The recent advances in in vitro 3D culture technologies, such as organoids, have opened new avenues for the development of novel, more physiological human cancer models. Such preclinical models are essential for more efficient translation of basic cancer research into novel treatment regimens for patients with cancer. Wild-type organoids can be grown from embryonic and adult stem cells and display self-organizing capacities, phenocopying essential aspects of the organs they are derived from. Genetic modification of organoids allows disease modelling in a setting that approaches the physiological environment. Additionally, organoids can be grown with high efficiency from patient-derived healthy and tumour tissues, potentially enabling patient-specific drug testing and the development of individualized treatment regimens. In this Review, we evaluate tumour organoid protocols and how they can be utilized as an alternative model for cancer research.
Over the past decades, our knowledge of the origin of cancer has increased immensely. Despite substantial progress in the treatment of (certain types of) cancer, it remains a major worldwide health problem1. The number of cancer deaths may be reduced by preventive measurements and early detection. In addition, the development of new, more targeted therapies offers opportunities. One of the major hurdles for the development of novel treatment regimens is the challenge of translating scientific knowledge from bench to bedside, which is mainly due to the fact that many cancer models only poorly recapitulate the patient’s tumour2, and as a consequence, many drugs that perform well in cancer models ultimately fail in clinical trials3. Although animal cancer models have provided important insights into the basics of cancer, their generation is time consuming, and it is argued that these models often do not faithfully recapitulate pathogenic processes in patients. For example, the histological complexity and genetic heterogeneity of human cancers are typically not reflected in genetically engineered mouse models of cancer4.
Commonly used human cancer models include cancer cell lines and primary patient-derived tumour xenografts (PDTXs). Cancer cell lines are derived from primary patient material and have contributed tremendously to cancer research. However, they have several drawbacks. For instance, their generation from primary patient material is very inefficient and involves extensive adaptation and selection to in vitro 2D culture conditions. As only rare clones are able to expand and can be maintained over many passages, the derived cell lines may have undergone substantial genetic changes and no longer recapitulate the genetic heterogeneity of the original tumours. Other limitations of cell lines include the absence of normal tissue-derived control cell lines as reference and the lack of stromal compartments (Table 1). A recently developed method called ‘conditional reprogramming’ facilitates the establishment of 2D cell cultures from normal and tumour epithelial cells with high efficiency5. These cultures can be maintained long term and retain a stable karyotype. The procedure involves the presence of a RHO kinase inhibitor and fibroblast feeder cells5 (Table 1). PDTXs have the advantage of mimicking the biological characteristics of the human tumour much better than in vitro culture models. PDTXs are generated by transplanting freshly derived patient material subcutaneously or orthotopically into immunodeficient mice. The ability to serially transplant tumour tissues into increasing numbers of animals allows for preclinical testing of novel therapies for cancer treatment (Table 1). Limitations of PDTXs include the use of animals and limited engraftment efficiencies for subsets of patient tumours. Moreover, the approach is expensive, time consuming and resource consuming, and PDTXs may undergo mouse-specific tumour evolution6,7.
Recently developed 3D culture technologies have led to the development of novel and more physiological human healthy tissue and cancer models. Upon embedment into a 3D matrix, tissue-derived adult stem cells can be grown with high efficiencies into self-organizing organotypic structures, termed organoids. In 2009, Sato et al.8 demonstrated that 3D epithelial organoids can be established from a single leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ intestinal stem cell. Upon embedment into Matrigel, cells are cultured under serum-free conditions mimicking the in vivo stem cell niche (involving R-spondin 1 (a WNT agonist and ligand of LGR5 (refs9,10)), epidermal growth factor (EGF) and the bone morphogenetic protein (BMP) inhibitor noggin). Consequently, LGR5+ intestinal stem cells grow out as organotypic, highly polarized epithelial structures with proliferative crypt and differentiated villus compartments8. This culture protocol formed the starting point for other organoid culture protocols of multiple mouse and human epithelia, including colon11,12, liver13, pancreas14, prostate15,16, stomach17, fallopian tube18, taste buds19, salivary glands20, oesophagus21, lung22, endometrium23 and breast24 (reviewed in ref.25). Organoids can be expanded long term, can be cryopreserved and genetically modified and remain genetically and phenotypically stable. This allows for a wide range of applications in cancer research. Instead of culturing organoids in medium containing niche-recapitulating and tissue-specific growth factors, the Kuo laboratory26 pioneered an organoid culture system using an air–liquid interface with stromal support cells as a source of essential growth factors. Organoids can also be derived from induced pluripotent stem cells (iPSCs)25. However, the efficiency of generating iPSC-based cancer models from patients may depend on cancer type and the presence or absence of specific oncogenic mutations, potentially selecting for outgrowth of tumour subclones and loss of the genetic heterogeneity of the tumour it is derived from27. Generally, it appears more practical to grow tumour organoids directly from cancers than to involve an intermediate iPSC step.
