Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells.
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ABSTRACT:
SUBMITTER: Bonsergent E
PROVIDER: S-EPMC7994380 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundExtracellular vesicles are small vesicles that contain cytoplasmic and membrane components from their paternal cells. They enter target cells through uptake to transfer their biological cargo. In this study, we investigated the process of endothelial EV internalization and created a 3D visualization of their intracellular distribution.Methods and resultsTwo immortalized endothelial cell lines that express h-TERT (human telomerase) were used for EV release: microvascular TIME and macrovascular HUVEC. EVs were isolated from the cell culture medium via differential centrifugation and used for the uptake experiments. The size distribution of the EVs was measured using TRPS technology on a qNano instrument. Internalization of EVs was observed using a Zeiss LSM 710 confocal laser microscope after staining of the EVs with PKH26. EVs were observed intracellularly and distributed in the perinuclear region of the target cells. The distribution patterns were similar in both cell lines.ConclusionThe perinuclear localization of the internalized EVs shows their biological stability after their uptake to the endothelial cells. The 3D visualization allows the determination of a more accurate location of EVs relative to the donor cell nucleus.
| S-EPMC6296015 | biostudies-literature
Project description:Extracellular vesicles (EVs) are thought to mediate intercellular communication by transferring cargoes from donor to acceptor cells. The EV content-delivery process within acceptor cells is still poorly characterized and debated. CD63 and CD9, members of the tetraspanin family, are highly enriched within EV membranes and are respectively enriched within multivesicular bodies/endosomes and at the plasma membrane of the cells. CD63 and CD9 have been suspected to regulate the EV uptake and delivery process. Here we used two independent assays and different cell models (HeLa, MDA-MB-231 and HEK293T cells) to assess the putative role of CD63 and CD9 in the EV delivery process that includes uptake and cargo delivery. Our results suggest that neither CD63, nor CD9 are required for this function.
| S-EPMC10192366 | biostudies-literature
Project description:Extracellular vesicles (ECVs) facilitate intercellular communication along the nephron, with the potential to change the function of the recipient cell. However, it is not known whether this is a regulated process analogous to other signaling systems. We investigated the potential hormonal regulation of ECV transfer and report that desmopressin, a vasopressin analogue, stimulated the uptake of fluorescently loaded ECVs into a kidney collecting duct cell line (mCCDC11) and into primary cells. Exposure of mCCDC11 cells to ECVs isolated from cells overexpressing microRNA-503 led to downregulated expression of microRNA-503 target genes, but only in the presence of desmopressin. Mechanistically, ECV entry into mCCDC11 cells required cAMP production, was reduced by inhibiting dynamin, and was selective for ECVs from kidney tubular cells. In vivo, we measured the urinary excretion and tissue uptake of fluorescently loaded ECVs delivered systemically to mice before and after administration of the vasopressin V2 receptor antagonist tolvaptan. In control-treated mice, we recovered 2.5% of administered ECVs in the urine; tolvaptan increased recovery five-fold and reduced ECV deposition in kidney tissue. Furthermore, in a patient with central diabetes insipidus, desmopressin reduced the excretion of ECVs derived from glomerular and proximal tubular cells. These data are consistent with vasopressin-regulated uptake of ECVs in vivo We conclude that ECV uptake is a specific and regulated process. Physiologically, ECVs are a new mechanism of intercellular communication; therapeutically, ECVs may be a vehicle by which RNA therapy could be targeted to specific cells for the treatment of kidney disease.
| S-EPMC5084879 | biostudies-other
Project description:Extracellular vesicles (EVs) are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.
| S-EPMC4122821 | biostudies-literature
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.
