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1.
Nat Commun ; 14(1): 4631, 2023 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-37532704

RESUMEN

Plasmodium falciparum (Pf) parasite development in liver represents the initial step of the life-cycle in the human host after a Pf-infected mosquito bite. While an attractive stage for life-cycle interruption, understanding of parasite-hepatocyte interaction is inadequate due to limitations of existing in vitro models. We explore the suitability of hepatocyte organoids (HepOrgs) for Pf-development and show that these cells permitted parasite invasion, differentiation and maturation of different Pf strains. Single-cell messenger RNA sequencing (scRNAseq) of Pf-infected HepOrg cells has identified 80 Pf-transcripts upregulated on day 5 post-infection. Transcriptional profile changes are found involving distinct metabolic pathways in hepatocytes with Scavenger Receptor B1 (SR-B1) transcripts highly upregulated. A novel functional involvement in schizont maturation is confirmed in fresh primary hepatocytes. Thus, HepOrgs provide a strong foundation for a versatile in vitro model for Pf liver-stages accommodating basic biological studies and accelerated clinical development of novel tools for malaria control.


Asunto(s)
Malaria Falciparum , Malaria , Humanos , Plasmodium falciparum/genética , Hígado/metabolismo , Hepatocitos/metabolismo , Malaria/parasitología , Organoides/metabolismo , Malaria Falciparum/parasitología
2.
Int J Mol Sci ; 24(6)2023 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-36982422

RESUMEN

Wnt ligands are secreted signaling proteins that display a wide range of biological effects. They play key roles in stimulating Wnt signaling pathways to facilitate processes such as tissue homeostasis and regeneration. Dysregulation of Wnt signaling is a hallmark of many cancers and genetic alterations in various Wnt signaling components, which result in ligand-independent or ligand-dependent hyperactivation of the pathway that have been identified. Recently, research is focusing on the impact of Wnt signaling on the interaction between tumor cells and their micro-environment. This Wnt-mediated crosstalk can act either in a tumor promoting or suppressing fashion. In this review, we comprehensively outline the function of Wnt ligands in different tumor entities and their impact on key phenotypes, including cancer stemness, drug resistance, metastasis, and immune evasion. Lastly, we elaborate approaches to target Wnt ligands in cancer therapy.


Asunto(s)
Neoplasias , Proteínas Wnt , Humanos , Ligandos , Proteínas Wnt/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Vía de Señalización Wnt/genética , Transporte Biológico , Microambiente Tumoral
3.
Nat Commun ; 13(1): 3135, 2022 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-35668108

RESUMEN

Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression of LGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.


Asunto(s)
Neoplasias Colorrectales , Organoides , Neoplasias Colorrectales/genética , Humanos , Organoides/patología , Fenotipo , Transducción de Señal
4.
Biomolecules ; 12(3)2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35327645

RESUMEN

Targeting cancer hallmarks is a cardinal strategy to improve antineoplastic treatment. However, cross-talk between signaling pathways and key oncogenic processes frequently convey resistance to targeted therapies. The p53 and Wnt pathway play vital roles for the biology of many tumors, as they are critically involved in cancer onset and progression. Over recent decades, a high level of interaction between the two pathways has been revealed. Here, we provide a comprehensive overview of molecular interactions between the p53 and Wnt pathway discovered in cancer, including complex feedback loops and reciprocal transactivation. The mutational landscape of genes associated with p53 and Wnt signaling is described, including mutual exclusive and co-occurring genetic alterations. Finally, we summarize the functional consequences of this cross-talk for cancer phenotypes, such as invasiveness, metastasis or drug resistance, and discuss potential strategies to pharmacologically target the p53-Wnt interaction.


Asunto(s)
Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Humanos , Neoplasias/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismo
5.
Stem Cell Reports ; 16(9): 2379-2394, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34358453

RESUMEN

The skin epidermis is a highly compartmentalized tissue consisting of a cornifying epithelium called the interfollicular epidermis (IFE) and associated hair follicles (HFs). Several stem cell populations have been described that mark specific compartments in the skin but none of them is specific to the IFE. Here, we identify Troy as a marker of IFE and HF infundibulum basal layer cells in developing and adult human and mouse epidermis. Genetic lineage-tracing experiments demonstrate that Troy-expressing basal cells contribute to long-term renewal of all layers of the cornifying epithelium. Single-cell transcriptomics and organoid assays of Troy-expressing cells, as well as their progeny, confirmed stem cell identity as well as the ability to generate differentiating daughter cells. In conclusion, we define Troy as a marker of epidermal basal cells that govern interfollicular epidermal renewal and cornification.


