RESUMEN
There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias/diagnóstico , Animales , Biomarcadores de Tumor/sangre , Línea Celular , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Aprendizaje Automático , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Neoplasias/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Proteómica/métodos , Sensibilidad y Especificidad , Tetraspanina 29/metabolismo , Proteínas de Unión al GTP rap/metabolismoRESUMEN
Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.
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Vesículas Extracelulares , Ácidos Grasos , Hígado Graso , Hígado , Neoplasias Pancreáticas , Animales , Ratones , Sistema Enzimático del Citocromo P-450/genética , Vesículas Extracelulares/metabolismo , Ácidos Grasos/metabolismo , Hígado Graso/tratamiento farmacológico , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado Graso/prevención & control , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias Hepáticas/secundario , Humanos , Inflamación/metabolismo , Ácido Palmítico/metabolismo , Macrófagos del Hígado , Fosforilación Oxidativa , Proteínas rab27 de Unión a GTP/deficienciaRESUMEN
Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
Asunto(s)
Encéfalo/metabolismo , Exosomas/metabolismo , Integrinas/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Tropismo , Animales , Biomarcadores/metabolismo , Encéfalo/citología , Línea Celular Tumoral , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Genes src , Humanos , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/antagonistas & inhibidores , Integrina alfa6beta4/metabolismo , Cadenas beta de Integrinas/metabolismo , Integrina beta4/metabolismo , Integrinas/antagonistas & inhibidores , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Hígado/citología , Pulmón/citología , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Fosforilación , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/metabolismo , Proteínas S100/genéticaRESUMEN
BACKGROUND: Exosomes are nanovesicles that have been shown to mediate carcinogenesis in pancreatic ductal adenocarcinoma (PDAC). Given the direct communication of pancreatic duct fluid with the tumor and its relative accessibility, we aimed to determine the feasibility of isolating and characterizing exosomes from pancreatic duct fluid. METHODS: Pancreatic duct fluid was collected from 26 patients with PDAC (n = 13), intraductal papillary mucinous neoplasm (IPMN) (n = 8) and other benign pancreatic diseases (n = 5) at resection. Exosomes were isolated by serial ultracentrifugation, proteins were identified by mass spectrometry, and their expression was evaluated by immunohistochemistry. RESULTS: Exosomes were isolated from all specimens with a mean concentration of 5.9 ± 1 × 108 particles/mL and most frequent size of 138 ± 9 nm. Among the top 35 proteins that were significantly associated with PDAC, multiple carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and extracellular matrix (ECM) proteins were identified. Interestingly, CEACAM 1/5 expression by immunohistochemistry was seen only on tumor epithelia whereas tenascin C positivity was restricted to stroma, suggesting that both tumor and stromal cells contributed to exosomes. CONCLUSION: This is the first study showing that exosome isolation is feasible from pancreatic duct fluid, and that exosomal proteins may be utilized to diagnose patients with PDAC.
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Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/química , Moléculas de Adhesión Celular/análisis , Exosomas/química , Proteínas de la Matriz Extracelular/análisis , Conductos Pancreáticos/química , Neoplasias Intraductales Pancreáticas/química , Jugo Pancreático/química , Neoplasias Pancreáticas/química , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/patología , Estudios de Factibilidad , Femenino , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Conductos Pancreáticos/patología , Neoplasias Intraductales Pancreáticas/patología , Neoplasias Pancreáticas/patología , Proyectos Piloto , Valor Predictivo de las Pruebas , UltracentrifugaciónRESUMEN
Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms. For complete details on the use and execution of this protocol, please refer to Hoshino et al.1.
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Vesículas Extracelulares , Proteómica , Humanos , Animales , Ratones , Western Blotting , Biología Computacional , Espectrometría de MasasRESUMEN
Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.
RESUMEN
Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.
