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1.
Cell ; 183(5): 1420-1435.e21, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33159857

RESUMO

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.


Assuntos
Bancos de Espécimes Biológicos , Tumores Neuroendócrinos/patologia , Organoides/patologia , Animais , Cromossomos Humanos/genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Masculino , Camundongos , Modelos Genéticos , Mutação/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transcriptoma/genética , Sequenciamento Completo do Genoma
2.
Cell ; 174(4): 856-869.e17, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096312

RESUMO

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.


Assuntos
Adenocarcinoma/patologia , Organoides/patologia , Neoplasias Gástricas/patologia , Estômago/patologia , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Carcinogênese , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Organoides/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Trombospondinas/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nature ; 592(7852): 99-104, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33627870

RESUMO

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.


Assuntos
Colo/citologia , Íleo/transplante , Mucosa Intestinal/citologia , Organoides/transplante , Regeneração , Medicina Regenerativa/métodos , Síndrome do Intestino Curto/terapia , Animais , Colo/irrigação sanguínea , Colo/inervação , Colo/cirurgia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Íleo/citologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/inervação , Mucosa Intestinal/cirurgia , Masculino , Técnicas de Cultura de Órgãos , Organoides/citologia , Ratos , Ratos Endogâmicos Lew , Síndrome do Intestino Curto/patologia , Síndrome do Intestino Curto/cirurgia
4.
Nature ; 577(7789): 254-259, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31853059

RESUMO

With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.


Assuntos
Colite Ulcerativa/genética , Epitélio/metabolismo , Interleucina-17/genética , Mutação , Colite Ulcerativa/metabolismo , Humanos , Interleucina-17/metabolismo , Fenótipo , Transdução de Sinais
5.
Nat Chem Biol ; 18(6): 605-614, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35273398

RESUMO

Precision oncology presumes an accurate prediction of drug response on the basis of the molecular profile of tumors. However, the extent to which patient-derived tumor organoids recapitulate the response of in vivo tumors to a given drug remains obscure. To gain insights into the pharmacobiology of human colorectal cancer (CRC), we here created a robust drug screening platform for patient-derived colorectal organoids. Application of suspension culture increased organoid scalability, and a refinement of the culture condition enabled incorporation of normal and precursor organoids to high-throughput drug screening. Drug screening identified bromodomain and extra-terminal (BET) bromodomain protein inhibitor as a cancer-selective growth suppressor that targets genes aberrantly activated in CRC. A multi-omics analysis identified an association between checkpoint with forkhead and ring finger domaines (CHFR) silencing and paclitaxel sensitivity, which was further validated by gene engineering of organoids and in xenografts. Our findings highlight the utility of multiparametric validation in enhancing the biological and clinical fidelity of a drug screening system.


Assuntos
Neoplasias Colorretais , Organoides , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer , Epigênese Genética , Humanos , Organoides/patologia , Medicina de Precisão
6.
Gastroenterology ; 163(5): 1391-1406.e24, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35963362

RESUMO

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.


Assuntos
Receptores Acoplados a Proteínas G , Células-Tronco , Humanos , Camundongos , Animais , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , Fluoruracila , Fatores de Crescimento Transformadores/metabolismo
7.
Gastroenterology ; 158(3): 638-651.e8, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31622618

RESUMO

BACKGROUND & AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids. METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells. RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells. CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.


Assuntos
Adenoma/genética , Neoplasias do Colo/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Organoides/patologia , Trombospondinas/genética , Adenoma/patologia , Animais , Sistemas CRISPR-Cas , Neoplasias do Colo/patologia , Fator de Iniciação 3 em Eucariotos/genética , Fusão Gênica , Engenharia Genética , Humanos , Masculino , Camundongos , Modelos Biológicos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Receptores Acoplados a Proteínas G/genética , Proteína Supressora de Tumor p53/genética , Via de Sinalização Wnt
8.
Cancer Sci ; 105(10): 1254-60, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25117675

RESUMO

Non-alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus-independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH-HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18 weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR-122 in non-tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks. Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18 weeks. Expression of miR-122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR-122 is an early event during hepatocarcinogenesis from NASH, and that miR-122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.


Assuntos
Carcinoma Hepatocelular/etiologia , Inativação Gênica , Neoplasias Hepáticas/etiologia , MicroRNAs/genética , Hepatopatia Gordurosa não Alcoólica/complicações , Animais , Sequência de Bases , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Metilação de DNA , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Regiões Promotoras Genéticas
9.
Nat Commun ; 15(1): 2192, 2024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38467634

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.


Assuntos
Carcinoma Ductal Pancreático , Imunoconjugados , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Microambiente Tumoral , Antígenos CD , Moléculas de Adesão Celular , Proteínas Ligadas por GPI
10.
NPJ Precis Oncol ; 8(1): 117, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38789724

RESUMO

E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.

11.
Nat Cell Biol ; 26(10): 1759-1772, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39232216

RESUMO

Human pancreatic cancer is characterized by the molecular diversity encompassing native duct-like and squamous cell-like identities, but mechanisms underlying squamous transdifferentiation have remained elusive. To comprehensively capture the molecular diversity of human pancreatic cancer, we here profiled 65 patient-derived pancreatic cancer organoid lines, including six adenosquamous carcinoma lines. H3K27me3-mediated erasure of the ductal lineage specifiers and hijacking of the TP63-driven squamous-cell programme drove squamous-cell commitment, providing survival benefit in a Wnt-deficient environment and hypoxic conditions. Gene engineering of normal pancreatic duct organoids revealed that GATA6 loss and a Wnt-deficient environment, in concert with genetic or hypoxia-mediated inactivation of KDM6A, facilitate squamous reprogramming, which in turn enhances environmental fitness. EZH2 inhibition counterbalanced the epigenetic bias and curbed the growth of adenosquamous cancer organoids. Our results demonstrate how an adversarial microenvironment dictates the molecular and histological evolution of human pancreatic cancer and provide insights into the principles and significance of lineage conversion in human cancer.


