation of pancreatic progenitor organoids in vitro and in vivo.
Figure 4: Progenitor organoids model early PDAC.
Figure 5: TP53 mutational status, localization of SOX9 and clinical outcome.
Figure 6: Establishment of tumor organoids that conserve patient-specific traits.

References

  1. Ghaneh, P., Costello, E. & Neoptolemos, J.P. Biology and management of pancreatic cancer. Postgrad. Med. J. 84, 478–497 (2008).

    Article  CAS  Google Scholar 

  2. Kanji, Z.S. & Gallinger, S. Diagnosis and management of pancreatic cancer. CMAJ 185, 1219–1226 (2013).

    Article  Google Scholar 

  3. Vincent, A., Herman, J., Schulick, R., Hruban, R.H. & Goggins, M. Pancreatic cancer. Lancet 378, 607–620 (2011).

    Article  Google Scholar 

  4. Agbunag, C., Lee, K.E., Buontempo, S. & Bar-Sagi, D. Pancreatic duct epithelial cell isolation and cultivation in two-dimensional and three-dimensional culture systems. Methods Enzymol. 407, 703–710 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Li, X. et al. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nat. Med. 20, 769–777 (2014).

    Article  CAS  Google Scholar 

  7. Pagliuca, F.W. et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428–439 (2014).

    Article  CAS  Google Scholar 

  8. Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121–1133 (2014).

    Article  CAS  Google Scholar 

  9. Schiesser, J.V. & Wells, J.M. Generation of beta cells from human pluripotent stem cells: are we there yet? Ann. NY Acad. Sci. 1311, 124–137 (2014).

    Article  CAS  Google Scholar 

  10. Jennings, R.E. et al. Development of the human pancreas from foregut to endocrine commitment. Diabetes 62, 3514–3522 (2013).

    Article  CAS  Google Scholar 

  11. Pan, F.C. & Wright, C. Pancreas organogenesis: from bud to plexus to gland. Dev. Dyn. 240, 530–565 (2011).

    Article  CAS  Google Scholar 

  12. McCracken, K.W. & Wells, J.M. Molecular pathways controlling pancreas induction. Semin. Cell Dev. Biol. 23, 656–662 (2012).

    Article  CAS  Google Scholar 

  13. Hick, A.C. et al. Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1. BMC Dev. Biol. 9, 66 (2009).

    Article  Google Scholar 

  14. Riedel, M.J. et al. Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas. Diabetologia 55, 372–381 (2012).

    Article  CAS  Google Scholar 

  15. Lyttle, B.M. et al. Transcription factor expression in the developing human fetal endocrine pancreas. Diabetologia 51, 1169–1180 (2008).

    Article  CAS  Google Scholar 

  16. Outzen, H.C. & Leiter, E.H. Transplantation of pancreatic islets into cleared mammary fat pads. Transplantation 32, 101–105 (1981).

    Article  CAS  Google Scholar 

  17. Laitio, M., Lev, R. & Orlic, D. The developing human fetal pancreas: an ultrastructural and histochemical study with special reference to exocrine cells. J. Anat. 117, 619–634 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Nielsen, S.K. et al. Characterization of primary cilia and Hedgehog signaling during development of the human pancreas and in human pancreatic duct cancer cell lines. Dev. Dyn. 237, 2039–2052 (2008).

    Article  CAS  Google Scholar 

  19. Kolodecik, T., Shugrue, C., Ashat, M. & Thrower, E.C. Risk factors for pancreatic cancer: underlying mechanisms and potential targets. Front. Physiol. 4, 415 (2013).

    PubMed  Google Scholar 

  20. Chang, D.K., Grimmond, S.M. & Biankin, A.V. Pancreatic cancer genomics. Curr. Opin. Genet. Dev. 24, 74–81 (2014).

    Article  CAS  Google Scholar 

  21. Chakravarty, G., Rider, B. & Mondal, D. Cytoplasmic compartmentalization of SOX9 abrogates the growth arrest response of breast cancer cells that can be rescued by trichostatin A treatment. Cancer Biol. Ther. 11, 71–83 (2011).