In this Review, we discuss the use of the adult stem cell-derived organoid technology in both basic and translational cancer research. We highlight the approaches to exploit patient-derived tumour organoid biobanks for drug development and personalized medicine and evaluate the application of organoid technology as an experimental tumour model. Finally, we discuss the limitations and potential of exploiting organoids for cancer research.
Organoids for translational research
Living organoid biobanks
The ability to grow organoids with high efficiency from healthy human adult stem cells has paved the way to grow organoids from patient-derived tumour tissue. So far, we and others have shown that long-term organoid cultures can be established from primary colon11,28,29, oesophagus11, pancreas14,30, stomach17, liver31, endometrium23 and breast24 cancer tissues, as well as from metastatic colon32, prostate33,34 and breast24 cancer biopsy samples. Importantly, these studies have shown that tumour-derived organoids — both phenotypically and genetically — resemble the tumour epithelium they were derived from. Tumour organoids do not grow faster per se than their matching normal organoid counterparts and, counterintuitively, in many cases even grow at slower rates, possibly owing to higher rates of mitotic failures and subsequent cell death35,36. Therefore, the overgrowth of tumour organoids by healthy epithelial organoids derived from remaining normal tissue present in tumour biopsy samples needs to be avoided. Hence, it is essential to initiate tumour organoid cultures using either pure tumour material or to grow the samples under selective culture conditions. For example, in the vast majority of colorectal cancers (CRCs), activating mutations in the WNT signalling pathway are present37. In these cases, pure tumour organoid cultures can be obtained by using culture medium lacking WNT and R-spondins11, which are essential growth factors for healthy tissue-derived colon organoids. Similarly, tumours harbouring mutations in the EGF receptor (EGFR) signalling pathway can be selected by EGF withdrawal29,35,38. The small molecule nutlin-3, which stabilizes p53 by disrupting the binding of p53 to its negative regulator E3 ubiquitin ligase MDM2, has been used as a strategy to remove TP53-wild-type (WT) healthy cells from TP53-mutant cancer organoid cultures24,35. When such selection methods are not available, using pure tumour cells as starting material is a prerequisite.
Currently, large collections of patient-derived tumour and matching healthy organoids are generated and biobanked. These resources can be used to determine whether organoids have predictive value for drug responses for individual patients (Fig. 1). One of the earliest efforts to store large collections of primary tumour cultures was undertaken by Inoue and colleagues39. A collection of cultures derived from patients with CRC was assembled using a method to expand tumour tissues as tumour spheroids in vitro. This culture method did not sustain the growth of healthy colon epithelium39. More recently, a tumour organoid biobank derived from patients with CRC was generated that consists of a set of 20 genetically diverse tumour organoid cultures and their matching normal tissue-derived organoids28. Integrating genomic and monotherapy drug-response data demonstrated that only one of the tumour organoid cultures was sensitive to the inhibitor of WNT secretion LGK974 (ref.40) owing to a mutation in the WNT antagonist E3 ubiquitin ligase RNF43 (refs28,41,42). Besides several other known correlations between drug responses and the presence of mutations, several compounds were identified with differential cytotoxicities among patient-derived tumour organoids without an apparent genetic marker28. Increasing the number of biobanked organoids will be necessary to increase the statistical power to that required to correlate genetic markers with differences in drug sensitivity. In a separate study, a subset of CRC organoids from this same biobank were used for proteomic analyses and comparative transcriptomic analysis43. Substantially different protein profiles were observed between tumour organoids and matching healthy organoids as well as among tumour organoids from individual patients displaying distinct personalized profiles43. This finding points towards an important role for proteomic profiling in personalized medicine. However, a direct comparison should be made between the proteomic profiles of tumour organoids and the primary tumour tissue to ensure preservation of protein expression profiles following expansion in vitro.
In line with the previous study28, Sato and colleagues29 generated an organoid biobank derived from 55 patients with CRC. For certain CRC subtypes, organoids could be established only when cultured under specific conditions. For instance, some organoid lines were maintained in medium lacking an inhibitor of p38 MAPK, a component of the original human colon organoid culture medium, or under hypoxic conditions29. These dependencies may reflect differences in the mutational backgrounds of tumours. As the genetic background of tumours is in many cases not determined when establishing organoid cultures from fresh patient-derived tissues, high efficiencies may result from culturing under various media conditions that differ in their combinations of growth factors. Importantly, it was shown that tumour organoids preserve the histopathological features of the original tumours not only in vitro but also following xenotransplantation under the kidney capsule of immunodeficient mice29. This finding suggests that organoid transplantations can be used to validate in vitro drug responses in a more complex in vivo environment. Another study exploited a biobank consisting of both CRC-derived PDTXs and organoids to identify biomarkers to predict sensitivity towards EGFR inhibitors44.
