scholarly journals Extracellular Vesicles Carry Distinct Proteo-Transcriptomic Signatures That are Different from Their Cancer Cell of Origin

2021 ◽  
Author(s):  
Tzu-Yi Chen ◽  
Edgar Gonzalez-Kozlova ◽  
Taliah Soleymani ◽  
Sabrina La Salvia ◽  
Natasha Kyprianou ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Extracellular Proteins ◽  
Small Rna Sequencing ◽  
Cell Of Origin ◽  
Non Invasive ◽  
Donor Cells ◽  
Delivery Mechanism

AbstractCirculating extracellular vesicles (EVs) contain molecular footprints from their cell of origin and may provide potential non-invasive access for detection, characterization, and monitoring of numerous diseases. Despite their growing promise, the integrated proteo-transcriptomic landscape of EVs and their donor cells remain poorly understood. To assess their cargo, we conducted small RNA sequencing and mass spectrometry (LC-MS/MS) of EVs isolated from in vitro cancer cell culture and prostate cancer patients’ serum. Here, we report that EVs enrich for distinct molecular cargo, and their proteo-transcriptome is predominantly different from their cancer cell of origin, implicating a coordinated disposal and delivery mechanism. We have discovered that EVs package their cargo in a non-random fusion, as their most enriched RNAs and proteins are not the most abundant cargo from their donor cells. We show that EVs enrich for 4 times more cytoskeletal and 2 times extracellular proteins than their donor cells. While the donor cells carry 10 times more mitochondrial and 3 times nuclear proteins than their EVs. EVs predominantly (40-60%) enrich for small RNA (~15-200 nucleotides) molecules that implicate cell differentiation, development, and signaling signatures. Finally, our integrated proteo-transcriptomic analyses reveal that EVs are enriched of RNAs (RNY3, vtRNA, and MIRLET-7) and their complementary proteins (YBX1, IGF2BP2, SRSF1/2), implicating an interrelated mechanism that may protect and regulate transcripts until a biological function is achieved. Based on these results, we envision that the next-generation clinical assays will take an integrative multi-omic (proteomic and transcriptomic) approach for liquid biopsy in numerous diseases.

scholarly journals xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

2018 ◽  
Author(s):  
Avi Z. Rosenberg ◽  
Carrie Wright ◽  
Karen Fox-Talbot ◽  
Anandita Rajpurohit ◽  
Courtney Williams ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Small Rna ◽  
Mirna Expression ◽  
Low Cost ◽  
Expression Patterns ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Cell Type

AbstractAccurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

scholarly journals Cancer Alters the Metabolic Fingerprint of Extracellular Vesicles

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3292
Author(s):  
Mari Palviainen ◽  
Kirsi Laukkanen ◽  
Zeynep Tavukcuoglu ◽  
Vidya Velagapudi ◽  
Olli Kärkkäinen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Extracellular Vesicles ◽  
Western Blotting ◽  
Cell Metabolism ◽  
Cancer Cell Lines ◽  
Cancerous Cell ◽  
Metabolic Fingerprint ◽  
And Control

Cancer alters cell metabolism. How these changes are manifested in the metabolite cargo of cancer-derived extracellular vesicles (EVs) remains poorly understood. To explore these changes, EVs from prostate, cutaneous T-cell lymphoma (CTCL), colon cancer cell lines, and control EVs from their noncancerous counterparts were isolated by differential ultracentrifugation and analyzed by nanoparticle tracking analysis (NTA), electron microscopy (EM), Western blotting, and liquid chromatography-mass spectrometry (LC-MS). Although minor differences between the cancerous and non-cancerous cell-derived EVs were observed by NTA and Western blotting, the largest differences were detected in their metabolite cargo. Compared to EVs from noncancerous cells, cancer EVs contained elevated levels of soluble metabolites, e.g., amino acids and B vitamins. Two metabolites, proline and succinate, were elevated in the EV samples of all three cancer types. In addition, folate and creatinine were elevated in the EVs from prostate and CTCL cancer cell lines. In conclusion, we present the first evidence in vitro that the altered metabolism of different cancer cells is reflected in common metabolite changes in their EVs. These results warrant further studies on the significance and usability of this metabolic fingerprint in cancer.

scholarly journals Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽  
2019 ◽  
Vol 147 (8) ◽  
pp. 855-864
Author(s):  
Collette Britton ◽  
Roz Laing ◽  
Eileen Devaney
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Parasitic Nematodes ◽  
Small Rna Sequencing ◽  
Small Interfering Rnas ◽  
Rna Sequences ◽  
C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

scholarly journals Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro

2020 ◽  
Vol 21 (17) ◽  
pp. 5951
Author(s):  
Laura Patras ◽  
Marcel H. A. M. Fens ◽  
Pieter Vader ◽  
Arjan Barendrecht ◽  
Alina Sesarman ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Extracellular Vesicles ◽  
Hypoxia Inducible Factor ◽  
B Cell Lymphoma ◽  
Tumour Microenvironment ◽  
Hypoxia Inducible Factor 1 ◽  
Apoptotic Protein ◽  
Donor Cells

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour–stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1α) induction and B-cell lymphoma–extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1α induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance.

scholarly journals The Biological Function of Extracellular Vesicles during Fertilization, Early Embryo—Maternal Crosstalk and Their Involvement in Reproduction: Review and Overview

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1510
Author(s):  
Emanuele Capra ◽  
Anna Lange-Consiglio
Keyword(s):  
Extracellular Vesicles ◽  
Spatial Interaction ◽  
Cell Communication ◽  
Early Embryo ◽  
Molecular Profile ◽  
Cell Of Origin ◽  
Non Invasive ◽  
Temporal And Spatial ◽  
Mediate Cell