Asunto(s)
Diferenciación Celular/genética , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Folículo Piloso/embriología , Folículo Piloso/metabolismo , Organogénesis/genética , Receptores del Factor de Necrosis Tumoral/genética , Animales , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Inmunofenotipificación , Ratones , Organoides , Receptores del Factor de Necrosis Tumoral/metabolismo , Análisis de la Célula Individual/métodos
6.
EMBO J ; 39(18): e103932, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32965059

RESUMEN

Wnt/ß-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce ß-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing ß-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.


Asunto(s)
Quinasa de la Caseína I/metabolismo , Neoplasias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , Quinasa de la Caseína I/genética , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , beta Catenina/genética , beta Catenina/metabolismo
8.
Nat Cell Biol ; 21(8): 924-932, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31358966

RESUMEN

The sebaceous gland (SG) is an essential component of the skin, and SG dysfunction is debilitating1,2. Yet, the cellular bases for its origin, development and subsequent maintenance remain poorly understood. Here, we apply large-scale quantitative fate mapping to define the patterns of cell fate behaviour during SG development and maintenance. We show that the SG develops from a defined number of lineage-restricted progenitors that undergo a programme of independent and stochastic cell fate decisions. Following an expansion phase, equipotent progenitors transition into a phase of homeostatic turnover, which is correlated with changes in the mechanical properties of the stroma and spatial restrictions on gland size. Expression of the oncogene KrasG12D results in a release from these constraints and unbridled gland expansion. Quantitative clonal fate analysis reveals that, during this phase, the primary effect of the Kras oncogene is to drive a constant fate bias with little effect on cell division rates. These findings provide insight into the developmental programme of the SG, as well as the mechanisms that drive tumour progression and gland dysfunction.


Asunto(s)
Proliferación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/inmunología , Homeostasis/fisiología , Células Madre/citología , Animales , Progresión de la Enfermedad , Ratones Transgénicos
9.
Proc Natl Acad Sci U S A ; 116(29): 14630-14638, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31253707

RESUMEN

Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.


Asunto(s)
Células Madre Adultas/fisiología , Técnicas de Cultivo de Célula/métodos , Epidermis/fisiología , Queratinocitos/fisiología , Organoides/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Técnicas de Sustitución del Gen , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Acoplados a Proteínas G/genética , Factores de Tiempo
10.
Vet Dermatol ; 29(5): 375-e126, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29963730

RESUMEN

BACKGROUND: Perturbations of epidermal and follicular homeostasis have been attributed to a variety of skin diseases affecting dogs. The availability of an in vitro system to investigate these diseases is important to understand underlying pathomechanisms. OBJECTIVES: To establish an accurate and reliable in vitro 3D system of canine keratinocyte organoids to lay the basis for studying functional defects in interfollicular epidermis (IFE) and hair follicle (HF) morphogenesis, reconstitution and differentiation that lead to alopecic and epidermal diseases. ANIMALS: Skin biopsies were obtained from freshly euthanized dogs of different breeds with no skin abnormalities. METHODS: Cells derived from microdissected IFE and HFs were seeded in Matrigel and keratinocyte organoids were grown and characterized using immunohistochemistry, RT-qPCR and RNA sequencing. RESULTS: Both organoid lines develop into a basal IFE-like cell type. Gene and protein expression analysis revealed high mRNA and protein levels of keratins 5 and 14, IFE differentiation markers and intercellular molecules. Key markers of HF stem cells were lacking. Withdrawal of growth factors resulted in upregulation of markers such as KRT16, Involucrin, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. CONCLUSION AND CLINICAL IMPORTANCE: Our 3D in vitro culture system provides the basis to explore epidermal function, to investigate the culture conditions necessary for the development of organoids with a HF signature and to address cutaneous disorders in dogs. However, for induction of HF signatures or hair growth, addition of different growth factors or co-culture with dermal papilla will be required.


Asunto(s)
Perros/anatomía & histología , Queratinocitos/citología , Técnicas de Cultivo de Órganos/veterinaria , Animales , Biopsia/veterinaria , Células Cultivadas/citología , Células Epidérmicas , Queratinocitos/patología , Técnicas de Cultivo de Órganos/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Análisis de Secuencia de ARN/veterinaria , Piel/citología , Piel/patología , Enfermedades de la Piel/patología , Enfermedades de la Piel/veterinaria
11.
Nat Microbiol ; 3(7): 814-823, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29946163

RESUMEN

Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.


Asunto(s)
Criptosporidiosis/genética , Cryptosporidium/patogenicidad , Perfilación de la Expresión Génica/métodos , Organoides/parasitología , Criptosporidiosis/parasitología , Cryptosporidium/crecimiento & desarrollo , Regulación de la Expresión Génica , Humanos , Intestino Delgado/parasitología , Pulmón/parasitología , Modelos Biológicos , Técnicas de Cultivo de Órganos , Análisis de Secuencia de ARN , Análisis Espacio-Temporal
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