Adult stem cell–based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-derived cancer organoids therefore hold great promise for personalized medicine. Here, we provide an overview of the protocols used by different groups to establish organoids from various epithelial tissues and cancers, plus the different protocols subsequently used to test the in vitro therapy sensitivity of these patient-derived organoids. We also provide an in-depth protocol for the generation of head and neck squamous cell carcinoma organoids and their subsequent use in semi-automated therapy screens. Establishment of organoids and subsequent screening can be performed within 3 months, although this timeline is highly dependent on a.o. starting material and the number of therapies tested. The protocol provided may serve as a reference to successfully establish organoids from other cancer types and perform drug screenings thereof.
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We thank S. Kolders and S. Spelier for their help in optimizing the drug-screening protocol. We thank S. Boi and T. Dalton for critically reading the manuscript. We thank M. Putker for her valuable contributions to Tables 1–3. We thank P. van der Groep, A. Brousali, A. Snelting and O. Kranenburg of the Utrecht Platform for Organoid Technology (U-PORT; UMC Utrecht) for patient inclusion and tissue acquisition. We thank C. Ammerlaan, J. Bernink, G. Busslinger, T. Dalton, I. Franken and K. Lõhmussaar for providing images of organoid cultures. This work was supported by the gravitation program CancerGenomiCs.nl from the Netherlands Organisation for Scientific Research (NWO), the Oncode Institute (partly financed by the Dutch Cancer Society), the European Research Council under ERC Advanced Grant Agreement no. 67013 (H.C.), the Koerber Foundation (H.C.), ZonMw grant 116.006.10 (H.C.) and the German Cancer Aid (K.K.). K.K. was the recipient of a VENI grant from the Netherlands Organisation for Scientific Research (NWO-ZonMW, 016.166.140) and was a long-term fellow of the Human Frontier Science Program Organization (HFSPO, LT771/2015).
H.C., E.D. and K.K. are named inventors on multiple patents or patents pending related to organoids.
Peer review information Nature Protocols thanks Takanori Takebe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key reference using this protocol
Driehuis, E. et al. Cancer Discov. 9, 852–871 (2019): https://doi.org/10.1158/2159-8290.CD-18-1522
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Driehuis, E., Kretzschmar, K. & Clevers, H. Establishment of patient-derived cancer organoids for drug-screening applications. Nat Protoc 15, 3380–3409 (2020). https://doi.org/10.1038/s41596-020-0379-4
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