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

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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Figure 1: Generation and characterization of mouse mammary tumor organoids.
Figure 2: Comparison of PARPi response of original GEMM tumors, tumor-derived organoids and organoid-derived tumors.
Figure 3: PARPi-resistant GEMM-derived cancer organoids preserve functional properties of the original tumors.
Figure 4: Mouse mammary cancer organoids for in vivo validation of PARPi resistance factors.

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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.).

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Authors

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

Correspondence to Jos Jonkers or Sven Rottenberg.

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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. Source data

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. Source data

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.

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

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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

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