RESUMO
Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three- to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.
Assuntos
Vesículas Extracelulares , Soro/química , Ultracentrifugação , Biomarcadores , Centrifugação com Gradiente de Concentração , Exossomos/metabolismo , Exossomos/ultraestrutura , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , Masculino , MicroRNAs , RNA não Traduzido , Ultracentrifugação/métodosRESUMO
High-risk human papillomavirus (hrHPV) is an emerging risk factor for sinonasal squamous cell carcinoma (SNSCC). The goal of this study was to assess the prevalence of hrHPV and subtype distribution in SNSCC and correlation with patient and clinical characteristics. This retrospective cohort study included 43 cases diagnosed with incident primary SNSCC at the University of Cincinnati Medical Center from 2010 to 2015. The prevalence of hrHPV was interrogated using a multi-assay approach that included p16 immunohistochemistry (IHC), RNA in-situ hybridization (ISH), and hrHPV DNA sequencing. The association of hrHPV with 5-year overall survival (OS) and 2-year disease-free survival (DFS) was assessed. Fourteen cases (32.6â¯%) were classified as hrHPV positive, based on the a priori definition of having either a positive RNAScope™ ISH test or hrHPV DNA and p16-positive IHC; 9 cases (20.9â¯%) were positive for all three tests. All cases that arose from an inverted sinonasal papilloma (ex-ISP) were negative for hrHPV. HPV16 was the most common subtype among hrHPV positive cases (58.8â¯%), followed by HPV18 (17.6â¯%). No significant association was observed between hrHPV and OS or DFS after adjusting for potential confounding. hrHPV is prevalent in a sizable fraction of SNSCC. Additional studies are needed to better elucidate the relationship with patient survival outcomes and determine the optimal testing modality for prognostication.
Assuntos
Carcinoma de Células Escamosas , Infecções por Papillomavirus , Neoplasias dos Seios Paranasais , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/complicações , Idoso , Estudos Retrospectivos , Neoplasias dos Seios Paranasais/virologia , Neoplasias dos Seios Paranasais/patologia , Carcinoma de Células Escamosas/virologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/mortalidade , Adulto , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Imuno-Histoquímica , Papillomavirus HumanoRESUMO
Nano-scale extracellular vesicles are lipid-bilayer delimited particles that are naturally secreted by all cells and have emerged as valuable biomarkers for a wide range of diseases. Efficient isolation of small extracellular vesicles while maintaining yield and purity is crucial to harvest their potential in diagnostic, prognostic, and therapeutic applications. Most conventional methods of isolation suffer from significant shortcomings, including low purity or yield, long duration, need for large sample volumes, specialized equipment, trained personnel, and high costs. To address some of these challenges, our group has reported a novel insulator-based dielectrophoretic device for rapid isolation of small extracellular vesicles from biofluids and cell culture media based on their size and dielectric properties. In this study, we report a comprehensive characterization of small extracellular vesicles isolated from cancer-patients' biofluids at a twofold enrichment using the device. The three-fold characterization that was performed using conventional flow cytometry, advanced imaging flow cytometry, and microRNA sequencing indicated high yield and purity of the isolated small extracellular vesicles. The device thus offers an efficient platform for rapid isolation while maintaining biomolecular integrity.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Biomarcadores , Neoplasias/diagnóstico , Dispositivos Lab-On-A-ChipRESUMO
BACKGROUND: The objective was to assess secretion of small extracellular vesicular microRNA (exo-miRNA) in head and neck squamous cell carcinoma (HNSCC) according to human papillomavirus (HPV) status, and determine the translational potential as a liquid biopsy for early detection. METHODS: This study employed a combination of cell culture and case-control study design using archival pretreatment serum. Small extracellular vesicles (sEV) were isolated from conditioned culture media and human serum samples via differential ultracentrifugation. miRNA-sequencing was performed on each sEV isolate. RESULTS: There were clear exo-miRNA profiles that distinguished HNSCC cell lines from nonpathologic oral epithelial control cells. While there was some overlap among profiles across all samples, there were apparent differences in exo-miRNA profiles according to HPV-status. Importantly, differential exo-miRNA profiles were also apparent in serum from early-stage HNSCC cases relative to cancer-free controls. CONCLUSIONS: Our findings indicate that exo-miRNA are highly dysregulated in HNSCC and support the potential of exo-miRNA as biomarkers for HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço , MicroRNAs , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , MicroRNAs/genética , Infecções por Papillomavirus/genética , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/genética , Estudos de Casos e Controles , Biópsia Líquida , Papillomaviridae/genéticaRESUMO
Human milk-derived extracellular vesicles (HMEVs) are crucial functional components in breast milk, contributing to infant health and development. Maternal conditions could affect HMEV cargos; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study evaluated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. Milk samples (9 prenatal SARS-CoV-2 vs. 9 controls) were retrieved from the IMPRINT birth cohort. After defatting and casein micelle disaggregation, 1 mL milk was subjected to a sequential process of centrifugation, ultrafiltration, and qEV-size exclusion chromatography. Particle and protein characterizations were performed following the MISEV2018 guidelines. EV lysates were analyzed through proteomics and miRNA sequencing, while the intact EVs were biotinylated for surfaceomic analysis. Multi-Omics was employed to predict HMEV functions associated with prenatal SARS-CoV-2 infection. Demographic data between the prenatal SARS-CoV-2 and control groups were similar. The median duration from maternal SARS-CoV-2 test positivity to milk collection was 3 months (range: 1-6 months). Transmission electron microscopy showed the cup-shaped nanoparticles. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1e11 particles from 1 mL milk. Western immunoblots detected ALIX, CD9 and HSP70, supporting the presence of HMEVs in the isolates. Thousands of HMEV cargos and hundreds of surface proteins were identified and compared. Multi-Omics predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming and mucosal tissue development, while mitigating inflammation and lower EV transmigration potential. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era.
