scholarly journals exRNA Signatures in Extracellular Vesicles and their Tumor-Lineage from Prostate Cancer

2020 ◽  
Author(s):  
Navneet Dogra ◽  
Mehmet Eren Ahsen ◽  
Edgar Gonzalez Kozlova ◽  
Tzu-yi Chen ◽  
kimaada allette ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Differential Expression ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Tumor Tissue ◽  
Biomarker Discovery ◽  
De Novo ◽  
Culture Media ◽  
Tumor Resection ◽  
Small Rna Sequencing

Circulating extracellular vesicles (EVs) present in the bodily fluids of patients with cancer may provide non-invasive access to the tumor tissue. Yet, the transcriptomic lineage of tumor-derived EVs before and after tumor-resection remains poorly understood. Here, we established 60 total small RNA-sequencing profiles from 17 aggressive prostate cancer (PCa) patients tumor and adjacent normal tissue, and EVs isolated from urine, serum, and cancer cell culture media. We interrogated the key satellite alteration in tumor-derived EVs and found that resection of tumor prostate tissue leads to differential expression of reactive oxygen species (ROS), P53 pathways, inflammatory/cytokines, oncogenes, and tumor suppressor genes in the EV nanosatellites. Furthermore, we provide a set of novel EV-specific RNA signature, which are present in cancer but are nonexistent in post-resection patients with undetectable cancer. Finally, using a de novo RNAseq assembly followed by characterization of the small RNA landscape, we found novel small RNA clusters (smRCs) in the EVs, which reside in the unannotated regions. Novel smRCs were orthogonally validated for their differential expression in the biomarker discovery cohort using RT-qPCR. We demonstrate that circulating tumor EVs provide a glimpse of the tumor tissue biology, resolving a major bottleneck in the current liquid biopsy efforts. Secretory vesicles appear to be playing a key role in non-canonical Wnt signaling and miRNA pathways, similar to the circulating tumor cells (CTCs), hence, we propose that such vesicles be called circulating tumor extracellular vesicles (CTEVs).

scholarly journals High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Anson Ku ◽  
Jacob Fredsøe ◽  
Karina D. Sørensen ◽  
Michael Borre ◽  
Mikael Evander ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Small Rna Sequencing ◽  
High Grade ◽  
Enrichment Technique ◽  
Diagnostic Potential

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.

scholarly journals Glycosylation of Cancer Extracellular Vesicles: Capture Strategies, Functional Roles and Potential Clinical Applications

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 109
Author(s):  
Álvaro M. Martins ◽  
Cátia C. Ramos ◽  
Daniela Freitas ◽  
Celso A. Reis
Keyword(s):  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
De Novo ◽  
Recipient Cell ◽  
Cell Communication ◽  
Cancer Biomarkers ◽  
Functional Roles ◽  
Molecular Information ◽  
Glycosylation Pathway ◽  
Organ Tropism

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies.

scholarly journals Proteomics in Diagnosis of Prostate Cancer/ Протеомика Во Дијагноза На Простатниот Карцином

PRILOZI ◽  
2015 ◽  
Vol 36 (1) ◽  
pp. 5-36 ◽  
Author(s):  
Katarina Davalieva ◽  
Momir Polenakovic
Keyword(s):  
Prostate Cancer ◽  
Tumor Tissue ◽  
Biomarker Discovery ◽  
Molecular Level ◽  
Specific Antigen ◽  
Shotgun Proteomics ◽  
Comparative Proteomics ◽  
Label Free ◽  
The Past ◽  
Proteomics Research

Abstract Prostate cancer (PCa) is the second most frequently diagnosed malignancy in men worldwide. The introduction of prostate specific antigen (PSA) has greatly increased the number of men diagnosed with PCa but at the same time, as a result of the low specificity, led to overdiagnosis, resulting to unnecessary biopsies and high medical cost treatments. The primary goal in PCa research today is to find a biomarker or biomarker set for clear and effecttive diagnosis of PCa as well as for distinction between aggressive and indolent cancers. Different proteomic technologies such as 2-D PAGE, 2-D DIGE, MALDI MS profiling, shotgun proteomics with label-based (ICAT, iTRAQ) and label-free (SWATH) quantification, MudPIT, CE-MS have been applied to the study of PCa in the past 15 years. Various biological samples, including tumor tissue, serum, plasma, urine, seminal plasma, prostatic secretions and prostatic-derived exosomes were analyzed with the aim of identifying diagnostic and prognostic biomarkers and developing a deeper understanding of the disease at the molecular level. This review is focused on the overall analysis of expression proteomics studies in the PCa field investigating all types of human samples in the search for diagnostics biomarkers. Emphasis is given on proteomics platforms used in biomarker discovery and characterization, explored sources for PCa biomarkers, proposed candidate biomarkers by comparative proteomics studies and the possible future clinical application of those candidate biomarkers in PCa screening and diagnosis. In addition, we review the specificity of the putative markers and existing challenges in the proteomics research of PCa.

Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Kasey C Vickers ◽  
Michael G Levin ◽  
Michael P Anderson ◽  
Qing Xu ◽  
Joshua Anzinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Cellular Response ◽  
Culture Media ◽  
Recipient Cell ◽  
Inflammatory Cells ◽  
High Density Lipoproteins ◽  
Small Rna Sequencing

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.

scholarly journals Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

2020 ◽  
Vol 21 (24) ◽  
pp. 9425
Author(s):  
Sebastian Sjoqvist ◽  
Kentaro Otake ◽  
Yoshihiko Hirozane
Keyword(s):  
Cerebrospinal Fluid ◽  
Extracellular Vesicles ◽  
Proteomic Analysis ◽  
Magnetic Beads ◽  
Biomarker Discovery ◽  
De Novo ◽  
Size Exclusion ◽  
Cell Regulation ◽  
Promising Source ◽  
Proximity Extension Assay

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

Performance of a high-intensity 508-gene circulating-tumor DNA (ctDNA) assay in patients with metastatic breast, lung, and prostate cancer.

2017 ◽  
Vol 35 (18_suppl) ◽  
pp. LBA11516-LBA11516 ◽  
Author(s):  
Pedram Razavi ◽  
Bob T. Li ◽  
Wassim Abida ◽  
Alex Aravanis ◽  
Byoungsok Jung ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Detection ◽  
High Intensity ◽  
Tumor Tissue ◽  
De Novo ◽  
Early Cancer ◽  
Metastatic Breast ◽  
Early Cancer Detection ◽  
Driver Mutations ◽  
Castration Resistant Prostate Cancer

LBA11516 Background: ctDNA assays can noninvasively assess tumor burden and biology by identifying tumor-derived somatic alterations. For broad applicability, including early cancer detection, an unprecedented high-intensity approach (ultra-deep sequencing of plasma cell-free DNA (cfDNA) with broad genomic coverage) is needed to address intra-patient and population-level heterogeneity. We present initial results with this approach in patients (pts) with metastatic breast (BC), non-small cell lung (NSCLC), and castration-resistant prostate cancer (CRPC). Methods: Blood and tissue were prospectively collected w/in 6 wks with no intervening therapy change from pts with de novo or progressive cancer. cfDNA and white blood cell (WBC) genomic DNA from each pt were sequenced with a 508-gene panel (2 Mb; >60,000X raw depth). cfDNA variant calling used molecular barcoding for error correction and filtering for WBC variants. Tissue was sequenced using the MSK-IMPACT assay (410 genes, 1.4 Mb, >500X depth) blinded to plasma/WBC sequencing. Variants were classified as clonal or subclonal based on tumor sequencing in BC and NSCLC. Results: Of 161 eligible pts, 124 (39 BC, 41 NSCLC, and 44 CRPC) were evaluable for concordance. In tissue, 864 variants were detected across the 3 tumor types, with 627 (73%) also detected in plasma: single nucleotide variants/indels - 75%, fusions - 67%, and copy number alterations - 58%. In 90% of pts, at least 1 of the variants detected in tumor tissue was also detected in plasma: BC - 97%, NSCLC - 85%, CRPC - 84%. Most actionable mutations detected in tissue were also detected in plasma (54/71, 76%; SNVs only: 28/31, 90%). A subset of driver mutations (eg. in ESR1, PIK3CA, ERBB2, EGFR) were observed in plasma but not tissue. Clonal variants in tissue were more likely to be detected in plasma than subclonal variants (p<.001). Conclusions: This novel, high-intensity ctDNA assay enabled broad detection of genomic variants in plasma at high rates of concordance with corresponding tumor tissue, providing strong evidence for tumor-derivation of these signals. This study will inform development of a high-intensity sequencing approach for early cancer detection.

