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1.
Science ; 382(6669): 451-458, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37883554

RESUMO

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.


Assuntos
Sistemas CRISPR-Cas , Linhagem da Célula , Células Enteroendócrinas , Regulação da Expressão Gênica , Proteínas Repressoras , Dedos de Zinco , Humanos , Diferenciação Celular/genética , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Organoides , Adulto , Linhagem da Célula/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
2.
Cancer Res Commun ; 3(10): 2158-2169, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37823774

RESUMO

Novel therapeutic strategies are urgently needed for patients with high-risk Ewing sarcoma and for the reduction of severe side effects for all patients. Immunotherapy may fill this need, but its successful application has been hampered by a lack of knowledge on the composition and function of the Ewing sarcoma immune microenvironment. Here, we explore the immune microenvironment of Ewing sarcoma, by single-cell RNA sequencing of 18 Ewing sarcoma primary tissue samples. Ewing sarcoma is infiltrated by natural killer, T, and B cells, dendritic cells, and immunosuppressive macrophages. Ewing sarcoma-associated T cells show various degrees of dysfunction. The antigen-presenting cells found in Ewing sarcoma lack costimulatory gene expression, implying functional impairment. Interaction analysis reveals a clear role for Ewing sarcoma tumor cells in turning the Ewing sarcoma immune microenvironment into an immunosuppressive niche. These results provide novel insights into the functional state of immune cells in the Ewing sarcoma tumor microenvironment and suggest mechanisms by which Ewing sarcoma tumor cells interact with, and shape, the immune microenvironment. SIGNIFICANCE: This study is the first presenting a detailed analysis of the Ewing sarcoma microenvironment using single-cell RNA sequencing. We provide novel insight into the functional state of immune cells and suggests mechanisms by which Ewing tumor cells interact with, and shape, their immune microenvironment. These insights provide help in understanding the failures and successes of immunotherapy in Ewing sarcoma and may guide novel targeted (immuno) therapeutic approaches.


Assuntos
Tumores Neuroectodérmicos Primitivos Periféricos , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/genética , Análise da Expressão Gênica de Célula Única , Linhagem Celular Tumoral , Células Apresentadoras de Antígenos/metabolismo , Microambiente Tumoral/genética
3.
Oncogene ; 42(20): 1661-1671, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37020038

RESUMO

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.


Assuntos
Neoplasias Encefálicas , Tumor Rabdoide , Teratoma , Criança , Humanos , Teratoma/tratamento farmacológico , Teratoma/genética , Proteína SMARCB1/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Receptores Notch , Epigenômica , DNA Helicases , Proteínas Nucleares , Fatores de Transcrição/genética
4.
EMBO Mol Med ; 14(10): e16001, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-35916583

RESUMO

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.


Assuntos
Organoides , Rabdomiossarcoma , Criança , Humanos , Organoides/patologia , Rabdomiossarcoma/diagnóstico , Rabdomiossarcoma/patologia
5.
Nat Cancer ; 3(4): 418-436, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35469014

RESUMO

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.


Assuntos
Anticorpos Biespecíficos , Neoplasias Epiteliais e Glandulares , Anticorpos Biespecíficos/farmacologia , Receptores ErbB/metabolismo , Humanos , Imidazóis , Neoplasias Epiteliais e Glandulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismo
6.
Cancers (Basel) ; 13(23)2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34885181

RESUMO

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft tissue sarcoma with a lack of effective treatment options and a poor prognosis. DSRCT is characterized by a chromosomal translocation, resulting in the EWSR1-WT1 gene fusion. The molecular mechanisms driving DSRCT are poorly understood, and a paucity of preclinical models hampers DSRCT research. Here, we establish a novel primary patient-derived DSRCT in vitro model, recapitulating the original tumor. We find that EWSR1-WT1 expression affects cell shape and cell survival, and we identify downstream target genes of the EWSR1-WT1 fusion. Additionally, this preclinical in vitro model allows for medium-throughput drug screening. We discover sensitivity to several drugs, including compounds targeting RTKs. MERTK, which has been described as a therapeutic target for several malignancies, correlates with EWSR1-WT1 expression. Inhibition of MERTK with the small-molecule inhibitor UNC2025 results in reduced proliferation of DSRCT cells in vitro, suggesting MERTK as a therapeutic target in DSRCT. This study underscores the usefulness of preclinical in vitro models for studying molecular mechanisms and potential therapeutic options.

7.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33500353

RESUMO

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.