A collection of organoid lines was established from the primary pancreatic ductal adenocarcinoma (PDAC) tissue of 17 patients30. Although mutation analysis to validate tumour origin was not performed, organoids maintained similar histological features to the parental tumours both in vitro as well as following xenotransplantation. Organoids could not be established from poorly differentiated to moderately differentiated PDACs and from intraductal papillary mucinous cystic neoplasms, suggesting that — as for different CRC subtypes29 — different tumour subtypes require specific compositions of culture media. Similarly, Seino et al.45 generated a genetically characterized organoid biobank derived from 39 patients with PDAC. Three functional PDAC organoid subtypes were identified as having distinct dependencies on WNT signalling, which were also associated with different gene expression subtypes45.
Recently, a biobank of breast cancer organoids was generated from >100 patients representing all major breast cancer subtypes24. Besides preservation of histopathological and genetic features, the majority of the breast cancer organoids also retained expression of the breast cancer biomarkers oestrogen receptor (ER), progesterone receptor (PR) and human EGFR2 (HER2; also known as ERBB2). The receptor status of the parental tumour was lost in only a small number of organoid cases, yet this highlights the need to adequately characterize tumour-derived organoids before their utilization in downstream experimental procedures and drug screening platforms. A proof-of-principle drug screen using various drugs that target the HER signalling pathway family demonstrated that drug sensitivity levels generally correlated with HER2 status24.
Besides these living biobanks, smaller tumour organoid collections from advanced prostate cancer (seven lines)33, CRC metastases32 (eight lines), primary pancreatic intraepithelial neoplasms (eight lines)14 and primary liver cancer (seven lines)31 have been generated (Table 2). Notably, prostate cancer organoids carrying an androgen receptor (AR) amplification showed higher sensitivity towards the AR signalling inhibitor enzalutamide than AR– prostate cancer organoids33
, again demonstrating that tumour organoid biobanks enable physiologically relevant drug screens.
Currently, a combined effort of the US National Cancer Institute (NCI), Cancer Research UK, the UK Wellcome Trust Sanger Institute and the foundation Hubrecht Organoid Technology, Netherlands — which is known as the Human Cancer Models Initiative (HCMI) — is ongoing to generate a large, globally accessible bank of new cancer cell culture models, including organoids, available for the research community.
Drug development and personalized cancer treatment
Although drug screens on large 2D cancer cell line collections have provided major insights into genetic predictions of drug response46, their fairly poor reflection of native tumour tissue may have contributed to the high failure rate of newly discovered drugs in clinical trials2,3. Patient-derived tumour organoids better recapitulate native tumours and may be superior models to identify and test novel anticancer drugs. The development of high-throughput drug screening methods in patient-derived organoids is just beginning to be explored. As discussed above, small-scale drug screens on organoid biobanks performed so far have yielded promising results24,28,33.
Profiling of patient-derived organoids may reveal causal epigenetic and/or genetic changes that underlie drug resistance, which could be used to stratify individual patients to specific treatment regimens. To test the potential of organoids to evaluate drug responses in a preclinical setting, Verissimo et al.36 utilized a CRC organoid biobank to investigate the effect of different RAS pathway inhibitors, either as single agents or in combinations, that are currently used in the clinic. Using this strategy, the authors confirm earlier observations in cancer cell lines that combinatorial targeting of MEK and ERK, or of the HER family and MEK in RAS-mutant CRCs, effectively suppressed growth. These treatments merely forced organoids into cell cycle arrest rather than apoptosis, and cells rapidly re-initiated growth when the treatment was stopped36. These results therefore question the effectiveness of this combination therapy in the treatment of RAS-mutant CRCs. However, combinatorial EGFR pathway inhibition sensitized RAS-mutant CRCs to low levels of anti-apoptosis inhibitors36, possibly providing an alternative treatment strategy. More recently, patient-derived organoid models of metastatic gastrointestinal cancer (CRC and gastroesophageal cancer) were generated and used to determine whether organoids can predict patient treatment response47. Using a compound library of drugs either used in the clinic or currently in clinical trials, organoid drug sensitivities were tested and compared with the responses of patients. The authors report a positive predictive value (predicting that a particular drug works) of 88% and a negative predictive value (predicting that a particular drug does not work) of 100%, suggesting that patient-derived organoids recapitulate patient responses in clinical trials and can be used for personalized medicine programmes47.
