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BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance

Nature Methods 15, 134–140 (2018) | Download Citation

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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.

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European Nucleotide Archive

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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

    • 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

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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 figures

  1. 1.

    Comparison of mouse mammary organoids derived from malignant and healthy tissues.

  2. 2.

    Genetic characterization of mouse mammary tumor organoids.

  3. 3.

    Genetic characterization of mouse mammary tumor organoids by DNA copy number profiling.

  4. 4.

    Histological characterization of KB1P(M)/KB2P mammary tumor organoids.

  5. 5.

    Lentiviral transduction of KB1P mammary tumor organoids.

  6. 6.

    Response of KB1P(M) and KB2P tumors to PARPi treatment.

  7. 7.

    Comparison of in vitro PARPi response of GEMM tumor-derived organoids.

  8. 8.

    ORG-KB1P4R.1-derived tumors preserve PARPi resistance mechanism of the original tumor.

  9. 9.

    Response of genetically modified organoid-derived mammary tumors to olaparib treatment.

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)

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DOI

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

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