RESUMO
MicroRNAs (miRNAs) are small non-coding RNA that are powerful regulators of gene expression and can affect the expression of hundreds of genes. miRNAs can be packed in small extracellular vesicles (SEV) and released into the extracellular space by neurons and microglia to act locally as well as pass through the blood-brain barrier and act systemically. We sought to understand the differences in neuronal SEV miRNA expression between frontotemporal dementia (FTD), Alzheimer's disease (AD), and healthy aging. Plasma was obtained from FTD, AD, and healthy aging participants that were matched based on age, sex, and race/ethnicity. Additionally, a subset of participants also provided paired cerebrospinal fluid samples to compare neuronal SEV miRNAs in plasma and cerebrospinal fluid. Neuronal SEV were isolated using differential ultracentrifugation and antibody conjugated Dynabeads® for the neuronal surface marker, L1CAM. RNA sequencing was performed. 12 FTD, 11 with AD, and 10 healthy aging participants were enrolled in the study. In FTD, SEV miRNA-181c was downregulated compared to healthy controls. In AD, miRNA-122 and miRNA-3591 were downregulated compared to those in healthy controls and FTD. Using an FDR <0.2, only miRNA-21-5p was found to have increased expression in the cerebrospinal fluid compared to plasma in a group of AD and FTD participants. SEV miRNA-181c is significantly downregulated in FTD compared to healthy controls and may mediate its effects through microglial-directed neuroinflammation and interaction with TAR DNA-binding protein 43 (TDP-43) based on pathway analysis. Additionally, the FOXO and Hippo pathways may be important mediators of FTD, based on pathway analysis. Lastly, because only one SEV miRNA was differentially expressed between the plasma and cerebrospinal fluid in paired samples, plasma represents an appropriate biofluid for studying neuronal SEV miRNA.
Assuntos
Doença de Alzheimer , Vesículas Extracelulares , Demência Frontotemporal , MicroRNAs , Molécula L1 de Adesão de Célula Nervosa , Doença de Alzheimer/genética , Atrofia , Proteínas de Ligação a DNA , Vesículas Extracelulares/genética , Demência Frontotemporal/líquido cefalorraquidiano , Demência Frontotemporal/genética , Humanos , MicroRNAs/genética , NeurôniosRESUMO
Small extracellular vesicles (sEV) are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by essentially all cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA) and other small non-coding RNA (ncRNA), that can alter the phenotype of recipient cells. sEV are present in essentially all extracellular biofluids, including serum, urine and saliva, and offer a new avenue for discovery and development of novel biomarkers of various disease states and exposures. The objective of this study was to systematically interrogate similarities and differences between sEV ncRNA derived from saliva, serum and urine, as well as cell-free small ncRNA (cf-ncRNA) from serum. Saliva, urine and serum were concomitantly collected from 4 healthy donors to mitigate potential bias that can stem from interpersonal and temporal variability. sEV were isolated from each respective biofluid, along with cf-RNA from serum. sEV were isolated from the respective biofluids via differential ultracentrifugation with a 30% sucrose cushion to minimize protein contamination. Small RNA-sequencing was performed on each sample, and cluster analysis was performed based on ncRNA profiles. While some similarities existed in terms of sEV ncRNA cargo across biofluids, there are also notable differences in ncRNA class and ncRNA secretion, with sEV in each biofluid bearing a unique ncRNA profile, including major differences in composition by ncRNA class. We conclude that sEV ncRNA cargo varies according to biofluid, so thus should be carefully selected and interpreted when designing or contrasting translational or epidemiological studies.