Assuntos
Carcinoma Ductal Pancreático , Proteína Potenciadora do Homólogo 2 de Zeste , Fator de Transcrição GATA6 , Organoides , Neoplasias Pancreáticas , Microambiente Tumoral , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Organoides/metabolismo , Organoides/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Wnt , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Animais , Transdiferenciação Celular/genética , Reprogramação Celular/genética , Epigênese Genética , Histonas/metabolismo , Histonas/genética , Camundongos , Proteínas Supressoras de Tumor , Histona Desmetilases
12.
Cell Rep ; 42(3): 112212, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36870059

RESUMO

Human lung cancer is a constellation of tumors with various histological and molecular properties. To build a preclinical platform that covers this broad disease spectrum, we obtained lung cancer specimens from multiple sources, including sputum and circulating tumor cells, and generated a living biobank consisting of 43 lines of patient-derived lung cancer organoids. The organoids recapitulated the histological and molecular hallmarks of the original tumors. Phenotypic screening of niche factor dependency revealed that EGFR mutations in lung adenocarcinoma are associated with the independence from Wnt ligands. Gene engineering of alveolar organoids reveals that constitutive activation of EGFR-RAS signaling provides Wnt independence. Loss of the alveolar identity gene NKX2-1 confers Wnt dependency, regardless of EGFR signal mutation. Sensitivity to Wnt-targeting therapy can be stratified by the expression status of NKX2-1. Our results highlight the potential of phenotype-driven organoid screening and engineering for the fabrication of therapeutic strategies to combat cancer.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/metabolismo , Bancos de Espécimes Biológicos , Receptores ErbB/metabolismo , Genótipo , Neoplasias Pulmonares/patologia , Organoides/metabolismo , Fenótipo
13.
Cell Rep ; 35(10): 109218, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34038715

RESUMO

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system precludes the development of a clinically relevant SARS-CoV-2 infection model. Here, we establish an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibit indolent growth in a Wnt- and R-spondin-dependent manner. Gene expression, immunofluorescence, and electron microscopy analyses reveal the presence of alveolar cells in alveolospheres. Alveolospheres express ACE2 and allow SARS-CoV-2 to propagate nearly 100,000-fold in 3 days of infection. Whereas lopinavir and nelfinavir, protease inhibitors used for the treatment of human immunodeficiency virus (HIV) infection, have a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, shows an anti-viral effect at the concentration comparable with the circulating drug level. These results demonstrate the validity of the alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/patogenicidade , Esferoides Celulares , Fatores de Tempo , Replicação Viral/efeitos dos fármacos , Via de Sinalização Wnt
14.
Cell Stem Cell ; 23(6): 787-793.e6, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30526881

RESUMO

Cellular diversity that shapes tissue architecture and function is governed by multiple niche signals. Nonetheless, maintaining cellular diversity in human intestinal organoids has been challenging. Based on niche ligands present in the natural stem cell milieu, we establish a refined organoid culture condition for intestinal epithelia that allows human intestinal organoids to concurrently undergo multi-differentiation and self-renewal. High-throughput screening reveals that the combination of insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) enhances the clonogenic capacity and CRISPR-genome engineering efficiency of human intestinal stem cells. The combination equally enables long-term culture of a range of intestinal organoids, including rat small intestinal organoids. Droplet-based single-cell RNA sequencing further illustrates the conservation of the native cellular diversity in human small intestinal organoids cultured with the refined condition. The modified culture protocol outperforms the conventional method and offers a viable strategy for modeling human intestinal tissues and diseases in an in vivo relevant context.


Assuntos
Técnicas de Cultura de Células , Autorrenovação Celular , Intestinos/citologia , Organoides/citologia , Nicho de Células-Tronco , Animais , Células Cultivadas , Humanos , Masculino , Ratos , Ratos Endogâmicos Lew
15.
Cell Stem Cell ; 22(3): 454-467.e6, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29337182

RESUMO

Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression.


Assuntos
Progressão da Doença , Organoides/patologia , Neoplasias Pancreáticas/patologia , Nicho de Células-Tronco , Sistemas CRISPR-Cas/genética , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Fator de Transcrição GATA6/metabolismo , Regulação Neoplásica da Expressão Gênica , Engenharia Genética , Humanos , Ligantes , Neoplasias Pancreáticas/genética , Via de Sinalização Wnt
16.
Sci Rep ; 6: 25311, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27143627

RESUMO

Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established organoids from intestinal tumors of Apc(Min/+) (Min) mice and subjected them to 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-responsive genes were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers.


Assuntos
Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Organoides/efeitos dos fármacos , Animais , Azacitidina/análogos & derivados , Azacitidina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Decitabina , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Análise em Microsséries , Células Tumorais Cultivadas
17.
Cell Stem Cell ; 18(6): 827-838, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27212702

RESUMO

Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development.


Assuntos
Carcinogênese/patologia , Neoplasias Colorretais/patologia , Organoides/patologia , Nicho de Células-Tronco , Animais , Carcinogênese/metabolismo , Neoplasias Colorretais/genética , Fator de Crescimento Epidérmico/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Genoma Humano , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação/genética , Organoides/metabolismo , Nicho de Células-Tronco/genética , Fator de Crescimento Transformador beta/metabolismo , Transplante Heterólogo , Células Tumorais Cultivadas , Proteínas Wnt/metabolismo
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