2015-03-05 | E-GEOD-66488 | biostudies-arrayexpress
Project description:Extracellular vesicles (EVs) are nanometer-scale particles that are secreted by cells and mediate intercellular communication by transferring biomolecules between cells. Harnessing this mechanism for therapeutic biomolecule delivery represents a promising frontier for regenerative medicine and other clinical applications. One challenge to realizing this goal is that to date, our understanding of which factors affect EV uptake by recipient cells remains incomplete. In this study, we systematically investigated such delivery questions in the context of breast cancer cells, which are one of the most well-studied cell types with respect to EV delivery and therefore comprise a facile model system for this investigation. By displaying various targeting peptides on the EV surface, we observed that although displaying GE11 on EVs modestly increased uptake by MCF-7 cells, neuropeptide Y (NPY) display had no effect on uptake by the same cells. In contrast, neurotensin (NTS) and urokinase plasminogen activator (uPA) display reduced EV uptake by MDA-MB-231 cells. Interestingly, EV uptake rate did not depend on the source of the EVs; breast cancer cells demonstrated no increase in uptake on administration of breast cancer-derived EVs in comparison to HEK293FT-derived EVs. Moreover, EV uptake was greatly enhanced by delivery in the presence of polybrene and spinoculation, suggesting that maximal EV uptake rates are much greater than those observed under basal conditions in cell culture. By investigating how the cell's environment might provide cues that impact EV uptake, we also observed that culturing cells on soft matrices significantly enhanced EV uptake, compared to culturing on stiff tissue culture polystyrene. Each of these observations provides insights into the factors impacting EV uptake by breast cancer cells, while also providing a basis of comparison for systematically evaluating and perhaps enhancing EV uptake by various cell types.
| S-EPMC5689128 | biostudies-literature
Project description:The majority of colorectal cancer (CRC) patients carry mutations in the APC gene, which lead to the unregulated activation of the Wnt pathway. Extracellular vesicles (EV) are considered potential therapeutic tools. Although CRC is a genetically heterogeneous disease, the significance of the intra-tumor heterogeneity in EV uptake of CRC cells is not yet known. By using mouse and patient-derived organoids, the currently available best model of capturing cellular heterogeneity, we found that Apc mutation induced the expression of interferon-induced transmembrane protein 1 (Ifitm1), a membrane protein that plays a major role in cellular antiviral responses. Importantly, organoids derived from IFITM1high CRC cells contained more proliferating cells and they had a markedly reduced uptake of fibroblast EVs as compared to IFITM1low/- cells. In contrast, there was no difference in the intensity of EV release between CRC subpopulations with high and low IFITM1 levels. Importantly, the difference in cell proliferation between these two subpopulations disappeared in the presence of fibroblast-derived EVs, proving the functional relevance of the enhanced EV uptake by IFITM1low CRC cells. Furthermore, inactivating IFITM1 resulted in an enhanced EV uptake, highlighting the importance of this molecule in establishing the cellular difference for EV effects. Collectively, we identified CRC cells with functional difference in their EV uptake ability that must be taken into consideration when using EVs as therapeutic tools for targeting cancer cells.
| S-EPMC8558170 | biostudies-literature
Project description:The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.
| S-EPMC7249041 | biostudies-literature
Project description:Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads important in the intercellular signaling that coordinates the response of multiple cell types in cutaneous wound healing. Here we focus on the use of EV reporters to address cell type specificity and central questions regarding EV source cells, target cells, heterogeneity, payload, and activity using a combination of molecular approaches to address some of the barriers to translation in the use of engineered EVs. We identify a role for EVs released by resident macrophages present in adipose tissue and dermis to stimulate proliferation of overlying basal layer of keratinocytes. For these studies we use an allograft model, where donor EVs are harvested from subcutaneous implants, then purified for transplantation into the wound bed of recipient mice with a defined impaired wound healing phenotype. We demonstrate that EVs isolated from diabetic obese mice decreased keratinocyte proliferation and delayed wound closure, and that EVs loaded with specific miRNAs like miR-425-5p increased proliferation and accelerated wound closure. Utilizing a CD9-GFP reporter system, we show that fibroblasts uptake macrophage-derived EVs. In this work, we show that cell type-specific approaches can be used to unravel the biochemical activity of EVs in the complex microenvironment of the wound bed and form the basis for developing targeted pro-reparative EV payloads.
2024-02-16 | GSE242497 | GEO
Project description:Extracellular vesicles (EVs) are a group of nanoscale membrane-bound organelles including exosomes, microvesicles (MVs), membrane particles, and apoptotic bodies, which are released from almost all eukaryotic cells. Owing to their ingredients, EVs can be employed as biomarkers for human diseases. Interestingly, EVs show favorable features as candidates for targeted drug delivery and thus, they are suggested as ideal drug carriers as well as good vaccines for various human diseases including cancer. Among various drugs loaded in EVs for targeted drug delivery, immune checkpoint inhibitors (ICIs), including antibodies against programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), have attracted an increasing attention for cancer researchers and clinicians. Animal and clinical studies have shown combination of EVs and immunotherapy antibodies to improve the efficacy and reduce possible side effects in systemic administration of ICIs. In this review, we discuss the EVs and their significance in drug delivery with a focus on cancer immunotherapy agents.
| S-EPMC10234250 | biostudies-literature