Asunto(s)
Neoplasias Hepáticas , Hígado , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirugía , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Masculino , Femenino , Persona de Mediana Edad , Anciano , Hígado/patología , Hígado/metabolismo , Biopsia , Estadificación de Neoplasias , Pancreatectomía , Trampas Extracelulares/metabolismo , PronósticoRESUMEN
Plasma extracellular vesicles and particles (EVPs) are enriched in biomolecules that reflect individuals' physiological and pathological states. Several studies have demonstrated the potential of human plasma EVPs as a novel liquid biopsy. Here we describe a protocol for human plasma EVPs isolation and proteomic characterization. We isolated human plasma EVPs by the classical ultracentrifugation method and performed mass spectrometry-based proteomic profiling. Using this protocol, researchers can reveal the plasma EVPs proteome and explore the clinical application of plasma EVPs proteins for developing disease biomarkers.
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Vesículas Extracelulares , Proteómica , Humanos , Proteómica/métodos , Espectrometría de Masas , Ultracentrifugación , Proteínas Sanguíneas/metabolismo , Vesículas Extracelulares/metabolismo , Proteoma/metabolismoRESUMEN
BACKGROUND: Ischemic preconditioning (IPC) of the liver decreases liver injury secondary to ischemia and reperfusion. An attractive alternative to IPC is remote ischemic preconditioning (R-IPC), but these two methods have not previously been compared. MATERIAL AND METHODS: Eighty-seven rats were randomized into four groups: sham operated (n = 15), 1 h segmental ischemia (IRI, n = 24), preceded by IPC (n = 24), or R-IPC (n = 24) (to the left hindleg). IPC and R-IPC were performed with 10 min ischemia and 10 min of reperfusion. Analyses of liver microdialysate (MD), serum transaminase levels, and liver histology were made. RESULTS: Rats treated with IPC and R-IPC had significantly lower AST, 71.5 (19.6) IU/L respective 96.6 (12.4) at 4 h reperfusion than those subjected to IRI alone, 155 (20.9), P = 0.0004 and P = 0.04 respectively. IPC also had lower ALT levels, 41.6 (11.3) IU/L than had IRI 107.4 (15.5), P = 0.003. The MD glycerol was significantly higher during ischemia in the R-IPC [759 (84) µM] and the IRI [732 (67)] groups than in the IPC 514 (70) group, P = 0.022 and P = 0.046 respectively. The MD glucose after ischemia was lower in the IPC group 7.1 (1.2) than in the IRI group 12.7 (1.6), P = 0.005. Preconditioning to the liver caused an direct increase in lactate, glucose and glycerol in the ischemic segment compared with the control segment an effect not seen in the R-IPC and IRI groups. CONCLUSIONS: IPC affects glucose metabolism in the rat liver, observed with MD. IPC reduces liver cell injury during ischemic and reperfusion in rats. R-IPC performed over the same length of time as IPC does not have the same effect as the latter on ALT levels and MD glycerol; this may suggest that R-IPC does not offer the same protection as IPC in this setting of rat liver IRI.
Asunto(s)
Precondicionamiento Isquémico/métodos , Hígado/irrigación sanguínea , Hígado/metabolismo , Microdiálisis/métodos , Daño por Reperfusión/prevención & control , Animales , Glucemia/metabolismo , Glicerol/metabolismo , Lactatos/metabolismo , Hígado/patología , Masculino , Modelos Animales , Piruvatos/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transaminasas/sangreRESUMEN
OBJECTIVE: N-acetylcysteine (NAC) is an antioxidative molecule known to protect liver tissue from oxygen radical species generated during ischemia and reperfusion (IR). Nutritional and toxicology studies have shown that NAC also improves glucose metabolism and glycogen stores. We hypothesized that NAC improves glycogenesis and that impaired glycogenesis is a key element in IR injury. MATERIAL AND METHODS: In an experimental model, 80 min of segmental liver ischemia was induced in 16 pigs and the reperfusion was followed for 360 min. Eight animals received NAC 150 mg/kg as a bolus injection followed by an infusion of NAC 50 mg/kg/h intravenously. RESULTS: AST and leukocyte density were lower in the NAC-treated animals, unrelated to the glutathione levels or apoptosis. Glycogen stores returned to a higher degree in the NAC-treated animals and microdialysis revealed lower levels of lactate during the reperfusion phase. Nitrite/Nitrate levels in the NAC group were lower in both serum and microdialysates, indicating that NAC scavenges radical nitrosative species. CONCLUSIONS: NAC treatment improves glycogenesis after liver IR injury and reduces the level of intraparenchymal lactate during reperfusion, possibly due to the scavenging of radical nitrosative species.