By performing drug screens on human primary liver cancer organoids, Huch and colleagues31 identified ERK inhibition as a potential therapeutic approach for the treatment of primary liver cancers. As another example, patient-derived castration-resistant prostate cancer organoids (either WT or deficient for chromodomain helicase DNA-binding protein 1 (CHD1)) were used to demonstrate that CHD1 deletions sensitize to DNA-damaging agents48. One patient with prostate cancer demonstrated rapidly progressing disease. Following tumour organoid development, CHD1-deficiency was identified as resulting in an improved response to carboplatin. This patient was subsequently successfully treated with this DNA-damaging agent48. Lastly, Chen and colleagues49 developed colonic organoids from iPSCs from patients with familial adenomatous coli (FAP), which is caused by mutations in the adenomatous polyposis coli (APC) gene, a negative regulator of the WNT pathway. They used these organoids as a platform for drug testing and found that cells with hyperactive WNT signalling were more sensitive to the ribosome-binding antibiotic geneticin. They additionally identified two compounds that decreased proliferation in organoids derived from patients with FAP but also affected cell proliferation in WT organoids, thereby highlighting the value of organoids for drug testing before clinical therapeutic application49.
Another major advantage of exploiting organoid technology for drug development is that organoids from both healthy and tumour tissue can be generated, which allows for screening for drugs that specifically target tumour cells while leaving healthy cells unharmed. This approach may result in reduced toxicities in patients. Drug-induced liver failure caused by hepatotoxicity is a major reason for drug failure in clinical trials50. Recently established hepatic organoid cultures could provide a relevant model for preclinical testing of the hepatotoxicity of experimental compounds51,52 (Fig. 1). The most common mechanisms of drug-induced hepatotoxicity are mediated through cytochrome P450 enzymes, and it is encouraging that hepatic organoids express these enzymes at near physiological levels upon induced differentiation13,52. On the same note, iPSC-derived cardiac organoids could be used for testing cardiotoxicity53,54, and iPSC-derived kidney organoids were recently exploited for toxicological studies55. For patients with cystic fibrosis (CF; a disease caused by a spectrum of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel), intestinal organoids are already successfully used to predict patient drug response. Readout is based on in vitro assays to test CFTR functionalities in patient-derived rectal organoids56. Beekman and colleagues57 exploited this assay to test different CFTR potentiators on rectal organoids derived from patients with CF that differ in CFTR genotypes. This work revealed that in vitro measurement of CFTR function in such organoids is useful to identify patients who would benefit from a particular type of drug treatment57. On the basis of these promising results, this organoid application is currently being implemented in the diagnostic pipeline in the Netherlands.
The emergence of resistance against single-agent targeted therapies is another important rate-limiting factor in the successful development of new anticancer drugs. Therefore, combination strategies have to be considered to minimize development of resistance to single compounds58,59. A very promising strategy is to exploit the patient’s own immune system to eradicate malignant cells. Since its initial successes in the clinic, cancer immunotherapy has seen a major revival60,61. However, it is essential that tumour cells display sufficient immunogenicity to elicit a proper immune response62,63,64. For tumour cells, this is — at least partially — determined by the mutational load of a tumour, as immune responses are dependent on antigens that result from these mutations (so-called neo-antigens)62,63,65. In many cases, the potency of the immune response elicited by the neo-antigens of the tumour is inadequate. In vitro activation and expansion of immune cells could provide a way to generate sufficient numbers of cytotoxic immune cells for in vivo use in patients.
Indeed, several recent studies successfully established culture systems in which haematopoietic cells are expanded in vitro in co-cultures with organoids. Nakamura and colleagues66 demonstrated short-term maintenance of intraepithelial lymphocytes (IELs) in a co-culture with mouse intestinal organoids. When the culture medium was supplemented with interleukin-2 (IL-2), IL-7 and IL-15, IELs were maintained and expanded over several weeks while retaining their functionality66. Finnberg et al.67 demonstrated that CD45+ lymphocytes can be maintained up to 8 days in co-culture with human air–liquid interface tumour organoids, whereas other haematopoietic cell populations were rapidly lost. Moreover, co-cultures of organoids together with a specific T cell subset, Vδ2+ T lymphocytes, can be directly established from primary human breast epithelia68. Interestingly, these T lymphocytes from the breast organoids can effectively kill triple-negative breast carcinoma cells68. It was recently demonstrated that naive T lymphocytes derived from healthy blood donors provide a source of T cells that recognize neo-antigens and target and kill melanoma cells harbouring such neo-antigens69. This finding reveals the exciting possibility of testing the cytotoxic potential of healthy donor-derived T cells on patient-derived tumour organoids. T cells displaying immune reactivity could be expanded in organoid co-cultures and subsequently used to treat patients. The recent development of thymus organoids could possibly provide a more physiological in vitro environment to efficiently maintain and expand tumour-specific T lymphocytes70.
