RESUMEN
BACKGROUND: Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed. METHODS: We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS. FINDINGS: We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (nâ¯=â¯2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (pâ¯=â¯0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (nâ¯=â¯4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (pâ¯=â¯0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2â¯â¯=â¯0.888). INTERPRETATION: Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.
Asunto(s)
Vesículas Extracelulares/metabolismo , Colorantes Fluorescentes/administración & dosificación , Glioma/metabolismo , Ácidos Levulínicos/administración & dosificación , Fármacos Fotosensibilizantes/metabolismo , Protoporfirinas/metabolismo , Adulto , Anciano , Animales , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Glioma/diagnóstico , Glioma/cirugía , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Imagen Óptica/métodos , Cirugía Asistida por Computador , Ácido AminolevulínicoRESUMEN
Hürthle cell carcinoma of the thyroid (HCC) is a form of thyroid cancer recalcitrant to radioiodine therapy that exhibits an accumulation of mitochondria. We performed whole-exome sequencing on a cohort of primary, recurrent, and metastatic tumors, and identified recurrent mutations in DAXX, TP53, NRAS, NF1, CDKN1A, ARHGAP35, and the TERT promoter. Parallel analysis of mtDNA revealed recurrent homoplasmic mutations in subunits of complex I of the electron transport chain. Analysis of DNA copy-number alterations uncovered widespread loss of chromosomes culminating in near-haploid chromosomal content in a large fraction of HCC, which was maintained during metastatic spread. This work uncovers a distinct molecular origin of HCC compared with other thyroid malignancies.
Asunto(s)
Aberraciones Cromosómicas , ADN Mitocondrial/genética , Mutación , Neoplasias de la Tiroides/genética , Variaciones en el Número de Copia de ADN , Haploidia , Humanos , Metástasis de la Neoplasia , Telomerasa/genética , Neoplasias de la Tiroides/patología , Secuenciación del ExomaRESUMEN
The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.
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Diferenciación Celular/inmunología , Homeostasis/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Animales , Técnicas de Cultivo de Célula , Harmina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Quinasas DyrKRESUMEN
The zinc finger transcriptional repressor Gfi-1b is essential for erythroid and megakaryocytic development in the embryo. Its roles in the maintenance of bone marrow erythropoiesis and thrombopoiesis have not been defined. We investigated Gfi-1b's adult functions using a loxP-flanked Gfi-1b allele in combination with a novel doxycycline-inducible Cre transgene that efficiently mediates recombination in the bone marrow. We reveal strict, lineage-intrinsic requirements for continuous adult Gfi-1b expression at two distinct critical stages of erythropoiesis and megakaryopoiesis. Induced disruption of Gfi-1b was lethal within 3 wk with severely reduced hemoglobin levels and platelet counts. The erythroid lineage was arrested early in bipotential progenitors, which did not give rise to mature erythroid cells in vitro or in vivo. Yet Gfi-1b(-/-) progenitors had initiated the erythroid program as they expressed many lineage-restricted genes, including Klf1/Eklf and Erythropoietin receptor. In contrast, the megakaryocytic lineage developed beyond the progenitor stage in Gfi-1b's absence and was arrested at the promegakaryocyte stage, after nuclear polyploidization, but before cytoplasmic maturation. Genome-wide analyses revealed that Gfi-1b directly regulates a wide spectrum of megakaryocytic and erythroid genes, predominantly repressing their expression. Together our study establishes Gfi-1b as a master transcriptional repressor of adult erythropoiesis and thrombopoiesis.
Asunto(s)
Plaquetas/fisiología , Médula Ósea/fisiología , Eritrocitos/fisiología , Regulación de la Expresión Génica/genética , Hematopoyesis/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Cartilla de ADN/genética , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Vectores Genéticos , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Microscopía Fluorescente , Mutagénesis , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genéticaRESUMEN
Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single-cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to address a variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T-cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by ImageStream technology.