Assuntos
Biomarcadores/análise , Vesículas Extracelulares/metabolismo , MicroRNAs/análise , Pequeno RNA não Traduzido/análise , Adulto , Idoso , Biomarcadores/sangue , Biomarcadores/urina , Líquidos Corporais/metabolismo , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , MicroRNAs/sangue , MicroRNAs/urina , Pessoa de Meia-Idade , Pequeno RNA não Traduzido/sangue , Pequeno RNA não Traduzido/urina , Saliva/metabolismo , Análise de Sequência de RNA , UltracentrifugaçãoRESUMO
Exosomes are nano-scale membrane-encapsulated vesicles produced by the majority of cells and have emerged as a rich source of biomarkers for a wide variety of diseases. Although many approaches have been developed for exosome isolation from biofluids, most of them have substantial shortcomings including long processing time, inefficiency, high cost, lack of specificity and/or surface marker-dependency. To address these issues, here we report a novel insulator-based dielectrophoretic (iDEP) device predicated on an array of borosilicate micropipettes to rapidly isolate exosomes from conditioned cell culture media and biofluids, such as plasma, serum, and saliva. The device is capable of exosome isolation from small sample volumes of 200 µL within 20 minutes under a relatively low (10 V cm-1) direct current (DC). This device is easy to fabricate thus, no cleanroom facility and expensive equipment are needed. Therefore, the iDEP device offers a rapid and cost-effective strategy for exosome isolation from biofluids in timely manner while maintaining the yield and purity.
Assuntos
Eletroforese/instrumentação , Vesículas Extracelulares/química , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Saliva/química , Adulto , Eletroforese/métodos , Humanos , MasculinoRESUMO
Aim: The goal of this study was to comprehensively interrogate and map DNA methylation across 16 CpG-dense regions previously associated with oral and pharyngeal squamous cell carcinoma (OPSCC). Materials & methods: Targeted multiplex bisulfite amplicon sequencing was performed on four OPSCC cell lines and primary non-neoplastic oral epithelial cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for a subset of associated genes. Results: There was clear differential methylation between one or more OPSCC cell lines and control cells for the majority of CpG-dense regions. Conclusion: Targeted multiplex bisulfite amplicon sequencing allowed us to efficiently map methylation across the entire region of interest with a high degree of sensitivity and helps shed light on novel differentially methylated regions that may have value as biomarkers of OPSCC.
Assuntos
Biomarcadores/análise , Carcinoma de Células Escamosas/genética , Ilhas de CpG/genética , Epigenômica , Neoplasias Bucais/genética , Neoplasias Faríngeas/genética , Biologia Computacional , Metilação de DNA , Análise de Sequência de DNARESUMO
BACKGROUND: Long non-coding RNA (lncRNA) has emerged as a new avenue of interest due to its various biological functions in cancer. Abnormal expression of lncRNA has been reported in other malignancies but has been understudied in head and neck squamous cell carcinoma (HNSCC). METHODS: The lncRNA expression was interrogated via quantitative real-time polymerase chain reaction (qRT-PCR) array for 19 human papillomavirus (HPV)-negative HNSCC tumor-normal pairs. The Cancer Genome Atlas (TCGA) was used to validate these results. The association between differentially expressed lncRNA and survival outcomes was analyzed. RESULTS: Differential expression was validated for 5 lncRNA (SPRY4-IT1, HEIH, LUCAT1, LINC00152, and HAND2-AS1). There was also an inverse association between MEG3 expression (not significantly differentially expressed in TCGA tumors but highly variable expression) and 3-year recurrence-free survival (RFS). CONCLUSION: We identified and validated differential expression of 5 lncRNA in HPV-negative HNSCC. Low MEG3 expression was associated with favorable 3-year RFS, although the significance of this finding remains unclear.
Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , RNA Longo não Codificante/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Bases de Dados de Ácidos Nucleicos , Intervalo Livre de Doença , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Papillomaviridae , Prognóstico , Modelos de Riscos Proporcionais , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de SobrevidaRESUMO
Exosomes are nano-scale, membrane encapsulated vesicles that are released by cells into the extracellular space and function as intercellular signaling vectors through horizontal transfer of biologic molecules, including microRNA (miRNA). There is evidence that cancer-derived exosomes enable the tumor to manipulate its microenvironment, thus contributing to the capacity of the tumor for immune evasion, growth, invasion, and metastatic spread. The objective of this study was to characterize differential secretion of exosomal miRNA by head and neck squamous cell carcinoma (HNSCC) and identify a set of candidate biomarkers that could be detected in non-invasive saliva samples. We isolated exosomes from conditioned media from 4 HNSCC cell lines and oral epithelial control cells and applied miRNA-sequencing to comprehensively characterize their miRNA cargo and compare transcript levels of each HNSCC cell line to that of oral epithelial control cells. A candidate set of miRNA differentially secreted by all 4 HNSCC cell lines was further evaluated in saliva collected from HNSCC patients and healthy controls. We observed extensive differences in exosomal miRNA content between HNSCC cells when compared to normal oral epithelial control cells, with a high degree of overlap in exosomal miRNA profiles between the 4 distinct HNSCC cell lines. Importantly, several of the exosomal miRNA secreted solely by cancer cells in culture were detected at substantially elevated levels in saliva from HNSCC patients relative to saliva from healthy controls. These findings provide important insight into tumor biology and yields a promising set of candidate HNSCC biomarkers for use with non-invasive saliva samples.