Organoids for basic research
Organoids to explore the link between infectious agents and cancer development
An estimated one out of five cancer cases have been linked to infectious agents71. However, in many cases, it is not known how a particular pathogen contributes to malignant transformation. As several studies have succeeded in establishing organoid co-culture systems with different types of pathogens, organoid technology could potentially be used to study these processes. Of particular interest for cancer researchers are organoid co-cultures with pathogens as potential risk factors for cancer development. For example, stomach organoids17 may be used to study the relationship between chronic Helicobacter pylori infections and gastric cancer72. Stomach organoids provoke strong primary inflammatory responses when micro-injected with H. pylori17 and have been used to demonstrate how H. pylori locates and colonizes the gastric epithelium73. Moreover, iPSC-derived stomach organoids have been used to model the acute response of the gastric epithelium to H. pylori infections74. However, to study the effects of chronic infection and its possible causal role in gastric tumorigenesis, a long-term co-culture system may have to be established and might need to involve immune elements as well. Scanu et al.75 used organoids of pre-transformed gallbladders derived from different transgenic mice as a model to study the contribution of Salmonella enterica infections to the development of gallbladder carcinoma. The authors demonstrated that S. enterica infection of gallbladder organoids with TP53 mutations and MYC amplifications induces growth factor-independent cell growth and neoplastic transformation through activation of AKT and MAPK signalling75. Moreover, viral infections can also be modelled in organoids76, suggesting that organoid–virus co-culture models can be exploited for cancer research as well. Liver or hepatocyte organoids could potentially be used to study the contribution of hepatitis infections to the development of hepatocellular carcinoma, whereas stomach organoids could be exploited to study the connection between Epstein–Barr virus infections and gastric cancer development.
Organoids to interrogate mutational processes underlying tumorigenesis
Cancer is caused by the gradual accumulation of mutations in disease-driving genes77. It is therefore important to gain insight into the mutational processes that are active during both tissue homeostasis and tumorigenesis. The high genetic stability of healthy organoid cultures over long periods of time11,13,78 enables the study of mutagenic processes in detail. Blokzijl et al.79 used single stem cell-derived (that is, clonal) organoid cultures to determine genome-wide mutation patterns in healthy stem cells of the small intestine, colon and liver over the course of life. The authors performed whole genome sequencing on clonal organoids derived from donors of various ages (Fig. 2). Surprisingly, although mutation rates did not differ among the different types of stem cell, the types of mutations or mutation signatures80 were distinct in small intestinal and colon stem cells from those of liver stem cells. The authors suggest that the rapid cycling of intestinal and colonic stem cells causes deamination-induced mutagenesis, resulting in the acquisition of a specific mutation signature, with frequent C>G to T>A transitions at CpG dinucleotides. Remarkably, this type of mutation is also found in common CRC driver genes, while driver mutations in the same genes arise through a different mechanism in liver cancer79. Overall, these findings suggest that organ-specific mutagenic processes result in the accumulation of specific types of somatic point mutations during malignant transformation. In an earlier study, genome sequencing data of clonally expanded mouse organoids derived from stomach, small intestine, colon and prostate were used to reconstruct developmental lineages on the basis of the presence of somatic point mutations78.
The importance of understanding the origin of mutational signatures in cancer was recently demonstrated by Davies et al.81, who showed that BRCA1 or BRCA2 deficiency can be predicted based on the mutational signature present in a tumour. Using this prediction, BRCA1-deficient or BRCA2-deficient tumour cells that lack BRCA1 or BRCA2 gene mutations can be identified and, akin to BRCA1-mutant or BRCA2-mutant tumours82,83,84, could benefit from poly(ADP-ribose) polymerase (PARP) inhibitors81. In cancer cells, many different mutational processes are active, which makes it challenging to study the origin of mutational signatures. The genetic stability of healthy organoid cultures provides a platform to causally attribute specific mutational processes to mutation signatures. We recently knocked out DNA repair genes in healthy human colon organoids using CRISPR–Cas9 genome editing; whole genome sequencing of these clonal knockout organoids demonstrated that a high contribution of signature 30 mutations in a tumour is indicative of (germline) mutations in the base excision repair gene nth like DNA glycosylase 1 (NTHL1), which could indicate a familial cause of the cancer85. Using a similar approach, it will be interesting to exploit organoids to study the effects of, for instance, chemotherapeutics and irradiation on the mutation rates and profiles of both healthy and tumour cells (Fig. 2). Such experiments may provide novel insights into the deleterious side effects of chemotherapeutics and the increased risk of secondary cancers later in life. Moreover, sporadic clones that develop resistance towards the treatment could be characterized to identify mutations as the cause of the acquired therapy resistance.
