Abstract

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

European Nucleotide Archive

References

  1. 1.

    et al. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc. Natl. Acad. Sci. USA 104, 12111–12116 (2007).

  2. 2.

    et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29, 418–425 (2001).

  3. 3.

    & Drug resistance in the mouse cancer clinic. Drug Resist. Updat. 15, 81–89 (2012).

  4. 4.

    et al. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer. Proc. Natl. Acad. Sci. USA 104, 12117–12122 (2007).

  5. 5.

    et al. High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc. Natl. Acad. Sci. USA 105, 17079–17084 (2008).

  6. 6.

    et al. Loss of 53BP1 causes PARP inhibitor resistance in Brca1-mutated mouse mammary tumors. Cancer Discov. 3, 68–81 (2013).

  7. 7.

    et al. BRCA2-deficient sarcomatoid mammary tumors exhibit multidrug resistance. Cancer Res. 75, 732–741 (2015).

  8. 8.

    et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature 521, 541–544 (2015).

  9. 9.

    et al. HELB is a feedback inhibitor of DNA end resection. Mol. Cell 61, 405–418 (2016).

  10. 10.

    et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).

  11. 11.

    et al. Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin. Clin. Cancer Res. 14, 3916–3925 (2008).

  12. 12.

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

  13. 13.

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

  14. 14.

    , & Hallmarks of 'BRCAness' in sporadic cancers. Nat. Rev. Cancer 4, 814–819 (2004).

  15. 15.

    et al. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 534, 47–54 (2016).

  16. 16.

    A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. J. Clin. Oncol. 26, 3785–3790 (2008).

  17. 17.

    et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat. Struct. Mol. Biol. 17, 688–695 (2010).

  18. 18.

    et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

  19. 19.

    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).

  20. 20.

    et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).

  21. 21.

    et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015).

  22. 22.

    et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun. 6, 8715 (2015).

  23. 23.

    , , & Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

  24. 24.

    , , & Genetic dissection of cancer development, therapy response, and resistance in mouse models of breast cancer. Cold Spring Harb. Symp. Quant. Biol. 81, 141–150 (2016).

  25. 25.

    et al. Efficacy of chemotherapy in BRCA1/2 mutation carrier ovarian cancer in the setting of PARP inhibitor resistance: a multi-institutional study. Clin. Cancer Res. 19, 5485–5493 (2013).

  26. 26.

    et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature 521, 489–494 (2015).

  27. 27.

    et al. A biobank of breast cancer explants with preserved intra-tumor heterogeneity to screen anticancer compounds. Cell 167, 260–274.e22 (2016).

  28. 28.

    et al. Screening out irrelevant cell-based models of disease. Nat. Rev. Drug Discov. 15, 751–769 (2016).

  29. 29.

    , & The role of tumour-stromal interactions in modifying drug response: challenges and opportunities. Nat. Rev. Drug Discov. 12, 217–228 (2013).

  30. 30.

    et al. BRCA-deficient mouse mammary tumor organoids to study cancer drug resistance. Protoc. Exch. (2017).

  31. 31.

    , , , & Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell 14, 570–581 (2008).

  32. 32.

    et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).

  33. 33.

    et al. Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland. Genes Dev. 30, 1470–1480 (2016).

  34. 34.

    et al. CopywriteR: DNA copy number detection from off-target sequence data. Genome Biol. 16, 49 (2015).

  35. 35.

    Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. ArXiv13033997 Q-Bio (2013).

  36. 36.

    ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  37. 37.

    et al. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy. Cell Death Dis. 5, e1228 (2014).

  38. 38.

    et al. PTPN11 is a central node in intrinsic and acquired resistance to targeted cancer drugs. Cell Rep. 12, 1978–1985 (2015).

  39. 39.

    , , , & Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222 (2000).

  40. 40.

    et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods 9, 81–83 (2011).

  41. 41.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

Download references

Acknowledgements

We wish to thank the members of the Preclinical Intervention Unit of the Mouse Clinic for Cancer and Ageing (MCCA) at the Netherlands Cancer Institute (NKI) N. Domanitskaia, N. Gerhards, G. Lakner, O. Levionnois, N. Regenscheit and M. Siffert (Vetsuisse Bern) for their technical support with the animal experiments. We are grateful to B. Evers (NKI) for providing the iKRUNC-Puro vector, H. van der Gulden (NKI) for her assistance with the genotyping procedure, and the NKI animal facility, animal pathology facility, flow cytometry facility and genomics core facility for their excellent service. Financial support came from the Dutch Cancer Society (KWF 2011-5220 and 2014-6532 to S.R. and J.J.), the Netherlands Organization for Scientific Research (VICI 91814643, Cancer Genomics Netherlands and a National Roadmap grant for Large-Scale Research Facilities to J.J., VENI 916.15.182 to N.S.), the Netherlands Genomics Initiative Zenith (93512009 to J.J.), the Swiss National Science Foundation (310030_156869 to S.R.), the Swiss Cancer Research Foundation (MD-PhD-3446-01-2014 to S.B.) and the European Union (ERC CoG-681572 to S.R. and ERC synergy grant 319661 COMBATCANCER to J.J.).

