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1.
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
2.
EMBO J ; 38(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30643021

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Fibrose Cística/patologia , Células Epiteliais/patologia , Técnicas de Cultura de Órgãos/métodos , Organoides/patologia , Infecções por Vírus Respiratório Sincicial/patologia , Sistema Respiratório/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Cultivadas , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Organoides/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Sistema Respiratório/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Cancer Cell ; 41(12): 2083-2099.e9, 2023 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-38086335

RESUMO

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Treatment options for patients with NENs are limited, in part due to lack of accurate models. We establish patient-derived tumor organoids (PDTOs) from pulmonary NETs and derive PDTOs from an understudied subtype of NEC, large cell neuroendocrine carcinoma (LCNEC), arising from multiple body sites. PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through hypothesis-driven drug sensitivity analyses, we identify ASCL1 as a potential biomarker for response of LCNEC to treatment with BCL-2 inhibitors. Additionally, we discover a dependency on EGF in pulmonary NET PDTOs. Consistent with these findings, we find that, in an independent cohort, approximately 50% of pulmonary NETs express EGFR. This study identifies an actionable vulnerability for a subset of pulmonary NETs, emphasizing the utility of these PDTO models.


Assuntos
Carcinoma Neuroendócrino , Neoplasias Pulmonares , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Humanos , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Carcinoma Neuroendócrino/tratamento farmacológico , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pancreáticas/patologia
4.
J Cell Mol Med ; 16(10): 2379-86, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22348515

RESUMO

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA-155 (miR-155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre-miR-155, anti-miR-155 or control-miR (ctrl-miR), followed by scratch- and transwell-assays. These functional assays displayed that miR-155 over-expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR-155 transfected cells was collected and zymography analysis showed a significant decrease in MMP-2 and MMP-9 activities. The predicted 3'-UTR of MMP-16, an activator of MMP-2 and -9, was cloned into the pMIR-REPORT vector and luciferase assays were performed. Introduction of miR-155 significantly reduced luciferase activity which could be abolished by cotransfection with anti-miR-155 or target site mutagenesis. By using MMP-16 siRNA to reduce MMP-16 levels or by using an MMP-16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP-16, miR-155 efficiently inhibits cell migration via a reduction in MMP-2 and -9 activities. Our study shows that miR-155 might be used to improve local retention of hCMPCs after intramyocardial delivery.


Assuntos
Movimento Celular , Metaloproteinase 16 da Matriz/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Clonagem Molecular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção
5.
Oncogene ; 41(21): 2932-2944, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35437308

RESUMO

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.


Assuntos
Neoplasias da Mama , Carcinoma Lobular , Animais , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patologia , Ciclo Celular/genética , Feminino , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Camundongos , Invasividade Neoplásica , Recidiva Local de Neoplasia
6.
J Cell Biochem ; 105(5): 1228-39, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18821573

RESUMO

Here, we report the isolation of progenitor cells from pig skeletal muscle tissue fragments. Muscle progenitor cells were stimulated to migrate from protease-digested tissue fragments and cultured in the presence of 5 ng/ml basic fibroblast growth factor. The cells showed a sustained long-term expansion capacity (>120 population doublings) while maintaining a normal karyotype. The proliferating progenitor cells expressed PAX3, DESMIN, SMOOTH MUSCLE ACTIN, VIMENTIN, CD31, NANOG and THY-1, while MYF5 and OCT3/4 were only expressed in the lower or higher cell passages. Myogenic differentiation of porcine progenitor cells was induced in a coculture system with murine C2C12 myoblasts resulting in the formation of myotubes. Further, the cells showed adipogenic and osteogenic lineage commitment when exposed to specific differentiation conditions. These observations were determined by Von Kossa and Oil-Red-O staining and confirmed by quantitative RT-PCR analysis. In conclusion, the porcine muscle-derived progenitor cells possess long-term expansion capacity and a multilineage differentiation capacity.


Assuntos
Fibras Musculares Esqueléticas/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Citometria de Fluxo , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Células-Tronco/metabolismo , Suínos
7.
Nat Med ; 23(1): 60-68, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869803

RESUMO

Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.


Assuntos
Anticorpos/farmacologia , Carcinoma Ductal Pancreático/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Receptores Frizzled/antagonistas & inibidores , Proteínas Oncogênicas/genética , Neoplasias Pancreáticas/genética , Via de Sinalização Wnt/efeitos dos fármacos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias Colorretais/metabolismo , Citometria de Fluxo , Imunofluorescência , Receptores Frizzled/metabolismo , Humanos , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Transplante de Neoplasias , Organoides/efeitos dos fármacos , Organoides/metabolismo , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases , Via de Sinalização Wnt/genética
10.
Cardiovasc Res ; 93(4): 655-65, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22227154

RESUMO

AIMS: Angiogenesis is a critical component of many pathological conditions in adult tissues and is essential for embryonic development. MicroRNAs are indispensable for normal vascular development, but their exact role in regulating angiogenesis remains unresolved. Previously, we have observed that miR-214 is differentially expressed in compensatory arteriogenesis. Here, we investigated the potential role of miR-214 in the process of angiogenesis. METHODS AND RESULTS: miR-214 is expressed in all major vascular cell types, and modulation of miR-214 levels in endothelial cells significantly affected tubular sprouting. In vivo silencing of miR-214 enhanced the formation of a perfused vascular network in implanted Matrigel plugs and retinal developmental angiogenesis in mice. miR-214 directly targets Quaking, a protein critical for vascular development. Quaking knockdown reduced pro-angiogenic growth factor expression and inhibited endothelial cell sprouting similar to miR-214 overexpression. In accordance, silencing of miR-214 increased the secretion of pro-angiogenic growth factors, including vascular endothelial growth factor, and enhanced the pro-angiogenic action of the endothelial cell-derived conditioned medium, whereas miR-214 overexpression had the opposite effect. CONCLUSION: Here, we report a novel role for miR-214 in regulating angiogenesis and identify Quaking as a direct target of miR-214. The anti-angiogenic effect of miR-214 is mediated through the down-regulation of Quaking and pro-angiogenic growth factor expression. This study presents miR-214 as a potential important target for pro- or anti-angiogenic therapies.


Assuntos
Proteínas Angiogênicas/metabolismo , Regulação para Baixo/fisiologia , MicroRNAs/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas de Ligação a RNA/genética , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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