    Article  CAS  Google Scholar 

  22. Chakravarty, G. et al. Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer. Exp. Biol. Med. (Maywood) 236, 145–155 (2011).

    Article  CAS  Google Scholar 

  23. Marusyk, A., Almendro, V. & Polyak, K. Intra-tumour heterogeneity: a looking glass for cancer? Nat. Rev. Cancer 12, 323–334 (2012).

    Article  CAS  Google Scholar 

  24. Waddell, N. et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 518, 495–501 (2015).

    Article  CAS  Google Scholar 

  25. van den Beucken, T. et al. Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER. Nat. Commun. 5, 5203 (2014).

    Article  CAS  Google Scholar 

  26. Johnson, A.B., Denko, N. & Barton, M.C. Hypoxia induces a novel signature of chromatin modifications and global repression of transcription. Mutat. Res. 640, 174–179 (2008).

    Article  CAS  Google Scholar 

  27. Viale, A. et al. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature 514, 628–632 (2014).

    Article  CAS  Google Scholar 

  28. Boj, S.F. et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 160, 324–338 (2015).

    Article  CAS  Google Scholar 

  29. Nostro, M.C. et al. Stage-specific signaling through TGF-β family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861–871 (2011).

    Article  CAS  Google Scholar 

  30. Nostro, M.C. et al. Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports 4, 591–604 (2015).

    Article  CAS  Google Scholar 

  31. Xiang, B. & Muthuswamy, S.K. Using three-dimensional acinar structures for molecular and cell biological assays. Methods Enzymol. 406, 692–701 (2006).

    Article  CAS  Google Scholar 

  32. Ritchie, M.E. et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

We thank members of S.K.M.'s laboratory for helpful suggestions and discussions, D. Barsyte-Lovejoy for helping with epigenetic drug screening experiments and members of the PanCuRx team, including D. Hedley, for support and assistance. This work was supported by the Ontario Institute for Cancer Research (OICR) PanCuRx program; Canadian Cancer Society; Lee K Margaret Lau Chair for Breast Cancer Research and Campbell Family Institute for Breast cancer research to S.K.M. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant 115766), Janssen, Merck and Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation (FAPESP), Takeda, and the Wellcome Trust. This was also funded in part by the Ontario Ministry of Health and Long Term Care (OMOHLTC). The views expressed do not necessarily reflect those of the OMOHLTC.

Author information

Authors and Affiliations

Authors

Contributions

S.K.M. conceived and coordinated the project, designed experiments and co-wrote the manuscript with L.H. L.H. also designed, performed and/or coordinated all experiments. G.K. and A.H. contributed to pancreatic lineage–committed precursor generation. I.J. contributed tumor organoid live imaging and manuscript preparation. M.B. contributed to tumor organoid immunofluorescence microscopy. I.L. contributed to collection of patient-derived xenograft tumors. N.N. contributed to organoid size measurement. C.N. contributed to pancreatic lineage–committed precursor generation. R.W. contributed to human fetal pancreas studies. L.B.M. contributed to bioinformatics analysis for gene expressions. H.C.C. contributed to experimental design. C.A. contributed to epigenetic drug screening. S.E.K., D.J.R., A.A.C., S.C. and D.F.S. contributed to studies of P53 and SOX9 localization in patient samples. M.R. contributed to pathological analysis on patient tumor and tumor organoids, and studies of P53 and SOX9 localization in patient samples. M.-S.T. contributed to pathological analysis on patient tumor and tumor organoids, and studies of P53 and SOX9 localization in patient samples. S.G. contributed to obtaining patient resections for tumor organoid.

Corresponding author

Correspondence to Senthil K Muthuswamy.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 1510 kb)

Source data to Supplementary Figures 1–5

Time-lapse imaging of UHN6 tumor-organoid (AVI 17147 kb)

Supplementary Data Set 1

Supplementary source data (XLSX 11 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, L., Holtzinger, A., Jagan, I. et al. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell– and patient-derived tumor organoids. Nat Med 21, 1364–1371 (2015). https://doi.org/10.1038/nm.3973

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3973

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing