RESUMEN
Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Modelos Biológicos , Técnicas de Cultivo de Órganos , Organoides/patología , Neoplasias Pancreáticas/patología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Páncreas/metabolismo , Páncreas/patologíaRESUMEN
Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.
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Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Hígado/metabolismo , Redes y Vías Metabólicas , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Animales , Animales Recién Nacidos , Dieta Alta en Grasa , Ayuno/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Proteína 2 Similar al Factor de Transcripción 7/genética , Activación TranscripcionalRESUMEN
Wnt/ß-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce ß-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing ß-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.
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Quinasa de la Caseína I/metabolismo , Neoplasias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , Quinasa de la Caseína I/genética , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Fibrosis Quística/patología , Células Epiteliales/patología , Técnicas de Cultivo de Órganos/métodos , Organoides/patología , Infecciones por Virus Sincitial Respiratorio/patología , Sistema Respiratorio/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Células Cultivadas , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Células Epiteliales/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Organoides/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/aislamiento & purificación , Sistema Respiratorio/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
[This corrects the article DOI: 10.1371/journal.pbio.1000539.].
RESUMEN
The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.
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Hepatocitos/citología , Hepatocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regeneración , Células Madre/citología , Células Madre/metabolismo , Vía de Señalización Wnt , Alelos , Animales , Conductos Biliares/citología , Conductos Biliares/metabolismo , Linaje de la Célula , Células Clonales/citología , Células Clonales/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Hepatocitos/patología , Hidrolasas/deficiencia , Hidrolasas/genética , Hígado/citología , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Hepatopatías/patología , Masculino , Ratones , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Organoides/citología , Organoides/trasplante , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Trombospondinas/deficiencia , Trombospondinas/genética , Trombospondinas/metabolismo , Tirosinemias/metabolismo , Tirosinemias/patologíaRESUMEN
Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.
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Células Madre Adultas/fisiología , Proliferación Celular , Páncreas/citología , Receptores Acoplados a Proteínas G/fisiología , Trombospondinas/fisiología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Embrión de Mamíferos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/fisiología , Páncreas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Trombospondinas/genética , Trombospondinas/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult human malignancies to treat. The 5-year survival rate of PDA patients is 7% and PDA is predicted to become the second leading cancer-related cause of death in the USA. Despite intensive efforts, the translation of findings in preclinical studies has been ineffective, due partially to the lack of preclinical models that faithfully recapitulate features of human PDA. Here, we review current preclinical models for human PDA (eg human PDA cell lines, cell line-based xenografts and patient-derived tumour xenografts). In addition, we discuss potential applications of the recently developed pancreatic ductal organoids, three-dimensional culture systems and organoid-based xenografts as new preclinical models for PDA.
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Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
BACKGROUND: Patient-derived intestinal organoids (PDIOs) show great potential as in vitro drug testing platform for personalised medicine in Cystic Fibrosis and oncology. PDIOs can be generated by culturing adult stem cells obtained through rectal forceps biopsy or suction biopsy, but the safety of these procedures and the success rates of generating organoids after shipment to a centralized lab using these procedures has not been studied in this context. We here report the safety and success rates of both biopsy procedures and the subsequent generation of PDIOs in the international multicentre HIT-CF Organoid Study. METHODS: 502 biopsy procedures were conducted, on 489 adult people with Cystic Fibrosis from 33 different hospitals across 12 countries. Depending on the preference of the hospital, either rectal forceps biopsies or suction biopsies were obtained and internationally shipped to a central laboratory for organoid generation. RESULTS: No adverse events were reported for 280 forceps biopsy procedures, while 222 rectal suction biopsy procedures resulted in 2 adverse events, namely continued bleeding and a probably nonrelated gastroenteritis. The success rate of organoid generation from all biopsies was 95%, and the main reason for failure was insufficient sample viability (3.2%). CONCLUSION: Our results indicate that both rectal suction biopsy and forceps biopsy procedures are safe procedures. The high success rates of PDIO generation from the obtained tissue samples demonstrate the feasibility of the organoid technology for personalised in vitro testing in an international setting.
RESUMEN
BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.
