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. 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:Bladder cancer is one of the most common cancers. Since prognosis ameliorates with early detection, it is a challenge to develop techniques that could replace or complement the current diagnosis protocols. The study of extracellular vesicles (EVs) that are present in urine samples has become an attractive alternative. The present study describes the mRNA content of vesicles isolated from voided urine samples within bladder cancer context. To discover a genetic signature of cancer, RNA associated to EVs was analyzed by microarray technique. Total RNA isolated from Extracellular Vesicles obtained from urine of bladder cancer patients was compared with RNA isolated from urinary vesicles of non-cancer patients.
Project description:Extracellular vesicles (EVs) function through their cargos. This study aims to identify active components of EVs generated from mouse adipose-derived stem cells (ASCs) by comparing the small RNA compositions between EVs and ASCs as well as EVs generated from naive ASCs and primed ASCs (iASCs).
Project description:Background: Renal cell carcinoma (RCC) accounts for about 2% of all cancers. Renal biopsy is the gold standard among the diagnostic tools, but it is invasive and not suitable for all patients. Therefore, new reliable and non-invasive biomarkers for ccRCC detection are required. Secretion of extracellular vesicles (EVs), containing RNA molecules that can be transferred between cells, seems to be a general characteristic of malignant transformation. Consistently, cancer-derived EVs are enriched in the blood, urine and various malignant effusions of cancer patients. Therefore, urinary samples can be a non-invasive approach for discovering diagnostic biomarkers. Methods: We enrolled 33 clear-cell RCC (ccRCC) patients and 22 healthy subjects (HS), age and sex-matched, for urine collection and extracellular vesicles isolation by differential centrifugation. Transcriptional profiles of urinary EVs from 12 patients with ccRCC and 11 HS were generated using the Illumina HumanHT-12 v4 BeadChip oligonucleotide arrays. Microarray analysis led to the identification of RNA that were then validated using RT-qPCR. Results: We showed for the first time that urinary exosomal shuttle RNA (esRNA) was significantly different in ccRCC patients compared to HS and we identified three EVs esRNA involved in the tumor biology that are potentially suitable as non-invasive biomarkers. GSTA1, CEBPA and PCBD1 RNA levels decreased in urinary EVs of patients compared to HS. After 1 month post-operation, the levels of RNA increased to reach the normal level. Conclusions: This study suggests, for the first time, the potential use of the RNA content of urinary EVs to provide a non-invasive first step to diagnose the ccRCC. Total RNA obtained from urinary extracellular vesicles isolated from ccRCC patients and healthy subjects.
Project description:Exosomes and microvesicles (i.e., extracellular vesicles; EVs) have been identified within ovarian follicular fluid, and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, it remains unknown if EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle. Examination of small RNA population in bovine follicular fluid extracellular vesicles isolated from antral follicles
Project description:Transcriptional comparison of B16F10 cells with B16F10 cell-derived extracellular vesicles (EVs) to identify transcripts enriched or de-enriched in EVs compared to their donor cells.
Project description:Analysis of the response of macrophages treated with brown adipocyte extracellular vesicles (EVs) to decipher their possible immunomodulatory role
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:MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146 overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs. The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Cells were transfected with a plasmid to direct overexpression of miR-146a. Extracellular vesicles were isolated by ultracentrifugation from untreated and transfected cells. RNA was isolated from one sample each of untreated and transfected cells and vesicles.Small RNA libraries were prepared for sequencing.
Project description:RNA-seq was performed on cultured human induced pluripotent cell derived cardiomyocytes (iPSC-CMs). There are four groups, with three samples per group: H1,H2,H3. Negative control. Healthy, normoxic iPSC-CMs D1,D2,D3. Positive control. iPSCs cultured under hypoxic (0-1% O2) for 48h with 2% v/v PBS as vehicle control EV1,EV2,EV3. Treatment 1. iPSCs cultured under hypoxic (0-1% O2) for 48h, with cardiac stromal cell derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) EV21, EV23, EV24. Treatment 2 iPSCs cultured under hypoxic (0-1% O2) for 48h, with bone marrow mesenchymal stromal cell (BM-MSC) derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) All EVs were isolated from cultured human cells using sequential centrifugation methods. Cells were cultured using commercial EV-depleted FBS to avoid contamination of bovine EVs. EVs were validated for CD81, CD9, ALIX positivity, and visualised by cryoEM. Particle to protein ratios were not different between cardiac and bone marrow EV isolates. Therefore EV doses were standardised so that the same dose by protein (67ng/µl) and EV number (~2,000 EVs per iPSC-CM) were added.
Project description:The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs with the potential to mediate conceptus-maternal communication during early pregnancy. In Study One, EVs were purified from uterine luminal fluid (ULF) of day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study Two, day 14 conceptuses were cultured ex vivo for 24 hours and found to release EVs into the culture medium. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. No evidence of EV escape from the uterine lumen was observed by analysis of the ovary and other maternal tissues. Proteomics analysis of the day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes including proteases, protease inhibitors, chaperones and chaperonins. RNA-sequencing of day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top expressed genes were overrepresented in ribosomal functions and components. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication during the establishment of pregnancy. Transcriptional profiles from day 14 conceptus extracellular vesicles isolated from 24 hour conceptus-conditioned culture media (n=3) were generated by sequencing on the Illumina HiSeq 2500 platform.