RESUMO
Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.
Assuntos
Proteínas Hedgehog , Células-Tronco Embrionárias Murinas , Camundongos , Animais , Proteínas Hedgehog/metabolismo , Glândulas Mamárias Animais , Células Epiteliais , Diferenciação Celular , OrganoidesRESUMO
Glioblastoma (GBM) remains lethal with no effective treatments. Despite the comprehensive identification of commonly perturbed molecular pathways, little is known about the disease's etiology, particularly in early stages. Several studies indicate that GBM is initiated in neural progenitor and/or stem cells. Here, we report that differentiated astrocytes are susceptible to GBM development when initiated by perturbation of the RB pathway, which induces a progenitor phenotype. In vitro and in vivo inactivation of Rb tumor suppression (TS) induces cortical astrocytes to proliferate rapidly, express progenitor markers, repress differentiation markers, and form self-renewing neurospheres that are susceptible to multi-lineage differentiation. This phenotype is sufficient to cause grade II astrocytomas which stochastically progress to GBM. Together with previous findings, these results demonstrate that cell susceptibility to GBM depends on the initiating driver.
RESUMO
Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.
Assuntos
Adenocarcinoma/terapia , Mesotelina/administração & dosagem , Mesotelioma/terapia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose , Proliferação de Células , Feminino , Engenharia Genética , Humanos , Masculino , Mesotelina/genética , Mesotelina/metabolismo , Mesotelioma/genética , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.
Assuntos
Proliferação de Células , Queratina-18/metabolismo , Proteína do Retinoblastoma/fisiologia , Linfócitos T/citologia , Timo/citologia , Animais , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Proteína do Retinoblastoma/genética , Células Estromais/metabolismo , Linfócitos T/imunologia , Timo/imunologia , Timo/metabolismo , TransgenesRESUMO
Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution.
Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Queratina-18/biossíntese , Queratina-18/genética , Queratina-19/biossíntese , Queratina-19/genética , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos , Proteína do Retinoblastoma/genéticaRESUMO
Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.
Assuntos
Astrocitoma/etiologia , Evolução Biológica , Modelos Animais de Doenças , Engenharia Genética/métodos , Glioblastoma/etiologia , Animais , Sequência de Bases , Progressão da Doença , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genéticaRESUMO
The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway, which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor sites in MITF or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Olho/metabolismo , Cor de Cabelo/genética , Fator de Transcrição Associado à Microftalmia/genética , Transgenes/genética , Processamento Alternativo , Animais , Sítios de Ligação/genética , Éxons/genética , Olho/crescimento & desenvolvimento , Feminino , Deleção de Genes , Masculino , Melanócitos/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Miocárdio/metabolismo , Fenótipo , Fosforilação , Serina/genética , Serina/metabolismo , Pele/crescimento & desenvolvimento , Pele/metabolismoRESUMO
Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two 'mimic' mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.