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BACKGROUND: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies. METHODS: We optimized drug screen methods on 5-11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient's response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness. RESULTS: Drug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003). CONCLUSIONS: Our study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.
Asunto(s)
Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Irinotecán/farmacología , Irinotecán/uso terapéutico , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Acetilcisteína/uso terapéutico , Medicina de Precisión , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Organoides , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversosRESUMEN
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/ß-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/ß-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and ß-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/ß-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a ß-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published ß-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Homeostasis/fisiología , Intestino Delgado/fisiología , Leucemia/fisiopatología , Metiltransferasas/fisiología , Factores de Transcripción/fisiología , beta Catenina/metabolismo , Animales , Línea Celular , Proliferación Celular , Metilación de ADN , N-Metiltransferasa de Histona-Lisina , Humanos , Intestino Delgado/patología , Metiltransferasas/genética , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción 4 , Factores de Transcripción/genética , Transcripción Genética , Pez Cebra/genéticaRESUMEN
The transcription of individual genes is determined by combinatorial interactions between DNA-binding transcription factors. The current challenge is to understand how such combinatorial interactions regulate broad genetic programs that underlie cellular functions and disease. The transcription factors Hnf1alpha and Hnf4alpha control pancreatic islet beta-cell function and growth, and mutations in their genes cause closely related forms of diabetes. We have now exploited genetic epistasis to examine how Hnf1alpha and Hnf4alpha functionally interact in pancreatic islets. Expression profiling in islets from either Hnf1a(+/-) or pancreas-specific Hnf4a mutant mice showed that the two transcription factors regulate a strikingly similar set of genes. We integrated expression and genomic binding studies and show that the shared transcriptional phenotype of these two mutant models is linked to common direct targets, rather than to known effects of Hnf1alpha on Hnf4a gene transcription. Epistasis analysis with transcriptomes of single- and double-mutant islets revealed that Hnf1alpha and Hnf4alpha regulate common targets synergistically. Hnf1alpha binding in Hnf4a-deficient islets was decreased in selected targets, but remained unaltered in others, thus suggesting that the mechanisms for synergistic regulation are gene-specific. These findings provide an in vivo strategy to study combinatorial gene regulation and reveal how Hnf1alpha and Hnf4alpha control a common islet-cell regulatory program that is defective in human monogenic diabetes.
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Epistasis Genética , Perfilación de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/fisiología , Factor Nuclear 4 del Hepatocito/fisiología , Animales , Haplotipos , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/genética , Islotes Pancreáticos/metabolismo , Ratones , Ratones Mutantes , Transcripción GenéticaRESUMEN
BACKGROUND: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier. METHODS: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib. RESULTS: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib. CONCLUSIONS: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.
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Enfermedades Inflamatorias del Intestino , Intestinos , Organoides , Humanos , Citocinas/metabolismo , Células Epiteliales/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Organoides/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Intestinos/fisiologíaRESUMEN
Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Proteínas de Neoplasias , Adulto , Humanos , Animales , Perros , Ratas , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Células CACO-2 , Organoides , Adenosina TrifosfatoRESUMEN
Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.
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Antineoplásicos , Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Vinorelbina/farmacología , Vinorelbina/uso terapéutico , Reposicionamiento de Medicamentos , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Organoides/metabolismoRESUMEN
BACKGROUND: The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. METHODS: We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. RESULTS: PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. CONCLUSIONS: By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.
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Neoplasias Colorrectales , Neoplasias Hepáticas , Neoplasias Peritoneales , Ratones , Animales , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Factor de Crecimiento Transformador beta3 , Peritoneo/metabolismo , Ascitis , Neoplasias Peritoneales/tratamiento farmacológico , Citocinas/metabolismo , Neoplasias Colorrectales/tratamiento farmacológicoRESUMEN
The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".
Asunto(s)
Ingeniería , Organoides , Humanos , Ingeniería de TejidosRESUMEN
Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias Glandulares y Epiteliales , Anticuerpos Biespecíficos/farmacología , Receptores ErbB/metabolismo , Humanos , Imidazoles , Neoplasias Glandulares y Epiteliales/metabolismo , Células Madre Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.