Assuntos
Glândulas Suprarrenais/patologia , Neuroblastoma/patologia , Células de Schwann/patologia , Análise de Célula Única , Medula Suprarrenal/patologia , Animais , Agregação Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Estadiamento de Neoplasias , Células-Tronco Neurais , Neuroblastoma/genética , Fenótipo
8.
Proc Natl Acad Sci U S A ; 116(52): 26599-26605, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31843916

RESUMO

Cycling intestinal Lgr5+ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

9.
EMBO J ; 38(15): e101654, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31282586

RESUMO

Patient-derived tumour xenografts and tumour organoids have become important preclinical model systems for cancer research. Both models maintain key features from their parental tumours, such as genetic and phenotypic heterogeneity, which allows them to be used for a wide spectrum of applications. In contrast to patient-derived xenografts, organoids can be established and expanded with high efficiency from primary patient material. On the other hand, xenografts retain tumour-stroma interactions, which are known to contribute to tumorigenesis. In this review, we discuss recent advances in patient-derived tumour xenograft and tumour organoid model systems and compare their promises and challenges as preclinical models in cancer research.


Assuntos
Neoplasias/patologia , Organoides/patologia , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Biológicos , Organoides/citologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Genet ; 51(5): 824-834, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036964

RESUMO

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.


Assuntos
Instabilidade Cromossômica , Neoplasias Colorretais/genética , Aneuploidia , Linhagem Celular Tumoral , Segregação de Cromossomos , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Humanos , Imageamento Tridimensional , Cariótipo , Cariotipagem , Instabilidade de Microssatélites , Mitose/genética , Mutação , Organoides/patologia , Análise de Célula Única
11.
Nature ; 556(7702): 457-462, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29643510

RESUMO

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.


Assuntos
Antineoplásicos/farmacologia , Células Clonais/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Evolução Molecular , Mutação , Análise de Célula Única , Proliferação de Células , Células Clonais/metabolismo , Células Clonais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Metilação de DNA , Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Taxa de Mutação , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Transcriptoma
12.
Proc Natl Acad Sci U S A ; 115(4): E610-E619, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29311336

RESUMO

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.


Assuntos
Ventrículos Laterais/citologia , Células-Tronco Neurais/fisiologia , Nicho de Células-Tronco , Animais , Linhagem da Célula , Proliferação de Células , Camundongos , Neurogênese , Receptores do Fator de Necrose Tumoral/metabolismo , Análise de Célula Única , Transcriptoma
13.
Gastroenterology ; 153(4): 1040-1053.e4, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28716720

RESUMO

BACKGROUND & AIMS: Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. METHODS: We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44-/-, Cd44s/s, or Cd44v4-10/v4-10 mice). RESULTS: Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44-/- mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. CONCLUSIONS: In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors.


Assuntos
Adenoma/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Receptores de Hialuronatos/metabolismo , Neoplasias Intestinais/metabolismo , Intestinos/enzimologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Regeneração , Células-Tronco/enzimologia , Adenoma/genética , Adenoma/patologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Genótipo , Fator de Crescimento de Hepatócito/farmacologia , Homeostase , Receptores de Hialuronatos/genética , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Intestinos/efeitos da radiação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Proteínas Proto-Oncogênicas c-met/genética , Regeneração/efeitos dos fármacos , Regeneração/efeitos da radiação , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Células-Tronco/efeitos da radiação , Fatores de Tempo , Técnicas de Cultura de Tecidos , Carga Tumoral
14.
Science ; 357(6346): 55-60, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28684519

RESUMO

The spread of cancer cells from primary tumors to regional lymph nodes is often associated with reduced survival. One prevailing model to explain this association posits that fatal, distant metastases are seeded by lymph node metastases. This view provides a mechanistic basis for the TNM staging system and is the rationale for surgical resection of tumor-draining lymph nodes. Here we examine the evolutionary relationship between primary tumor, lymph node, and distant metastases in human colorectal cancer. Studying 213 archival biopsy samples from 17 patients, we used somatic variants in hypermutable DNA regions to reconstruct high-confidence phylogenetic trees. We found that in 65% of cases, lymphatic and distant metastases arose from independent subclones in the primary tumor, whereas in 35% of cases they shared common subclonal origin. Therefore, two different lineage relationships between lymphatic and distant metastases exist in colorectal cancer.


