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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Liu, X. 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).
Jonkers, J. et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29, 418–425 (2001).
Rottenberg, S. & Borst, P. Drug resistance in the mouse cancer clinic. Drug Resist. Updat. 15, 81–89 (2012).
Rottenberg, S. 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).
Rottenberg, S. 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).
Jaspers, J.E. et al. Loss of 53BP1 causes PARP inhibitor resistance in Brca1-mutated mouse mammary tumors. Cancer Discov. 3, 68–81 (2013).
Jaspers, J.E. et al. BRCA2-deficient sarcomatoid mammary tumors exhibit multidrug resistance. Cancer Res. 75, 732–741 (2015).
Xu, G. et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature 521, 541–544 (2015).
Tkác, J. et al. HELB is a feedback inhibitor of DNA end resection. Mol. Cell 61, 405–418 (2016).
Ray Chaudhuri, A. et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).
Evers, B. et al. Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin. Clin. Cancer Res. 14, 3916–3925 (2008).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).
Turner, N., Tutt, A. & Ashworth, A. Hallmarks of 'BRCAness' in sporadic cancers. Nat. Rev. Cancer 4, 814–819 (2004).
Nik-Zainal, S. et al. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 534, 47–54 (2016).
Ashworth, A. 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).
Bouwman, P. 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).
Bunting, S.F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).
Schwank, G. 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).
Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).
Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015).
Freedman, B.S. et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun. 6, 8715 (2015).
Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Annunziato, S., Barazas, M., Rottenberg, S. & Jonkers, J. 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).
Ang, J.E. 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).
Patch, A.-M. et al. Whole-genome characterization of chemoresistant ovarian cancer. Nature 521, 489–494 (2015).
Bruna, A. et al. A biobank of breast cancer explants with preserved intra-tumor heterogeneity to screen anticancer compounds. Cell 167, 260–274.e22 (2016).
Horvath, P. et al. Screening out irrelevant cell-based models of disease. Nat. Rev. Drug Discov. 15, 751–769 (2016).
McMillin, D.W., Negri, J.M. & Mitsiades, C.S. The role of tumour-stromal interactions in modifying drug response: challenges and opportunities. Nat. Rev. Drug Discov. 12, 217–228 (2013).
Duarte, A.A. et al. BRCA-deficient mouse mammary tumor organoids to study cancer drug resistance. Protoc. Exch. http://dx.doi.org/10.1038/protex.2017.143 (2017).
Ewald, A.J., Brenot, A., Duong, M., Chan, B.S. & Werb, Z. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell 14, 570–581 (2008).
Wang, D. 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).
Annunziato, S. et al. Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland. Genes Dev. 30, 1470–1480 (2016).
Kuilman, T. et al. CopywriteR: DNA copy number detection from off-target sequence data. Genome Biol. 16, 49 (2015).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. ArXiv13033997 Q-Bio (2013).
ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
Grabinger, T. 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).
Prahallad, A. et al. PTPN11 is a central node in intrinsic and acquired resistance to targeted cancer drugs. Cell Rep. 12, 1978–1985 (2015).
Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222 (2000).
Koo, B.-K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods 9, 81–83 (2011).
Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
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
Authors and Affiliations
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.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Comparison of mouse mammary organoids derived from malignant and healthy tissues.
(a) Brightfield images of ORG-KB1PM7N.1/R.1 organoid cultures embedded in Basement Membrane Extract 2 days (passage 1) and 55 days (passage 5) following isolation. Scale bar, 100 μm. (b) Brightfield images of in vitro cultures of organoids derived from healthy mammary tissue (N-ORG) at the indicated time points. Scale bar, 100 μm. (c) In vitro proliferation of tumor (ORG-KP.5, ORG-KB1P4N.1, ORG-KB1PM7N.1 and KB2P17N.1) and healthy (N-ORG) mammary organoids, as determined by a cell viability assay.
Supplementary Figure 2 Genetic characterization of mouse mammary tumor organoids.
Genotyping PCR of genomic DNA from KB1PM7, KB1P4, KB2P17, KP.5 and KPM.1 GEMM tumors, tumor-derived organoids and organoid-derived tumors. Wild-type bands are indicated by red arrows and floxed or deletion bands are indicated by black arrows. Wild-type bands indicate the presence of stromal cells from the syngeneic wild-type mice in which tumors were transplanted. Spleen DNA from KB1PM or KB2P mice was used as a positive control for the floxed PCR products and liver DNA from a wildtype animal was used as a positive control for the wild-type PCR products.
Sample annotations:
(1) KB1P4 naïve original tumor, (2) ORG-KB1P4N.1, (3) ORG-KB1P4N.1-derived tumor, (4) KB1P4 PARPi-resistant original tumor, (5) ORG-KB1P4R.1, (6) ORG-KB1P4R.1-derived tumor, (7) KB1PM7 PARPi-naïve original tumor, (8) ORG-KB1PM7N.1, (9) ORG-KB1PM7N.1-derived tumor, (10) KB1PM7 PARPi-resistant original tumor, (11) ORG-KB1PM7R.1, (12) ORG-KB1PM7R.1-derived tumor, (13) KB2P17 PARPi-naïve original tumor, (14) ORG-KB2P17N.1, (15) ORG-KB2P17N.1-derived tumor, (16) KB2P17 PARPi-resistant original tumor, (17) ORG-KB2P17R.1, (18) ORG-KB2P17R.1-derived tumor, (19) KP.5 original tumor, (20) ORG-KP.5, (21) KPM.1 original spontaneous tumor, (22) ORG-KPM.1, (23) KB1PM tumor (positive control), (24) KB2P tumor (positive control), (25) KB1PM spleen (floxed control), (26) KB2P spleen (floxed control), (27) wild-type liver (wild-type control), (28) negative control (no DNA input).
