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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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).
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.
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): https://doi.org/10.1038/s41586-019-1526-3
Cheung, K. et al. Proc. Natl Acad. Sci. USA 113, E854–E863 (2016): https://doi.org/10.1073/pnas.1508541113
Cheung, K. et al. Cell 155, 1639–1651 (2013): https://doi.org/10.1016/j.cell.2013.11.029
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.
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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). https://doi.org/10.1038/s41596-020-0335-3