The majority of tumours harbour unstable genomes, and consequently, individual tumour cells within a tumour contain different sets of genetic alterations77,86. Although this intratumour heterogeneity is believed to play a key role in cancer progression and therapy resistance, knowledge of how tumour heterogeneity arises and contributes to disease progression is still limited87. The heterogeneous genetic composition of a tumour is retained in organoids that are derived from it14,28,29,32 and is maintained in culture over time31,44,88. However, clonal dynamics within tumour organoid cultures can occur29, suggesting that the genetic composition of a tumour-derived organoid culture changes over time. In a comprehensive genetic analysis of multiple patient-derived clonal organoids derived from different regions of the same treatment-naive tumour, Roerink et al.89 recently exploited organoid technology to study intratumour heterogeneity of CRC in great detail (Fig. 2). This strategy allowed an in-depth analysis of the genetic, epigenetic and transcriptomic profiles of different regions from the same tumour and to determine drug sensitivity of different tumour subclones. The authors observed a striking increase in mutation rates and the acquisition of novel mutational signatures during the development of a tumour. A similar observation was found when performing exome sequencing on organoids derived from normal tissue and adenomas of Apcmin/+ mice90. Thus, during the conversion of a normal cell into a malignant cell, the probability of accumulating additional driver mutations substantially increases and disease progression accelerates. In addition, Roerink et al.89 demonstrated that the contribution of mutational processes varies in different parts of the tumour. Another surprising observation came from drug treatment of the individual tumour clones with different anticancer drugs that are commonly used in clinical practice. This work revealed that within all three tumours examined, resistant cells already existed for each of the tested drugs before treatment89.
Genetic cancer modelling in organoids
Organoids can be used to model and study cancer initiation and progression in specific organs. Starting from healthy human intestinal organoids, two reports exploited CRISPR–Cas9 genome editing to introduce combinations of common CRC driver mutations to generate CRC progression models35,91. Using these models, it was shown that CRC driver mutations result in growth that is independent of intestinal stem cell niche factors, as organoids harbouring activating mutations in KRAS (KRASG12D) and inactivating mutations in APC, TP53 and SMAD4 grow independently of the niche factors EGF, WNT, R-spondin 1 and noggin35,91. We additionally demonstrated that loss of APC and TP53 are key drivers of chromosome instability and aneuploidy35, which are hallmarks of CRC92. Upon subcutaneous xenotransplantation into mice, the quadruple-mutant organoids grow as invasive adenocarcinomas35. Surprisingly, although organoids efficiently grew in vivo as invasive tumours, the developed tumours did not spontaneously metastasize, most likely owing to the lack of a native microenvironment. Indeed, when the same tumour organoids were orthotopically transplanted into the caecum of mice, spontaneous metastases developed in the liver and lungs93,94. This orthotopic organoid transplantation strategy was further used to demonstrate that a key contributor to the colonization of a metastatic site is the loss of dependency on stem cell niche signals93, thereby confirming earlier observations in patient-derived CRC organoids29.
Similar CRC progression models were generated in air–liquid interface mouse small intestinal and colonic organoids using RNAi-based strategies95. Remarkably, mouse small intestinal organoids showed a much more promiscuous transformation capacity than colonic organoids, as high-grade dysplasia developed with only two driver mutation combinations, KrasG12D and Apc–/– or Apc–/– and Trp53–/–, in small intestinal organoids95. This finding implies that mouse small intestinal organoids may not be a representative model to study multi-hit CRC tumorigenesis. The importance of transplanting tissue into its orthotopic environment was also demonstrated in a study by De Sauvage and colleagues96, where mouse small intestinal organoids carrying different combinations of CRC mutations were directly injected into the colonic epithelium. Mutations in Apc, Kras, Trp53 and Smad4 were required for progressive tumour growth and metastasis upon transplantation of organoids into their relevant physiological niche, while subcutaneous transplantation of the same organoids did not result in development of metastases, thereby confirming earlier studies35,93. Two other recent studies further underscore the need for an orthotopic transplantation environment for studying the metastatic cascade of CRC97,98.