Supplementary Figure 3 Genetic characterization of mouse mammary tumor organoids by DNA copy number profiling.
(a) Unsupervised hierarchical clustering of a panel of 18 organoid lines and respective original GEMM tumors (extended version of Fig. 1c). (b) Unsupervised hierarchical clustering of original GEMM tumor (KB1P), two organoid lines (ORG-KB1P.1, ORG-KB1P.2) and two cell lines derived from the same tumor (independent clones, KB1P-B11 and KB1P-G3, previously described7; correlation distance, complete linkage).
Supplementary Figure 4 Histological characterization of KB1P(M)/KB2P mammary tumor organoids.
(a-c) Representative images of hematoxylin & eosin (HE) stainings and immunohistochemical analyses of E-cadherin, Vimentin and Keratin-14 expression in KB1P4 (a), KB1PM7 (b) and KB2P17 (c) GEMM tumors, tumor-derived organoids and organoid-derived tumors. Scale bar, 100 μm.
Supplementary Figure 5 Lentiviral transduction of KB1P mammary tumor organoids.
Green fluorescent protein (GFP) was introduced into ORG-KB1P4N.1 organoids by lentiviral transduction using pLenti6-GFP at the indicated theoretical multiplicities of infection (MOI). GFP expression was analyzed by flow cytometry 3 days after transduction.
Supplementary Figure 6 Response of KB1P(M) and KB2P tumors to PARPi treatment.
(a-e) Mice orthotopically transplanted with olaparib-naïve or –resistant KB1P4 tumors (n = 2 naïve vehicle, n = 5 for other treatment groups) (a), ORG-KB1P4N.1/R.1 organoids (n = 5) (b), olaparib-naïve or –resistant KB1PM7 tumors (n = 4 resistant vehicle, n = 3 for other treatment groups) (c), ORG-KB1PM7N.1/R.1 organoids (n = 5) (d) or AZD2461-naïve or –resistant ORG-KB2P17N.1/R.1 organoids (n = 4 for vehicle, n = 6 for AZD2461) (e) were treated with either vehicle (a-e), olaparib (a-d) or AZD2461 (e) for 28 consecutive days. End of treatment is indicated by a dotted grid line. Graphs show relative tumor volume (ratio of tumor volume to initial size at start of treatment) as a function of time (see also Fig. 2).
Supplementary Figure 7 Comparison of in vitro PARPi response of GEMM tumor-derived organoids.
(a,b) In vitro response of ORG-KB1PM7N.1/R.1 (a) and ORG-KB1P4N.1/R.1 (b) organoids cultured in media depleted of R-spondin 1, Noggin and EGF as determined by a viability assay. Representative stainings of organoids are shown in duplicate. P values were determined by a non-linear regression model and extra sum-square F-test. (c) In vitro response of ORG-KB1PM7N.2/R.2 organoids, independently isolated from the original KB1PM7N/R tumors, and ORG-KB1PM7N.3/R.3 organoids, isolated from the vehicle-treated tumors that originated from transplantation of ORG-KB1PM7N.1/R.1 organoids. P values were determined as described for a,b. Data are presented as mean ± SD for at least 2 independent replicates.
Supplementary Figure 8 ORG-KB1P4R.1-derived tumors preserve PARPi resistance mechanism of the original tumor.
(a) RAD51 and 53BP1 ionizing radiation-induced foci (IRIF) formation in original KB1P4 olaparib-naïve and -resistant tumors and ORG-KB1P4N.1/R.1-derived tumors 2 hours after irradiation with 15 Gy (IR). A KP tumor was used as a positive control for RAD51 foci formation. Representative microscopic images are shown. Scale bar, 10 μm. See Fig. 3b,c for quantification. (b,c) In vivo response of tumors derived from ORG-KB1P4N.1/R.1 to cisplatin (b; n = 5 naïve vehicle, n = 3 resistant vehicle, n = 5 naïve/resistant cisplatin) and topotecan (c; n = 5 naïve vehicle, n = 3 resistant vehicle, n = 4 naïve topotecan, n = 5 resistant topotecan). Data presented as relative tumor volume over time.
Supplementary Figure 9 Response of genetically modified organoid-derived mammary tumors to olaparib treatment.
Mice bearing tumors derived from ORG-KB1PM7N.1 organoids modified in vitro by CRISPR/Cas9 using a gRNA targeting Trp53bp1 (Trp53bp1 gRNA) or a non-targeting gRNA (NT gRNA). Animals were treated with either vehicle or olaparib for 28 consecutive days (n = 10 per treatment group). End of treatment is indicated by a dotted grid line. Graphs show relative tumor volume (ratio of tumor volume to initial size at start of treatment) as a function of time. See also Fig. 4.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9 and Supplementary Table 1 (PDF 1851 kb)
Life Sciences Reporting Summary
Life Sciences Reporting Summary
Supplementary Protocol
BRCA-deficient mouse mammary tumor organoids - a tool to study cancer drug resistance (detailed protocol for in vitro procedures)
Rights and permissions
About this article
Cite this article
Duarte, A., Gogola, E., Sachs, N. et al. BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance. Nat Methods 15, 134–140 (2018). https://doi.org/10.1038/nmeth.4535
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.4535
This article is cited by
-
Exploring the interaction between extracellular matrix components in a 3D organoid disease model to replicate the pathophysiology of breast cancer
Journal of Experimental & Clinical Cancer Research (2023)
-
The role of organoids in cancer research
Experimental Hematology & Oncology (2023)
-
The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin
Nature Cell Biology (2022)
-
Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration
Scientific Reports (2022)
-
A living biobank of canine mammary tumor organoids as a comparative model for human breast cancer
Scientific Reports (2022)