Asunto(s)
Acetilcisteína/farmacología , Depuradores de Radicales Libres/farmacología , Glucógeno/biosíntesis , Hígado/metabolismo , Daño por Reperfusión/metabolismo , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Animales , Apoptosis , Aspartato Aminotransferasas/sangre , Glutatión/metabolismo , Ácido Láctico/metabolismo , Recuento de Leucocitos , Hígado/fisiopatología , Masculino , Microdiálisis , Neutrófilos , Nitratos/metabolismo , Nitritos/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , PorcinosRESUMEN
Cancer-related mortality is primarily a consequence of metastatic dissemination and associated complications. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and tends to metastasize early, especially in the liver. Emerging evidence suggests that organs that develop metastases exhibit microscopic changes that favor metastatic growth, collectively known as "pre-metastatic niches". By definition, a pre-metastatic niche is chronologically established before overt metastatic outgrowth, and its generation involves the release of tumor-derived secreted factors that modulate cells intrinsic to the recipient organ, as well as recruitment of additional cells from tertiary sites, such as bone marrow-all orchestrated by the primary tumor. The pre-metastatic niche is characterized by tumor-promoting inflammation with tumor-supportive and immune-suppressive features, remodeling of the extracellular matrix, angiogenic modulation and metabolic alterations that support growth of disseminated tumor cells. In this paper, we review the current state of knowledge of the hepatic pre-metastatic niche in PDAC and attempt to create a framework to guide future diagnostic and therapeutic studies.
RESUMEN
Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research.
Asunto(s)
Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Clusterina , Factores de Transcripción del Choque Térmico , Neoplasias Pancreáticas , Animales , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/patología , Clusterina/genética , Clusterina/metabolismo , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Neoplasias Pancreáticas/patología , Microambiente Tumoral/genética , Humanos , Neoplasias PancreáticasRESUMEN
We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
Asunto(s)
Líquidos Corporales/química , Centrifugación por Gradiente de Densidad , Vesículas Extracelulares/química , Fraccionamiento de Campo-Flujo , Proteómica , Animales , Línea Celular , Humanos , RatonesRESUMEN
The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.
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Neoplasias Encefálicas/genética , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Hialuronoglucosaminidasa/genética , Neovascularización Patológica/genética , Microambiente Tumoral/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Quimiocina CCL1/genética , Quimiocina CCL1/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Exosomas/patología , Humanos , Hialuronoglucosaminidasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Metástasis de la Neoplasia , Neovascularización Patológica/metabolismo , Neovascularización Patológica/mortalidad , Neovascularización Patológica/patología , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome's molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.
Asunto(s)
Alelos , Regulación de la Expresión Génica , Mutación/genética , Especificidad de Órganos/genética , ARN Ribosómico/genética , Animales , Secuencia de Bases , Cromosomas Humanos/genética , Secuencia Conservada/genética , ADN Ribosómico/genética , Evolución Molecular , Dosificación de Gen , Perfilación de la Expresión Génica , Genoma Humano , Células HEK293 , Humanos , Ratones , Operón/genética , Biosíntesis de Proteínas , Subunidades de Proteína/genética , Procesamiento Postranscripcional del ARN/genética , Ribosomas/metabolismoRESUMEN
The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.
