RESUMO
Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.
Assuntos
Células Epiteliais , Fatores de Transcrição Forkhead , Organoides , Timo , Animais , Organoides/citologia , Organoides/metabolismo , Timo/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Camundongos , Diferenciação Celular , Camundongos Nus , Linfócitos T/citologia , Linfócitos T/metabolismo , Camundongos Endogâmicos C57BL , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.
Assuntos
Túnica Conjuntiva , Células Caliciformes , Humanos , Camundongos , Animais , Túnica Conjuntiva/metabolismo , Células Caliciformes/metabolismo , Epitélio , Interleucina-13 , Homeostase , OrganoidesRESUMO
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/patologiaRESUMO
Cervical cancer is a common gynecological malignancy often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study the cervical epithelium and the cancers derived thereof. Here we describe a long-term culturing protocol for ecto- and endocervical epithelia that generates 3D organoids that stably recapitulate the two tissues of origin. As evidenced for HSV-1, organoid-based cervical models may serve to study sexually transmitted infections. Starting from Pap brush material, a small biobank of tumoroids derived from affected individuals was established that retained the causative human papillomavirus (HPV) genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutic agents and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches.
Assuntos
Neoplasias do Colo do Útero , Animais , Carcinogênese , Epitélio , Feminino , Humanos , Camundongos , Organoides , PapillomaviridaeRESUMO
The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland.
Assuntos
Síndromes do Olho Seco , Aparelho Lacrimal , Animais , Humanos , Camundongos , Organoides , Células-Tronco , LágrimasRESUMO
Pheochromocytomas and paragangliomas (PPGLs) caused by mutations in the B-subunit of the succinate dehydrogenase (SDHB) have the highest metastatic rate among PPGLs, and effective systemic therapy is lacking. To unravel underlying pathogenic mechanisms, and to evaluate therapeutic strategies, suitable in vivo models are needed. The available systemic Sdhb knock-out mice cannot model the human PPGL phenotype: heterozygous Sdhb mice lack a disease phenotype, and homozygous Sdhb mice are embryonically lethal. Using CRISPR/cas9 technology, we introduced a protein-truncating germline lesion into the zebrafish sdhb gene. Heterozygous sdhb mutants were viable and displayed no obvious morphological or developmental defects. Homozygous sdhb larvae were viable, but exhibited a decreased lifespan. Morphological analysis revealed incompletely or non-inflated swim bladders in homozygous sdhb mutants at day 6. Although no differences in number and ultrastructure of the mitochondria were observed. Clear defects in energy metabolism and swimming behavior were observed in homozygous sdhb mutant larvae. Functional and metabolomic analyses revealed decreased mitochondrial complex 2 activity and significant succinate accumulation in the homozygous sdhb mutant larvae, mimicking the metabolic effects observed in SDHB-associated PPGLs. This is the first study to present a vertebrate animal model that mimics metabolic effects of SDHB-associated PPGLs. This model will be useful in unraveling pathomechanisms behind SDHB-associated PPGLs. We can now study the metabolic effects of sdhb disruption during different developmental stages and develop screening assays to identify novel therapeutic targets in vivo. Besides oncological syndromes, our model might also be useful for pediatric mitochondrial disease caused by loss of the SDHB gene.
Assuntos
Larva/metabolismo , Paraganglioma/genética , Succinato Desidrogenase/metabolismo , Animais , Humanos , Peixe-ZebraRESUMO
High-grade serous ovarian cancer (HG-SOC)-often referred to as a "silent killer"-is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients.
Assuntos
Sistemas CRISPR-Cas/genética , Organoides , Neoplasias Ovarianas/etiologia , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína 9 Associada à CRISPR , Epitélio/patologia , Tubas Uterinas/patologia , Feminino , Edição de Genes/métodos , Camundongos , Mutação , Neurofibromatose 1/genética , Técnicas de Cultura de Órgãos/métodos , Organoides/efeitos dos fármacos , Organoides/fisiopatologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Ovário/patologia , PTEN Fosfo-Hidrolase/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs. SIGNIFICANCE: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Mucosa Bucal/patologia , Organoides/patologia , Medicina de Precisão/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Animais , Carboplatina/administração & dosagem , Cetuximab/administração & dosagem , Quimiorradioterapia , Cisplatino/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/efeitos da radiação , Organoides/efeitos dos fármacos , Organoides/efeitos da radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.
Assuntos
Organoides/patologia , Neoplasias Ovarianas/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Genômica , Xenoenxertos , Humanos , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Medicina de PrecisãoRESUMO
Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.
Assuntos
Organoides/patologia , Neoplasias da Bexiga Urinária/patologia , Animais , Camundongos , Medicina de PrecisãoRESUMO
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 XenoenxertoRESUMO
Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
Assuntos
Neoplasias da Mama/patologia , Heterogeneidade Genética , Organoides/patologia , Bancos de Tecidos , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Camundongos , Camundongos Nus , Organoides/efeitos dos fármacos , Medicina de Precisão/métodosRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.1000539.].
RESUMO
Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.
Assuntos
Neoplasias do Colo/etiologia , Regulação da Expressão Gênica , Integrases/genética , Camundongos Transgênicos , Adenoma/etiologia , Adenoma/metabolismo , Adenoma/patologia , Animais , Biomarcadores Tumorais , Anidrase Carbônica I/genética , Anidrase Carbônica I/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Técnicas de Introdução de Genes , Marcação de Genes , Genes APC , Genes ras , Loci Gênicos , Humanos , Imuno-Histoquímica , Integrases/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Mutação , Especificidade de Órgãos/genética , PesquisaRESUMO
Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration.
Assuntos
Linhagem da Célula , Enterócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Desdiferenciação Celular , Linhagem Celular , Proliferação de Células , Enterócitos/patologia , Integrases/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Mutação/genética , Organoides , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Regeneração/genética , Análise de Célula Única , beta-Galactosidase/metabolismoRESUMO
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/metabolismoRESUMO
Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos/genética , Anormalidades Congênitas/genética , Rearranjo Gênico , Genoma Humano , Mutação em Linhagem Germinativa , Animais , Pontos de Quebra do Cromossomo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Células HEK293 , Humanos , MicroRNAs/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Peixe-ZebraRESUMO
ENDOGLIN (ENG) is a co-receptor for transforming growth factor-ß (TGF-ß) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Endoglina , Citometria de Fluxo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Neovascularização Fisiológica/genéticaRESUMO
RATIONALE: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. OBJECTIVE: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. METHODS AND RESULTS: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1(+) lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C-mediated lymphangiogenesis in a corneal micropocket assay. CONCLUSIONS: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1(+) lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Endotélio Linfático/irrigação sanguínea , Endotélio Linfático/metabolismo , Linfangiogênese/fisiologia , Vasos Linfáticos/embriologia , Vasos Linfáticos/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Córnea/irrigação sanguínea , Córnea/citologia , Córnea/metabolismo , Endotélio Linfático/citologia , Humanos , Linfangiogênese/genética , Camundongos , Camundongos Knockout , Ligação Proteica/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/ß-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/ß-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and ß-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/ß-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a ß-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published ß-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.