Author information

Author notes

    • Norman Sachs

    Present address: Vertex Pharmaceuticals Incorporated, San Diego, California, USA.

    • Alexandra A Duarte
    •  & Ewa Gogola

    These authors contributed equally to this work.

Affiliations

  1. Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Alexandra A Duarte
    • , Ewa Gogola
    • , Marco Barazas
    • , Stefano Annunziato
    • , Julian R de Ruiter
    • , Julia M Houthuijzen
    • , Jos Jonkers
    •  & Sven Rottenberg
  2. Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands.

    • Norman Sachs
    •  & Hans Clevers
  3. Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Julian R de Ruiter
  4. Genomics Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Arno Velds
  5. Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

    • Sohvi Blatter
    •  & Sven Rottenberg
  6. Mouse Clinic for Cancer and Aging (MCCA), Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Marieke van de Ven
  7. Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

    • Piet Borst

Authors

  1. Search for Alexandra A Duarte in:

  2. Search for Ewa Gogola in:

  3. Search for Norman Sachs in:

  4. Search for Marco Barazas in:

  5. Search for Stefano Annunziato in:

  6. Search for Julian R de Ruiter in:

  7. Search for Arno Velds in:

  8. Search for Sohvi Blatter in:

  9. Search for Julia M Houthuijzen in:

  10. Search for Marieke van de Ven in:

  11. Search for Hans Clevers in:

  12. Search for Piet Borst in:

  13. Search for Jos Jonkers in:

  14. Search for Sven Rottenberg in:

Contributions

S.R., A.A.D. and E.G. conceived and designed the study. A.A.D., E.G. and N.S. developed and validated the mammary tumor organoid model with input from H.C. and help from J.M.H. For this purpose, they designed and conducted the experiments, and interpreted the results together with P.B., J.J. and S.R. M.B. and S.A. designed and performed the in vivo validation experiment using CRISPR/Cas9-mediated targeting of 53BP1, analyzed the data and contributed to the figures. J.R.d.R. and A.V. carried out the bioinformatical analyses and provided the corresponding figures. S.B. and M.v.d.V. helped with animal studies. P.B., J.J. and S.R. supervised the project. A.A.D. and E.G. prepared the manuscript with input from all of the authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jos Jonkers or Sven Rottenberg.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Supplementary Table 1

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    BRCA-deficient mouse mammary tumor organoids - a tool to study cancer drug resistance (detailed protocol for in vitro procedures)

Excel files

  1. 1.

    nmeth.4535-sf1.xlsx

  2. 2.

    nmeth.4535-sf7.xlsx

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.4535

Further reading

  • Organoid technology and applications in cancer research

    • Hanxiao Xu
    • , Xiaodong Lyu
    • , Ming Yi
    • , Weiheng Zhao
    • , Yongping Song
    •  & Kongming Wu

    Journal of Hematology & Oncology (2018)

  • The shieldin complex mediates 53BP1-dependent DNA repair

    • Sylvie M. Noordermeer
    • , Salomé Adam
    • , Dheva Setiaputra
    • , Marco Barazas
    • , Stephen J. Pettitt
    • , Alexanda K. Ling
    • , Michele Olivieri
    • , Alejandro Álvarez-Quilón
    • , Nathalie Moatti
    • , Michal Zimmermann
    • , Stefano Annunziato
    • , Dragomir B. Krastev
    • , Feifei Song
    • , Inger Brandsma
    • , Jessica Frankum
    • , Rachel Brough
    • , Alana Sherker
    • , Sébastien Landry
    • , Rachel K. Szilard
    • , Meagan M. Munro
    • , Andrea McEwan
    • , Théo Goullet de Rugy
    • , Zhen-Yuan Lin
    • , Traver Hart
    • , Jason Moffat
    • , Anne-Claude Gingras
    • , Alberto Martin
    • , Haico van Attikum
    • , Jos Jonkers
    • , Christopher J. Lord
    • , Sven Rottenberg
    •  & Daniel Durocher

    Nature (2018)

  • The ASCIZ-DYNLL1 axis promotes 53BP1-dependent non-homologous end joining and PARP inhibitor sensitivity

    • Jordan R. Becker
    • , Raquel Cuella-Martin
    • , Marco Barazas
    • , Rui Liu
    • , Catarina Oliveira
    • , Antony W. Oliver
    • , Kirstin Bilham
    • , Abbey B. Holt
    • , Andrew N. Blackford
    • , Jörg Heierhorst
    • , Jos Jonkers
    • , Sven Rottenberg
    •  & J. Ross Chapman

    Nature Communications (2018)