Organotypic culture assays for murine and human primary and metastatic-site tumors


Cancer invasion and metastasis are challenging to study in vivo since they occur deep inside the body over extended time periods. Organotypic 3D culture of fresh tumor tissue enables convenient real-time imaging, genetic and microenvironmental manipulation and molecular analysis. Here, we provide detailed protocols to isolate and culture heterogenous organoids from murine and human primary and metastatic site tumors. The time required to isolate organoids can vary based on the tissue and organ type but typically takes <7 h. We describe a suite of assays that model specific aspects of metastasis, including proliferation, survival, invasion, dissemination and colony formation. We also specify comprehensive protocols for downstream applications of organotypic cultures that will allow users to (i) test the role of specific genes in regulating various cellular processes, (ii) distinguish the contributions of several microenvironmental factors and (iii) test the effects of novel therapeutics.

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Fig. 1: Workflow diagram.
Fig. 2: Isolation of organoids from primary murine and human mammary tumors.
Fig. 3: Viral transduction of organoids.
Fig. 4: 3D assays for growth, invasion and dissemination of tumor organoids.
Fig. 5: 3D colony-formation assay to model metastasis formation.
Fig. 6: Isolation of metastatic organoids from murine and human mammary tumor-derived metastases.
Fig. 7: Downstream applications of 3D organotypic cultures: immunofluorescence, protein isolation and FACS.
Fig. 8: Application of methods to multiple model and organ systems.

Data availability

The data mentioned in the protocol are included. Any additional information may be provided by the corresponding author upon request. Source data are provided with this paper.


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We thank members of the Ewald laboratory for helpful comments on the manuscript and for sharing organoid yield information (data points in Fig. 2f). We also thank the Cooperative Human Tissue Network (CHTN) for providing patient samples used in this study. We thank Jin Zhu for assistance with FACS experiments. K.J.C. was supported by the Burroughs Welcome Fund Career Award for Medical Scientists 1013355.01. D.G. was supported by a Postdoctoral Fellowship Grant from the Susan G. Komen Foundation (PDF15332336). A.J.E. received support for this project through grants from The Breast Cancer Research Foundation (BCRF-18-048), the Metastatic Breast Cancer Network, Twisted Pink, Hope Scarves, Theresa’s Research Foundation and the National Institutes of Health/National Cancer Institute (U01CA217846, U01CA212007, U54CA2101732 and 3P30CA006973).

Author information




V.P., K.J.C., E.M.G, N.M.N., A.K.F., E.H., D.G. and A.J.E. contributed to the development and optimization of protocols described in this manuscript. W.M. and P.T.T. provided valuable advice on adaptations of these methods to different model systems. V.P., E.M.G. and A.J.E. wrote the manuscript with useful input from all authors.

Corresponding author

Correspondence to Andrew J. Ewald.

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Competing interests

A.J.E. and K.J.C. have a patent related to the use of keratin-14 as a biomarker for invasive cancer cell populations. A.J.E. and V.P. have a patent related to the use of antibodies for cancer therapy. A.J.E.’s spouse is an employee of Immunocore.

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Peer review information Nature Protocols thanks Johan de Rooij and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Padmanaban, V. et al. Nature 573, 439–444 (2019):

Cheung, K. et al. Proc. Natl Acad. Sci. USA 113, E854–E863 (2016):

Cheung, K. et al. Cell 155, 1639–1651 (2013):

Extended Data

Extended Data Fig. 1 Variables that affect organoid yield from mammary human tumor organoids.

a, Organoid yield increased as the protocol was optimized during the course of the study. b, Variations in organoid yield based on modifications to the protocol. Median with SD is represented.

Supplementary information

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 7

Unprocessed western blots.

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Padmanaban, V., Grasset, E.M., Neumann, N.M. et al. Organotypic culture assays for murine and human primary and metastatic-site tumors. Nat Protoc 15, 2413–2442 (2020).

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