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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Tuveson, D. & Clevers, H. Cancer modeling meets human organoid technology. Science 364, 952–955 (2019).
Kretzschmar, K. & Clevers, H. Organoids: modeling development and the stem cell niche in a dish. Dev. Cell 38, 590–600 (2016).
Dye, B. R. et al. In vitro generation of human pluripotent stem cell derived lung organoids. Elife 2015, 1–25 (2015).
Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Mullenders, J. et al. Mouse and human urothelial cancer organoids: a tool for bladder cancer research. Proc. Natl Acad. Sci. USA 116, 4567–4574 (2019).
Karthaus, W. R. et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159, 163–175 (2014).
Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721 (2013).
Lee, S. H. et al. Tumor evolution and drug response in patient-derived organoid models of bladder cancer. Cell 173, 515–528 (2018).
Sachs, N. et al. A living biobank of breast cancer organoids captures disease heterogeneity. Cell 172, 373–386 (2018).
Crespo, M. et al. Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing. Nat. Med. 23, 878–884 (2017).
Tiriac, H. et al. Organoid profiling identifies common responders to chemotherapy in pancreatic cancer. Cancer Discov. 8, 1112–1129 (2018).
Roerink, S. F. et al. Intra-tumour diversification in colorectal cancer at the single-cell level. Nature 556, 457–462 (2018).
Snover, D. C. Update on the serrated pathway to colorectal carcinoma. Hum. Pathol. 42, 1–10 (2011).
Boj, S. F. et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 160, 324–338 (2015).
Longmire, T. A. et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10, 398–411 (2012).
Bartfeld, S. et al. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology 148, 126–136.e6 (2015).
Ren, W. et al. Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo. Proc. Natl Acad. Sci. USA 111, 16401–16406 (2014).
Hill, S. J. et al. Prediction of DNA repair inhibitor response in short-term patient-derived ovarian cancer organoids. Cancer Discov. 8, 1404–1421 (2018).
Driehuis, E. et al. Oral mucosal organoids as a potential platform for personalized cancer therapy. Cancer Discov. 9, 852–871 (2019).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).
Gao, D. et al. Organoid cultures derived from patients with advanced prostate cancer. Cell 159, 176–187 (2014).
Seino, T. et al. Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression. Cell Stem Cell 22, 454–467.e6 (2018).
McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).
Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).
Takasato, M. et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 16, 118–126 (2014).
Takebe, T. et al. Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat. Protoc. 9, 396–409 (2014).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).
Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat. Biotechnol. 37, 303–313 (2019).
Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920–926 (2018).
Yao, Y. et al. Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer. Cell Stem Cell 26, 17–26.e6 (2020).
Ooft, S. N. et al. Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients. Sci. Transl. Med. 11, eaay2574 (2019).
Berkers, G. et al. Rectal organoids enable personalized treatment of cystic fibrosis. Cell Rep. 26, 1701–1708 (2019).
Haibe-Kains, B. et al. Inconsistency in large pharmacogenomic studies. Nature 504, 389–393 (2013).
Niepel, M. et al. A multi-center study on the reproducibility of drug-response assays in mammalian cell lines. Cell Syst. 9, 35–48.e5 (2019).
Hafner, M., Niepel, M., Chung, M. & Sorger, P. K. Growth rate inhibition metrics correct for confounders in measuring sensitivity to cancer drugs. Nat. Methods 13, 521–527 (2016).
Haverty, P. M. et al. Reproducible pharmacogenomic profiling of cancer cell line panels. Nature 533, 333–337 (2016).
Nanki, K. et al. Divergent routes toward Wnt and R-spondin niche independency during human gastric carcinogenesis. Cell 174, 856–869.e17 (2018).
Van De Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933–945 (2015).
Pauli, C. et al. Personalized in vitro and in vivo cancer models to guide precision medicine. Cancer Discov. 7, 462–477 (2017).
Fujii, M. et al. A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis. Cell Stem Cell 18, 827–838 (2016).
Schutte, M. et al. Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors. Nat. Commun. 8, 14262 (2017).
Li, X. et al. Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics. Nat. Commun. 9, 2983 (2018).
Kessler, M. et al. The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids. Nat. Commun. 6, 8989 (2015).
Boretto, M. et al. Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability. Development 144, 1775–1786 (2017).
Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015).
Broutier, L. et al. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat. Med. 23, 1424–1435 (2017).
Sachs, N. et al. Long‐term expanding human airway organoids for disease modeling. EMBO J. 38, e100300 (2019).