Organoids derived from mice harbouring the diphtheria toxin receptor knocked into the Lgr5 locus to enable targeted cell ablation were used to investigate the contribution of cancer stem cells (CSCs) to CRC growth. Remarkably, specific killing of CSCs did not result in regression of the primary tumour, but CSCs were required for the formation and maintenance of CRC-derived liver metastases96. In line with this, Shimokawa et al.99 performed renal capsule transplantations of human CRC organoids, in which an inducible caspase 9 (which is involved in the activation of apoptosis) was knocked into the LGR5 locus, thereby enabling the ablation of CSCs. Although elimination of CSCs initially resulted in reduction of the primary tumours, these eventually regrew owing to the reappearance of LGR5+ CSCs, demonstrating the extensive cellular plasticity within tumours99. Finally, human pancreatic organoids were recently used to model PDAC. Oncogenic mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A), KRAS, TP53 and SMAD4 resulted in transformation of pancreatic cells into cancer cells that, upon xenotransplantation, grew as tumours with characteristics of pancreatic intraepithelial neoplasia and invasive PDAC45.
Organoid technology can also be utilized to study cancer-related processes and signalling pathways. For instance, mouse small intestinal organoids were exploited to show that expression of recurrent hotspot mutations in RHOA, which were identified in gastric cancer, induces resistance to anoikis100. Moreover, serrated colon adenomas are thought to be driven by oncogenic BRAFV600E mutations101. CRISPR–Cas9-engineered BRAFV600E-expressing human colon organoids were therefore used as a model system to demonstrate that transforming growth factor-β (TGFβ) signalling drives serrated colon adenoma to CRCs of the mesenchymal subtype, which have a very poor prognosis102. Leung and colleagues exploited human serrated colon adenoma-derived organoids to demonstrate that RNF43 mutations, as identified in serrated neoplasia, activate WNT–β-catenin signalling103, thereby confirming earlier observations in mouse intestinal organoids104. The presence of RNF43 mutations in a subset of serrated colon neoplasias potentially provides therapeutic opportunities, as it was previously demonstrated that tumours harbouring mutations in RNF43 are sensitive to inhibitors of the O-acyltransferase porcupine, which inhibit WNT secretion28,105.
The establishment of organoids from transgenic mouse strains with particular oncogenic transgenes enables rapid testing of the effects of a particular mutation in the context of a specific genetic background. Kuo and colleagues demonstrated that TGFβ receptor 2 (Tgfbr2) loss in the context of inactivated cadherin 1 (Cdh1) and Trp53 induces metastatic diffuse gastric cancer by knocking down Tgfbr2 in stomach organoids derived from Cdh1–/–;Trp53–/– mice106. Transgenic mouse-derived organoids were also used to study the genetic interaction between loss of the transcription factor caudal type homeobox 2 (CDX2) and BRAFV600E mutations, alterations that frequently co-occur in human serrated CRCs38. By histologically and genetically analysing organoids derived from WT, Cdx2–/–, BrafV600E and Cdx2–/–;BrafV600E mice, the authors showed that these mutations cooperate to promote development of serrated CRC38.
The recent revolution in genome-editing technologies such as CRISPR–Cas9 provides a platform to investigate the effects of ‘repairing’ disease-causing genes. Organoids could presumably be used to test whether the repair of a particular oncogenic mutation reverts the tumorigenic phenotype. Previously, a mutated, dysfunctional CFTR allele was successfully repaired in rectal organoids derived from patients with CF104. Although cancer is genetically much more complex, with tumours typically harbouring hundreds of mutations, it was recently shown that restoration of APC expression recovers crypt homeostasis in a mouse CRC model and in organoids derived thereof97,107. Although restoration of a single driver mutation will likely result in the emergence of resistant tumour clones, it would be interesting to test whether restoring the activity of several driver genes simultaneously in patient-derived tumour organoids results in tumour regression upon xenotransplantation.
Limitations and perspectives
In this Review, we have discussed a spectrum of applications of organoid technology for cancer research. Many of these are just beginning to be explored, and although the first indications are promising, organoids have limitations as well. For example, compared with cancer cell lines, organoid culture consumes both time and resources (Table 1). One of the intrinsic limitations of organoid culture is the lack of stroma, blood vessels and immune cells. Future studies will address the possibility of developing co-culture systems incorporating additional cellular (and microbial) elements108. In a recent effort using iPSC-derived tissue engineering, intestinal organoid cultures with a functional enteric nervous system were developed109, demonstrating the potential of developing more complex organoid-based structures. Additionally, the Tuveson laboratory110 successfully established co-cultures of mouse pancreatic stellate cells (which differentiate into cancer-associated fibroblasts (CAFs)) with PDAC organoids. In line with this, Sato and colleagues45 demonstrated that patient-derived CAFs provide WNT ligands for PDACs using a co-culture of CAFs and PDAC organoids.