Assuntos
Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , Linfonodos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Neoplasias Colorretais/genética , DNA/genética , Humanos , Mutação INDEL , Metástase Linfática , Modelos Biológicos , Estadiamento de Neoplasias , Filogenia , Prognóstico
15.
Cell Rep ; 18(1): 263-274, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28052255

RESUMO

Diseases at the molecular level are complex and patient dependent, necessitating development of strategies that enable precision treatment to optimize clinical outcomes. Organoid technology has recently been shown to have the potential to recapitulate the in vivo characteristics of the original individual's tissue in a three-dimensional in vitro culture system. Here, we present a quantitative mass-spectrometry-based proteomic analysis and a comparative transcriptomic analysis of human colorectal tumor and healthy organoids derived, in parallel, from seven patients. Although gene and protein signatures can be derived to distinguish the tumor organoid population from healthy organoids, our data clearly reveal that each patient possesses a distinct organoid signature at the proteomic level. We demonstrate that a personalized patient-specific organoid proteome profile can be related to the diagnosis of a patient and with future development contribute to the generation of personalized therapies.


Assuntos
Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Variação Genética , Organoides/patologia , Proteoma/metabolismo , Proteômica/métodos , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Transcriptoma/genética , Via de Sinalização Wnt
16.
Nature ; 538(7624): 260-264, 2016 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-27698416

RESUMO

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.


Assuntos
Células-Tronco Adultas/metabolismo , Envelhecimento/genética , Acúmulo de Mutações , Taxa de Mutação , Especificidade de Órgãos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Colo/metabolismo , Análise Mutacional de DNA , Feminino , Genes Neoplásicos/genética , Humanos , Incidência , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Multipotentes/metabolismo , Neoplasias/epidemiologia , Neoplasias/genética , Organoides/metabolismo , Mutação Puntual/genética , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 112(43): 13308-11, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26460009

RESUMO

Tumor organoids are 3D cultures of cancer cells. They can be derived from the tumor of each individual patient, thereby providing an attractive ex vivo assay to tailor treatment. Using patient-derived tumor organoids for this purpose requires that organoids derived from biopsies maintain the genetic diversity of the in vivo tumor. In this study tumor biopsies were obtained from 14 patients with metastatic colorectal cancer (i) to test the feasibility of organoid culture from metastatic biopsy specimens and (ii) to compare the genetic diversity of patient-derived tumor organoids and the original tumor biopsy. Genetic analysis was performed using SOLiD sequencing for 1,977 cancer-relevant genes. Copy number profiles were generated from sequencing data using CopywriteR. Here we demonstrate that organoid cultures can be established from tumor biopsies of patients with metastatic colorectal cancer with a success rate of 71%. Genetic analysis showed that organoids reflect the metastasis from which they were derived. Ninety percent of somatic mutations were shared between organoids and biopsies from the same patient, and the DNA copy number profiles of organoids and the corresponding original tumor show a correlation of 0.89. Most importantly, none of the mutations that were found exclusively in either the tumor or organoid culture are in driver genes or genes amenable for drug targeting. These findings support further exploration of patient-derived organoids as an ex vivo platform to personalize anticancer treatment.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/genética , Variação Genética/genética , Metástase Neoplásica/genética , Organoides/citologia , Organoides/crescimento & desenvolvimento , Protocolos Antineoplásicos/normas , Sequência de Bases , Neoplasias Colorretais/tratamento farmacológico , Genes Neoplásicos/genética , Humanos , Dados de Sequência Molecular , Organoides/química , Medicina de Precisão/métodos , Análise de Sequência de DNA
18.
Cell ; 161(4): 933-45, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25957691

RESUMO

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.


Assuntos
Bancos de Espécimes Biológicos , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Organoides , Neoplasias Colorretais/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Oncogênicas/metabolismo , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Medicina de Precisão , Ubiquitina-Proteína Ligases
19.
Nature ; 521(7550): 43-7, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25924068

RESUMO

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Intestinos/patologia , Mutação/genética , Organoides/metabolismo , Organoides/patologia , Células-Tronco/patologia , Aneuploidia , Animais , Sistemas CRISPR-Cas , Criança , Pré-Escolar , Neoplasias Colorretais/metabolismo , Feminino , Genes APC , Genes p53/genética , Xenoenxertos , Humanos , Imidazóis , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transplante de Neoplasias , Piperazinas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Smad4/deficiência , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo
20.
J Pathol ; 236(2): 155-64, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25712196

RESUMO

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Deleção de Genes , Mutação em Linhagem Germinativa/genética , Proteínas Supressoras de Tumor/genética , Polipose Adenomatosa do Colo/metabolismo , Adulto , Estudos de Casos e Controles , Cromossomos Humanos Par 9/genética , Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA/genética , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Fases de Leitura Aberta/genética , Proteínas Supressoras de Tumor/metabolismo
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