Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.
TL;DR: The orthotopic engraftment of colon organoids is described, providing a broadly usable immunocompetent CRC model that recapitulates the entire adenoma–adenocarcinoma–metastasis axis in vivo.
Abstract: Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation.
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TL;DR: Increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype, and immunotherapies directed against TGF β signalling may have broad applications in treating patients with advanced colorectal cancer.
Abstract: Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (TH1) activity and reduced immune cytotoxicity or increased TGFβ levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFβ-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype. Immunotherapies directed against TGFβ signalling may therefore have broad applications in treating patients with advanced colorectal cancer.
1,176 citations
TL;DR: In this Review, Drost and Clevers discuss the recent advances in organoid models of cancer and how they can be exploited to drive the translation of basic cancer research into novel patient-specific treatment regimens in the clinic.
Abstract: 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.
955 citations
TL;DR: A method for transplanting human brain organoids into the adult mouse brain is established and progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain are shown.
Abstract: Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.
755 citations
TL;DR: The biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.
Abstract: Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients' tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.
271 citations
Dissertation•
01 Jan 2011
254 citations
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TL;DR: A model for the genetic basis of colorectal neoplasia that includes the following salient features is presented, which may be applicable to other common epithelial neoplasms, in which tumors of varying stage are more difficult to study.
11,576 citations
"Transplantation of engineered organ..." refers methods in this paper
...We envisaged an ideal model would: (1) develop focal tumors in the colon; (2) enable longitudinal analysis of disease progression and regression using endoscopy; (3) permit all stages of CRC progression; (4) allow rapid and iterative genetic manipulation; and (5) be broadly adaptable to laboratories that lack advanced surgical expertise....
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TL;DR: It is concluded that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
Abstract: The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
5,193 citations
TL;DR: This work shows that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells, and observes a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation.
Abstract: The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12 , as well as novel hits NF2 , CUL3 , TADA2B , and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
4,147 citations
TL;DR: A technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract is developed that might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia.
2,726 citations
TL;DR: This review gives an account of recent advances in the authors' knowledge of the molecular mechanisms in colorectal cancer.
Abstract: This review gives an account of recent advances in our knowledge of the molecular mechanisms in colorectal cancer. Genetic changes in the germ line, combined with somatic mutations, occur in familial syndromes of colorectal cancer, whereas somatic mutations are the outstanding feature of sporadic colorectal cancer. Genetic changes drive the progression from adenoma to carcinoma and probably influence individual susceptibility and response to treatment.
1,741 citations
"Transplantation of engineered organ..." refers methods in this paper
...We envisaged an ideal model would: (1) develop focal tumors in the colon; (2) enable longitudinal analysis of disease progression and regression using endoscopy; (3) permit all stages of CRC progression; (4) allow rapid and iterative genetic manipulation; and (5) be broadly adaptable to laboratories that lack advanced surgical expertise....
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