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Fraccionamiento Celular/métodos , Exosomas/metabolismo , Nanopartículas , Neoplasias/metabolismo , Proteínas/metabolismo , Animales , Biomarcadores/metabolismo , ADN/genética , ADN/metabolismo , Metabolismo Energético , Exosomas/clasificación , Exosomas/genética , Exosomas/patología , Femenino , Glicómica , Glicosilación , Células HCT116 , Humanos , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Metabolómica , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Neoplasias/genética , Neoplasias/patología , Células PC-3 , Fenotipo , Proteómica , ARN/genética , ARN/metabolismo , Transducción de Señal , Distribución TisularRESUMEN
Pancreatic stellate cells (PSCs) are the key precursor cells for cancer-associated fibroblasts (CAFs) in pancreatic tumor stroma. In this study, we explored miRNA as therapeutic targets in tumor stroma and found miR-199a-3p and miR-214-3p induced in patient-derived pancreatic CAFs and TGF-ß-activated human PSCs (hPSCs). Inhibition of miR-199a/-214 using hairpin inhibitors significantly inhibited TGFß-induced differentiation markers (e.g. α-SMA, collagen, PDGFßR), migration and proliferation. Furthermore, heterospheroids of Panc-1 and hPSCs attained smaller size with hPSCs transfected with anti-miR-199a/-214 compared to control anti-miR. The conditioned medium obtained from TGFß-activated hPSCs induced tumor cell growth and endothelial cell tube formation. Interestingly, these inductions were abrogated in hPSCs transfected with anti-miR-199a or miR-214. Moreover, IPA analyses revealed signaling pathways related to miR-199a (TP53, mTOR, Smad1) and miR-214 (PTEN, Bax, ING4). Taken together, this study reveals miR-199a-3p and miR-214-3p as major regulators of PSC activation and PSC-induced pro-tumoral effects, representing them as key therapeutic targets in pancreatic cancer.
Asunto(s)
MicroARNs/genética , Neoplasias Pancreáticas/genética , Células Estrelladas Pancreáticas/metabolismo , Antagomirs/genética , Apoptosis/genética , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hibridación in Situ , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
AIM: To investigate whether nitrite administered prior to ischemia/reperfusion (I/R) reduces liver injury. METHODS: Thirty-six male Sprague-Dawley rats were randomized to 3 groups, including sham operated (n = 8), 45-min segmental ischemia of the left liver lobe (IR, n = 14) and ischemia/reperfusion (I/R) preceded by the administration of 480 nmol of nitrite (n = 14). Serum transaminases were measured after 4 h of reperfusion. Liver microdialysate (MD) was sampled in 30-min intervals and analyzed for glucose, lactate, pyruvate and glycerol as well as the total nitrite and nitrate (NOx). The NOx was measured in serum. RESULTS: Aspartate aminotransferase (AST) at the end of reperfusion was higher in the IR group than in the nitrite group (40 ± 6.8 µkat/L vs 22 ± 2.6 µkat/L, P = 0.022). Similarly, alanine aminotransferase (ALT) was also higher in the I/R group than in the nitrite group (34 ± 6 µkat vs 14 ± 1.5 µkat, P = 0.0045). The NOx in MD was significantly higher in the nitrite group than in the I/R group (10.1 ± 2.9 µmol/L vs 3.2 ± 0.9 µmol/L, P = 0.031) after the administration of nitrite. During ischemia, the levels decreased in both groups and then increased again during reperfusion. At the end of reperfusion, there was a tendency towards a higher NOx in the I/R group than in the nitrite group (11.6 ± 0.7 µmol/L vs 9.2 ± 1.1 µmol/L, P = 0.067). Lactate in MD was significantly higher in the IR group than in the nitrite group (3.37 ± 0.18 mmol/L vs 2.8 ± 0.12 mmol/L, P = 0.01) during ischemia and the first 30 min of reperfusion. During the same period, glycerol was also higher in the IRI group than in the nitrite group (464 ± 38 µmol/L vs 367 ± 31 µmol/L, P = 0.049). With respect to histology, there were more signs of tissue damage in the I/R group than in the nitrite group, and 29% of the animals in the I/R group exhibited necrosis compared with none in the nitrite group. Inducible nitric oxide synthase transcription increased between early ischemia (t = 15) and the end of reperfusion in both groups. CONCLUSION: Nitrite administered before liver ischemia in the rat liver reduces anaerobic metabolism and cell necrosis, which could be important in the clinical setting.
