Characterization of tumor extracellular vesicle RNA cargo
Ontology highlight
ABSTRACT: Comparative RNA profiling between tumor cells and their secreted extracellular vesicles. Results revealed enrichment in genes involved in cellular migration and metastasis in extracellular vesicles, in agreement with their role as mediators of tumor progression.
Project description:Comparative RNA profiling between tumor cells and their secreted extracellular vesicles. Results revealed enrichment in genes involved in cellular migration and metastasis in extracellular vesicles, in agreement with their role as mediators of tumor progression. Mice were orthotoplically transplanted with MDA-MB-231 Breast Adenocarcinoma cells. Cells and extracellular vesicles (EVs) from the resulting tumors were isolated. EVs were characterized by electron microscopy and Nanoparticle Tracking Analysis before total RNA isolation for comparative analysis with cellular RNA. Three biological replicates were analyzed in (technical) duplicate.
Project description:A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether T. cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16M-bM-^@M-^S40 nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells. Small RNAs profiles (16-40 nt) of epimastigote-derived extracellular vesicles, metacyclic trypomastigote-derived extracellular vesicles and metacyclic trypomastigote parental cells.
Project description:Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA (miRNA) and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.
Project description:We set out to identify the contents of extracellular vesicles in epithelial cells and how they differ in vesicles with enhanced antiviral activity.
Project description:A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether T. cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16–40 nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells.
Project description:Extracellular vesicles (EVs) are key mediators of intercellular communication, with important roles in numerous physiological and pathological processes, including profound effects on bone metabolism. These small membrane-bound vesicles are produced and released in the extracellular environment by virtually all cell types, including cells in the osteogenic lineage such as bone marrow-derived mesenchymal stem cells (MSCs), osteoblasts, osteoclasts and osteocytes. EVs serve as potent carriers of bioactive molecules, such as nucleic acids, proteins, lipids and metabolites, where they can influence recipient cells through fusion with target cell membranes to deliver these functional biomolecules. However, once released, the source cell of the EV is difficult to ascertain with any certainty. To overcome this obstacle, we developed a conditional (e.g. Cre-mediated) mouse model that expresses an EV tag, containing a fusion of CD81 and multiple C-terminal tags, termed the “Snorkel-tag”. By crossing with a Cre of interest, representing a specific cell-type or tissue, the specific EV subpopulations that are released can be isolated using antibody affinity columns. We crossed the CAGS-Snorkel mouse with Prx1- and Ocn-Cre, representing cell-types in the early vs late stages of osteoblast differentiation, isolated EVs from bone marrow plasma, and treated mouse bone marrow stromal cells (mBMSCs) with Prx1-EVs, Ocn-EVs or All-EVs (isolated using a Pan EV Isolation Kit [Miltenyi Biotec]) for 3 days and performed bulk RNA-sequencing. We found unique transcriptional and pathway signatures elicited by the different EV subpopulations in the mBMSCs, suggesting that EVs from diverse sources have distinct biological activities.
Project description:The biogenesis of extracellular vesicles (EVs) is a regulated process, driven by mechanisms at specific subcellular milieus. Sphingomyelinases (SMases), which metabolize sphingomyelin in membranes, play a role in EV biogenesis. Their metabolic product, ceramide, induces invaginations at the endosome or blebbing from the plasma membrane, both important in EV generation. Here, we sought to evaluate the impact of SMase inhibition on EV protein and RNA cargoes. For this, we treated human MCF7 cells with the neutral SMase (NSM) inhibitor GW4869 or the acid SMase (ASM) inhibitor FTY720. EVs were then purified from the conditioned media of control or inhibitor-treated cells and characterized by a variety of approaches, including LC-MS/MS and RNA-sequencing. SMase inhibition resulted in morphological and phenotypic changes in the heterogeneous EV population. Strikingly, NSM inhibition resulted in a depletion of nanoparticles, as well as a decrease in the RNA and protein content of EVs, with a marked reduction in endosomal, spliceosomal, and translation-related proteins. Furthermore, we observed a reduction in the overall RNA-binding proteins (RBPs) in EVs released by cells treated with the NSM-inhibitor. In contrast, the ASM-inhibitor treatment, which appears to reduce plasma membrane-derived vesicles, elicited an inverse response, leading to an increase of RBP and associated machineries within the released EV population. RNA sequencing of these EV revealed changes in the RNA biotypes composition, with an increase in protein coding transcripts. Interestingly, ASM-inhibitor resulting EVs induced increased cell migration and protein translation in recipient MCF10A cells. These results suggest that SMase-dependent vesiculation represents a major route of RBP and RNA trafficking outside the cell, via endosomal pathways.
Project description:We newly invented the EV sheet, which is a unique nanofiber paper to capture extracellular vesicles from around 10 uL of bio-fluids. In this study, we investigated miRNA profiles of tumor-surface ascites, serum, and urine using the EV sheet.