Kim, M. et al. Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening. Nat. Commun. 10, 3991 (2019).
Kopper, O. et al. An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity. Nat. Med. 25, 838–849 (2019).
Pringle, S. et al. Human salivary gland stem cells functionally restore radiation damaged salivary glands. Stem Cells 34, 640–652 (2016).
Yan, H. H. N. et al. A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening. Cell Stem Cell 23, 882–897 (2018).
Seidlitz, T. et al. Human gastric cancer modelling using organoids. Gut 68, 207–217 (2019).
Tamura, H. et al. Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues. Oncol. Rep. 40, 635–646 (2018).
Grassi, L. et al. Organoids as a new model for improving regenerative medicine and cancer personalized therapy in renal diseases. Cell Death Dis. 10, 201 (2019).
Li, L. et al. Human primary liver cancer organoids reveal intratumor and interpatient drug response heterogeneity. JCI Insight 4, e121490 (2019).
Saito, Y. et al. Establishment of patient-derived organoids and drug screening for biliary tract carcinoma. Cell Rep. 27, 1265–1276.e4 (2019).
Driehuis, E. et al. Pancreatic cancer organoids recapitulate disease and allow personalized drug screening. Proc. Natl Acad. Sci. USA 116, 26580–26590 (2019).
Geurts, M. H. et al. CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell S1934-5909, 30019–30019 (2020).
Lee, G. Y., Kenny, P. A., Lee, E. H. & Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–365 (2007).
Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701–706 (2009).
de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).
Rheinwald, J. G. & Green, H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265, 421–424 (1977).
The Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 517, 576–582 (2015).
Haramis, A.-P. G. et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686 (2004).
Ganesh, K. et al. A rectal cancer model establishes a platform to study individual responses to chemoradiation. Nat. Med. 25, 1607–1614 (2019).
Boretto, M. et al. Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening. Nat. Cell Biol. 21, 1041–1051 (2019).
Lavitrano, M. et al. BTK inhibitors synergize with 5-FU to treat drug-resistant TP53-null colon cancers. J. Pathol. 250, 134–147 (2019).
Boehnke, K. et al. Assay establishment and validation of a high-throughput screening platform for three-dimensional patient-derived colon cancer organoid cultures. J. Biomol. Screen. 21, 931–941 (2016).
Lampis, A. et al. MIR21 drives resistance to heat shock protein 90 inhibition in cholangiocarcinoma. Gastroenterology 154, 1066–1079.e5 (2018).
Verissimo, C. S. et al. Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening. Elife 5, e18489 (2016).
Post, J. B. et al. Cancer modeling in colorectal organoids reveals intrinsic differences between oncogenic RAS and BRAF variants. Preprint at https://www.biorxiv.org/content/10.1101/860122v1 (2019).
Takahashi, N. et al. An in vitro system for evaluating molecular targeted drugs using lung patient-derived tumor organoids. Cells 8, 481 (2019).
Jabs, J. et al. Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations. Mol. Syst. Biol. 13, 955 (2017).
Dijkstra, K. K. et al. Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174, 1586–1598 (2018).
Bar-Ephraim, Y. E., Kretzschmar, K. & Clevers, H. Organoids in immunological research. Nat. Rev. Immunol. 20, 279–293 (2020).
Gao, M. et al. Development of patient-derived gastric cancer organoids from endoscopic biopsies and surgical tissues. Ann. Surg. Oncol. 25, 2767–2775 (2018).
Giobbe, G. G. et al. Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture. Nat. Commun. 10, 5658 (2019).
Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).
Vassilev, L. T. et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848 (2004).
Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015).
Michels, B. E. et al. Human colon organoids reveal distinct physiologic and oncogenic Wnt responses. J. Exp. Med. 216, 704–720 (2019).
Fujii, M. et al. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell 23, 787–793.e6 (2018).
Weeber, F. et al. Preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases. Proc. Natl Acad. Sci. USA 112, 13308–13311 (2015).
Driehuis, E. et al. Patient-derived head and neck cancer organoids recapitulate EGFR expression levels of respective tissues and are responsive to EGFR-targeted photodynamic therapy. J. Clin. Med. 8, E1880 (2019).
Sebaugh, J. L. Guidelines for accurate EC50/IC50 estimation. Pharm. Stat. 10, 128–134 (2011).
Bolhaqueiro, A. C. F. et al. Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nat. Genet. 51, 824–834 (2019).
Junttila, M. R. & de Sauvage, F. J. Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501, 346–354 (2013).
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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