RESUMEN
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.
Asunto(s)
Bancos de Muestras Biológicas , Neoplasias Colorrectales/patología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Organoides , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Oncogénicas/metabolismo , Técnicas de Cultivo de Órganos , Organoides/efectos de los fármacos , Medicina de Precisión , Ubiquitina-Proteína LigasasRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Carcinoma Neuroendocrino , Neoplasias Pulmonares , Tumores Neuroendocrinos , Neoplasias Pancreáticas , Humanos , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/metabolismo , Carcinoma Neuroendocrino/tratamiento farmacológico , Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pancreáticas/patologíaRESUMEN
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.
Asunto(s)
Movimiento Celular , Metaloproteinasa 16 de la Matriz/metabolismo , MicroARNs/metabolismo , Miocitos Cardíacos/citología , Células Madre/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Clonación Molecular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , MicroARNs/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
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.
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Neoplasias de la Mama , Carcinoma Lobular , Animales , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patología , Ciclo Celular/genética , Femenino , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Ratones , Invasividad Neoplásica , Recurrencia Local de NeoplasiaRESUMEN
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.
Asunto(s)
Fibras Musculares Esqueléticas/citología , Células Madre/citología , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Citometría de Flujo , Ratones , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Células Madre/metabolismo , PorcinosRESUMEN
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.
Asunto(s)
Anticuerpos/farmacología , Carcinoma Ductal Pancreático/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Receptores Frizzled/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Neoplasias Pancreáticas/genética , Vía de Señalización Wnt/efectos de los fármacos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias Colorrectales/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Receptores Frizzled/metabolismo , Humanos , Ratones , Ratones SCID , Terapia Molecular Dirigida , Trasplante de Neoplasias , Organoides/efectos de los fármacos , Organoides/metabolismo , Neoplasias Pancreáticas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas , Vía de Señalización Wnt/genéticaRESUMEN
This corrects the article DOI: 10.1038/nm.4219.
RESUMEN
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.