Secretory extracellular vesicles (EVs) are membrane-enclosed microparticles that mediate cell to cell communication in proximity to, or distant from, the cell of origin. Cells release a heterogeneous spectrum of EVs depending on their physiologic and metabolic state. Extracellular vesicles are generally classified as either exosomes or microvesicles depending on their size and biogenesis. Extracellular vesicles mediate temporal and spatial interaction during many events in sexual reproduction and supporting embryo-maternal dialogue. Although many omic technologies provide detailed understanding of the molecular cargo of EVs, the difficulty in obtaining populations of homogeneous EVs makes difficult to interpret the molecular profile of the molecules derived from a miscellaneous EV population. Notwithstanding, molecular characterization of EVs isolated in physiological and pathological conditions may increase our understanding of reproductive and obstetric diseases and assist the search for potential non-invasive biomarkers. Moreover, a more precise vision of the cocktail of biomolecules inside the EVs mediating communication between the embryo and mother could provide new insights to optimize the therapeutic action and safety of EV use.

miR-200c in extracellular vesicles as a circulating biomarker of EGFR-TKI response and a mediator of EGFR-TKI sensitivity in heterogeneous NSCLC.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e20547-e20547
Author(s):  
Chienchung Lin ◽  
Wu-Chou Su
Keyword(s):  
Extracellular Vesicles ◽  
Mutant Cell ◽  
Poor Response ◽  
Wild Type Cell ◽  
Egfr Mutations ◽  
Small Rna Sequencing ◽  
Wild Type ◽  
Egfr Mutant ◽  
Egfr Tki

e20547 Background: Through intercellular transfer of EV(extracellular vesicles) miRNA, tumor cells can confer drug resistance to each other and contribute to tumor heterogeneity. However, the mechanisms why EGFR-TKI can be effective in heterogeneous NSCLC with low abundances of EGFR mutations remain unknown. The aim of the study is to investigate the significance of EV miRNA in mediating the efficacy of EGFR-TKI in heterogeneous NSCLC and serving as the biomarker of response to EGFR-TKI. Methods: We first evaluate if EVs from EGFR mutant cell (PC9) can affect EGFR-TKI sensitivity of EGFR wild type cell (CL1-5, H1299) in vitro co-culture system and i n vivo. We then identified the differential miRNA panel by comparing EVs from PC9 to those from CL1-5. Finally, we verified if the expressions level of these miRNA are different in blood EVs from patient with good response compared to those with poor response to EGFR-TKI. Results: We first verified that CL1-5 can take up labelled EVs from PC9 under time-lapse microscope and EGFR mutant DNA can be detected in recipient EGFR wild-type cell using digital PCR. We found EVs from PC9 enhanced gefitinib sensitivity of CL1-5. And co-culturing PC9 with CL1-5 enhanced CL1-5 gefitinib sensitivity which was reversed by adding GW4789, the inhibitor of exosome secretion. In subcutaneous CL1-5 animal model, in comparison to treating with gefitinib or PC9 EVs alone, only the combination treatment with gefitinib and PC9 EVs delayed cancer growth. Using small RNA sequencing, we identified a unique miRNA profile allowing discriminating EV from PC9 cells to those from CL1-5; MiRNA 200 family including 200a, 200b, 200c and mir429 were up-regulated significantly in PC9 EV. From Aug 2015 to Sep 2017, sixteen patients with good response (PFS > 12M) or poor response (PFS < 6M) to EGFR-TKI were enrolled and blood were collected for EV miRNA isolation. Ten of these blood samples were qualified for miRNA analysis and mir200a and 200c were found up-regulated in good responder to EGFR-TKI. The transfection of mir200c in CL1-5 cells not only inhibited the oncogenic pathway contributing to EGFR-TKI resistance including Stat3, Akt, EMT and BIM pathway but also enhanced gefitinib sensitivity of CL1-5 cells. Conclusions: Our data suggested mir200c from blood EV serve as a biomarker of response to EGFR-TKI and mir200c may mediate EGFR-TKI sensitivity in heterogenous EGFR mutant NSCLC.

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 Integrated mRNA and small RNA sequencing for analyzing tea leaf spot pathogen Lasiodiplodia theobromae, under in vitro conditions and the course of infection

2020 ◽  
Author(s):  
Shilong Jiang ◽  
Qiaoxiu Yin ◽  
Dongxue Li ◽  
Xian Wu ◽  
Yong Wang ◽  
...  
Keyword(s):  
Small Rna ◽  
Leaf Spot ◽  
Guizhou Province ◽  
Phytopathogenic Fungus ◽  
Future Research ◽  
Small Rna Sequencing ◽  
Rna Sequences ◽  
Lasiodiplodia Theobromae ◽  
Tea Leaf

Lasiodiplodia theobromae (Pat.) Griffon & Maubl. is a phytopathogenic fungus, which can cause many different diseases on different crops. The pathogen can cause leaf spot on tea plants (Camellia sinensis), which negatively affects the productivity and quality of tea leaves in tea plantations in Guizhou Province, China. Although the genome sequence of L. theobromae has been published, no data on the transcriptome or small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf are available. Here, we report the high-quality transcriptome and small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf using the platform of Illumina HiSeq. This comprehensive expression profiling of the fungal pathogen will provide a valuable resource for future research on trait-specific genes of the pathogen, host-pathogen interactions and on disease resistance in the host.

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.

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).

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