RESUMEN
INTRODUCTION: Clinical trials provide fundamental evidence used to inform healthcare decisions at patient- and population levels and it is thus important that trials consider outcomes relevant to both patients and stakeholders. Although validated tools assessing other aspects of trial integrity exist, there is no tool for assessing the breadth and completeness of outcomes measured. Our objective was to develop a comprehensiveness of outcome reporting (COR) tool to assess this within trials in pregnancy. MATERIAL AND METHODS: We developed a tool that aids in visualizing outcome reporting through the automatic generation of a heatmap, enabling assessment of the range of maternal and fetal/neonatal outcomes included in clinical trials. Outcome reporting and measurement of each study is compared to a context-specific, user-determined, ideal standard set of outcomes, created by initially considering all domains within five core outcome areas. These include mortality, morbidity, functioning/life-impact, resource-use, and adverse events, as identified by the most recent taxonomy for outcomes in medical research. We tested the tool's functionality using trials previously identified as studies on obesity in pregnant patients, and further compared the utility of the COR Tool against Cochrane's Risk of Bias 2.0 Tool using correlational analysis. RESULTS: The pilot heatmap using clinical trials studying obesity in pregnancy (n = 15), illustrated a lack of comprehensiveness of reported outcomes in the majority of studies. Included trials were found to readily report physiological/clinical outcome but consistently neglected outcome areas related to functioning, delivery of care, resource-use, and adverse events. Outcome areas reported and measured were done so with largely varying degrees of quality. When the COR Tool was compared with Cochrane's RoB 2.0 Tool on a scatter plot, only a weak correlation was found (R = 0.2936, R2 = 0.0862) CONCLUSIONS: The COR Tool will promote transparency in clarifying what outcomes a trial's conclusions are based on, encourage trialists to consider outcomes related to all aspects of maternal and fetal/neonatal health, and support reviewers in appraising outcome reporting and measurement in the assessment of trial integrity. Used in tandem with RoB tools and core outcome sets, we hope the COR Tool will meaningfully contribute to improving maternal-infant health.
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Ensayos Clínicos como Asunto/normas , Evaluación de Resultado en la Atención de Salud/normas , Evaluación del Resultado de la Atención al Paciente , Atención Prenatal/normas , Informe de Investigación/normas , Investigación Biomédica/normas , Femenino , Humanos , Guías de Práctica Clínica como Asunto , Embarazo , Complicaciones del Embarazo/terapia , Resultado del Embarazo/epidemiologíaRESUMEN
Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ~1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment.
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Exosomas/fisiología , Proteoma/metabolismo , Transducción de Señal , Animales , Proteínas de Unión al Calcio/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales , Células Endoteliales/fisiología , Efrina-B2/metabolismo , Receptores ErbB/metabolismo , Exosomas/patología , Quinasas del Centro Germinal , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serrate-Jagged , Tenascina/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.
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Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Neoplasias del Colon/metabolismo , Exosomas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Exosomas/química , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Metilación , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteoma/análisis , Proteoma/genética , Proteoma/metabolismo , Proteómica , Regulación hacia Arriba , Vía de Señalización WntRESUMEN
Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.
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Anticuerpos/inmunología , Cromatografía de Afinidad/métodos , Neoplasias del Colon/metabolismo , Exosomas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Secuencias de Aminoácidos , Línea Celular Tumoral , Exosomas/ultraestructura , Humanos , Mastocitos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/orina , Especificidad de Órganos , Estructura Terciaria de ProteínaRESUMEN
Membranous vesicles are constitutively released by a multitude of cell types. Following fusion of multivesicular bodies with the plasma membrane, endocytic vesicles, 30-90 nm in size termed exosomes are released extracellularly. Whilst several groups have reported the presence of exosomes in cell-culture conditioned medium, their biological and physiological functions still remain unclear. In addition, exosomes have been detected in body fluids associated with disease, further demonstrating their potential as diagnostic biomarkers. This protocol employs size filtration followed by ultracentrifugation to isolate and purify exosomes from the colon carcinoma cell line LIM 1215. Morphological visualisation and characterisation is based on electron microscopy and western blotting, whilst protein identification is achieved using a combination of 1D SDS-PAGE and LC-MS/MS.
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Exosomas/química , Exosomas/ultraestructura , Proteínas de la Membrana/análisis , Proteómica/métodos , Western Blotting , Línea Celular Tumoral , Cromatografía Liquida , Medios de Cultivo Condicionados/química , Electroforesis en Gel de Poliacrilamida , Filtración , Humanos , Inmunohistoquímica , Microscopía Electrónica , Espectrometría de Masas en Tándem , UltracentrifugaciónRESUMEN
Exosomes are 40-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro. Recent studies have shown that exosomes are also found in vivo in body fluids such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk. While the biological function of exosomes is still unclear, they can mediate communication between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells. Exosome-like vesicles identified in Drosophila (referred to as argosomes) may be potential vehicles for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations. Interestingly, the recent observation that exosomes contain both mRNA and microRNA, which can be transferred to another cell, and be functional in that new environment, is an exciting new development in the unraveling exosome saga. The present review aims to summarize the physical properties that define exosomes as specific cell-type secreted membrane vesicles.
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Exosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Animales , Exosomas/ultraestructura , Humanos , Microscopía Electrónica , Modelos Biológicos , Transporte de ProteínasRESUMEN
Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras-transformed NIH3T3 cells. The vesicular nature and size (30-100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2-D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras-transformed NIH3T3 cells. LC-MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen alpha-1 (VI), 14-3-3 isoforms, guanine nucleotide-binding proteins (G proteins), the eukaryotic translation initiation factors elF-3 gamma and elF-5A accumulated (>2-fold) in exosomes upon Ras-induced oncogenic transformation. Significantly, the 10.4-fold increase in v-Ha-Ras p21 protein in exosomes derived from Ras-transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.
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Fibroblastos/metabolismo , Genes ras , Orgánulos/metabolismo , Proteoma/metabolismo , Animales , Línea Celular Transformada , Electroforesis en Gel Bidimensional , Fibroblastos/ultraestructura , Ratones , Microscopía Electrónica , Células 3T3 NIHRESUMEN
Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.