Asunto(s)
Hígado/irrigación sanguínea , Hígado/efectos de los fármacos , Daño por Reperfusión/prevención & control , Nitrito de Sodio/administración & dosificación , Animales , Biomarcadores/sangre , Citoprotección , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Inyecciones Intravenosas , Hígado/metabolismo , Hígado/patología , Hígado/cirugía , Masculino , Necrosis , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Daño por Reperfusión/patología , Factores de TiempoRESUMEN
AIM: To study the effects of preconditioning on inducible nitric oxide synthase (iNOS) and interleukin 1 (IL-1) receptor transcription in rat liver ischemia/reperfusion injury (IRI). METHODS: Seventy-two male rats were randomized into 3 groups: the one-hour segmental ischemia (IRI, n = 24) group, the ischemic preconditioning (IPC, n = 24) group or the remote ischemic preconditioning (R-IPC, n = 24) group. The IPC and R-IPC were performed as 10 min of ischemia and 10 min of reperfusion. The iNOS and the IL-1 receptor mRNA in the liver tissue was analyzed with real time PCR. The total Nitrite and Nitrate (NOx) in continuously sampled microdialysate (MD) from the liver was analyzed. In addition, the NOx levels in the serum were analyzed. RESULTS: After 4 h of reperfusion, the iNOS mRNA was significantly higher in the R-IPC (ΔCt: 3.44 ± 0.57) group than in the IPC (ΔCt: 5.86 ± 0.82) group (P = 0.025). The IL-1 receptor transcription activity was reduced in the IPC group (ΔCt: 1.88 ± 0.53 to 4.81 ± 0.21), but not in the R-IPC group, during reperfusion (P = 0.027). In the MD, a significant drop in the NOx levels was noted in the R-IPC group (12.3 ± 2.2 to 4.7 ± 1.2 µmol/L) at the end of ischemia compared with the levels in early ischemia (P = 0.008). A similar trend was observed in the IPC group (11.8 ± 2.1 to 6.4 ± 1.5 µmol/L), although this difference was not statistically significant. The levels of NOx rose quickly during reperfusion in both groups. CONCLUSION: IPC, but not R-IPC, reduces iNOS and IL-1 receptor transcription during early reperfusion, indicating a lower inflammatory reaction. NOx is consumed in the ischemic liver lobe.
Asunto(s)
Miembro Posterior/irrigación sanguínea , Precondicionamiento Isquémico/métodos , Hígado/irrigación sanguínea , Hígado/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Daño por Reperfusión/enzimología , Daño por Reperfusión/prevención & control , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Mediadores de Inflamación/metabolismo , Hígado/patología , Masculino , Microdiálisis , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo II/genética , Nitritos/sangre , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Factores de TiempoRESUMEN
The interplay between the tumor cells and the surrounding stroma creates inflammation, which promotes tumor growth and spread. The inflammation is a hallmark for pancreatic adenocarcinoma (PDAC) and is to high extent driven by IL-1α. IL-1α is expressed and secreted by the tumor cells and exerting its effect on the stroma, i.e. cancer associated fibroblasts (CAF), which in turn produce massive amount of inflammatory and immune regulatory factors. IL-1 induces activation of transcription factors such as nuclear factor-κß (NF-κß), but also activator protein 1 (AP-1) via the small G-protein Ras. Dysregulation of Ras pathways are common in cancer as this oncogene is the most frequently mutated in many cancers. In contrast, the signaling events leading up to the expression of IL-1α by tumor cells are not well elucidated. Our aim was to examine the signaling cascade involved in the induction of IL-1α expression in PDAC. We found p38MAPK, activated by the K-Ras signaling pathway, to be involved in the expression of IL-1α by PDAC as blocking this pathway decreased both the gene and protein expression of IL-1α. Blockage of the P38MAPK signaling in PDAC also dampened the ability of the tumor cell to induce inflammation in CAFs. In addition, the IL-1α autocrine signaling regulated the migratory capacity of PDAC cells. Taken together, the blockage of signaling pathways leading to IL-1α expression and/or neutralization of IL-1α in the PDAC microenvironment should be taken into consideration as possible treatment or complement to existing treatment of this cancer.