scholarly journals Analysis of small RNA changes in different Brassica napus synthetic allopolyploids

2019 ◽  
Author(s):  
Yunxiao Wei ◽  
Fei Li ◽  
Shujiang Zhang ◽  
Shifan Zhang ◽  
Hui Zhang ◽  
...  
Keyword(s):  
Differential Expression ◽  
Small Rna ◽  
Target Genes ◽  
Enrichment Analysis ◽  
Small Rna Sequencing ◽  
Go Enrichment ◽  
Polyploid Formation ◽  
Differentially Expressed Mirnas ◽  
New Perspective ◽  
Go Enrichment Analysis

Allopolyploidy is an evolutionary and mechanisticaly intriguing process involving the reconciliation of two or more sets of diverged genomes and regulatory interactions, resulting in new phenotypes. In this study, we explored the small RNA changes of eight F2 synthetic B. napus using small RNA sequencing. We found that a part of miRNAs and siRNAs were non-additively expressed in the synthesized B. napus allotetraploid. Differentially expressed miRNAs and siRNAs differed among eight F2 individuals, and the differential expression of miR159 and miR172 was consistent with that of flowering time trait. The GO enrichment analysis of differential expression miRNA target genes found that most of them were concentrated in ATP-related pathways, which might be a potential regulatory process contributing to heterosis. In addition, the number of siRNAs present in the offspring was significantly higher than that of the parent, and the number of high parents was significantly higher than the number of low parents. The results have shown that the differential expression of miRNA lays the foundation for solving the trait separation phenomenon, and the significant increase of siRNA alleviates the shock of the newly synthesized allopolyploidy. It provides a new perspective of small RNA changes and trait separation in the early stages of allopolyploid polyploid formation.

scholarly journals Extracellular vesicles secreted during blastulation show viability of bovine embryos

Reproduction ◽  
2019 ◽  
Vol 158 (6) ◽  
pp. 477-492 ◽  
Author(s):  
Edwin A Mellisho ◽  
Mario A Briones ◽  
Alejandra E Velásquez ◽  
Joel Cabezas ◽  
Fidel O Castro ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Differential Expression Analysis ◽  
Binary Logistic Regression ◽  
Small Rna Sequencing ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Group V ◽  
Mean Size

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86–91%) and snoRNA (9–14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

scholarly journals Relationships of Alzheimer’s disease and apolipoprotein E genotypes with small RNA and protein cargo of brain tissue extracellular vesicles

2020 ◽  
Author(s):  
Yiyao Huang ◽  
Tom A. P. Driedonks ◽  
Lesley Cheng ◽  
Andrey Turchinovich ◽  
Harinda Rajapaksha ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Apoe Genotype ◽  
Tau Proteins ◽  
Small Rna Sequencing ◽  
Health Crisis ◽  
Metabolic Functions ◽  
Apolipoprotein E Genotypes

ABSTRACTAlzheimer’s disease (AD) is a public health crisis that grows as populations age. Hallmarks of this neurodegenerative disease include aggregation of beta-amyloid peptides and hyperphosphorylated tau proteins in the brain. Variants of the APOE gene are the greatest known risk factors for sporadic AD. As emerging players in AD pathophysiology, extracellular vesicles (EVs) contain proteins, lipids, and RNAs and are involved in disposal of cellular toxins and intercellular communication. AD-related changes in the molecular composition of EVs may contribute to pathophysiology and lend insights into disease mechanisms. We recently adapted a method for separation of brain-derived EVs (bdEVs) from post-mortem tissues. Using this method, we isolated bdEVs from AD patients with different APOE genotypes (n=23) and controls (n=7). bdEVs were counted, sized, and subjected to parallel small RNA sequencing and proteomic analysis. Numerous bdEV-associated RNAs and proteins correlated with AD pathology and APOE genotype. Some of the identified entities have been implicated previously in important AD-related pathways, including amyloid processing, neurodegeneration, and metabolic functions. These findings provide further evidence that bdEVs and their molecular cargo modulate development and progression of AD.

Sign in / Sign up

Export Citation Format

Share Document