The requirement for mouse-derived extracellular matrix (ECM) substitutes (for example, Matrigel or basement membrane extract) and, in some organoid cultures, for fetal calf serum (required for the production of WNT-conditioned medium) incorporates undefined extrinsic factors that may influence the outcome of experiments, such as drug screens. For instance, the presence of serum was shown to be unfavourable for long-term growth of human pancreatic organoids45. Recently developed synthetic matrices that support the growth of mouse and human intestinal organoids could possibly replace mouse-derived ECM substitutes111. However, these matrices will have to be further optimized to increase efficiency and to sustain the growth of organoids derived from other organs as well. Garcia and colleagues112 developed water-soluble Frizzled (FZD)–low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6 heterodimerizers that function as surrogate WNT agonists. These surrogate WNT agonists were shown to efficiently support the growth of a range of human organoid cultures112, thereby providing a serum-free alternative for WNT-conditioned medium. Similarly, Mihara et al.113 demonstrated that water-soluble WNT can be produced when co-expressed with afamin, a component of fetal calf serum. Furthermore, lipid-mediated stabilization of WNT proteins may provide another means for serum-free growth of organoids114.
Another possible limitation may be that organoids derived from advanced cancers often grow more slowly than organoids derived from normal epithelium, possibly resulting in overgrowth of tumour organoids by organoids derived from contaminating normal epithelium. This finding is most likely due to a much higher rate of mitotic failure and subsequent cell death in tumour organoids. Future studies will provide additional insights into this counterintuitive observation. Finally, all organoids discussed above were grown from tumours of epithelial origin, that is, (adeno)carcinomas. It is currently unclear whether the organoid approach can be adapted for organoid culture systems from non-epithelial tumours. The recent advances in growing organoids from primary glioblastoma tissue115 may set the stage for growing other non-epithelial tumours.
Despite these limitations, organoids have emerged as a physiologically relevant in vitro model to study cancer. Organoids can be established with high efficiency from individual-patient-derived tumour tissue, making them a highly relevant model for translational applications and the development of personalized cancer treatments. It will be essential that patient-derived organoids can be generated and expanded efficiently to enable drug screening in a clinically meaningful time window. While anecdotal validation for the use of organoids as a diagnostic for CF already exists, solid validation of the predictive value of organoids for tumour drug responses will have to come from ongoing trials. Results of a first study comparing drug responses of patient-derived gastrointestinal organoids with the responses of the patients in the clinic are very encouraging47. The optimization of drug screening platforms in terms of sensitivity and robustness will be key before organoid-based personalized medicine can be implemented in the clinic.
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The authors thank K. Kretzschmar and J. Meijerink for critical reading of the manuscript. We are grateful for support from the Dutch Cancer Society (KWF) and the Alpe d’HuZes Bas Mulder Award to J.D. (KWF/Alpe d’HuZes, 10218) and for the support of Oncode Institute to H.C.
J.D. and H.C. are named as inventors on several patents related to leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ stem cell-based organoid technology.
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The number and appearance of chromosomes in the nucleus of a cell.
- Feeder cells
A layer of cells that is used to support the growth of a cell culture (that is, stem cell cultures) by secretion of important growth factors into the culture medium.
A mouse-derived ex vivo basement membrane substitute that is used to support 3D growth of organoid cultures.
- Intraepithelial lymphocytes
(IELs). Lymphocytes residing in the epithelial layer of mammalian mucosal linings, such as the small and large intestines, lungs, upper respiratory tract, reproductive tract and skin.
- Mutation signatures
Unique combinations of mutation types caused by different mutational processes.
- Base excision repair
A DNA repair mechanism that removes damaged bases (oxidized, alkylated or deaminated) that could otherwise cause mutations.
A pouch located between the small and large intestine that is considered to be the beginning of the large intestine and is thus part of the gastrointestinal tract.
A process of programmed cell death initiated by loss of cell–matrix interactions in anchorage-dependent cells.
- Serrated colon adenomas
A precursor colorectal cancer (CRC) subtype that is characterized by a serrated histopathological morphology. Serrated CRCs are genetically distinct from the classical adenocarcinomas. Whereas classical adenocarcinomas are typically initiated by mutations in the WNT pathway (for example, adenomatous polyposis coli (APC)), serrated CRCs are likely initiated by BRAF mutations.
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