Dataset Information

Renal Cell Carcinoma (Renca) Extracellular Vesicles under Hypoxia and Normoxia

ABSTRACT: Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles and provide a universal molecular cell-cell communication system. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support growth and invasion of tissues by tumor cells. EVs were purified from cell culture medium by ultracentrifugation followed by size exclusion chromatography with Exo-spin™ columns (Cell Guidance Systems Ltd). We performed proteomic analysis of five hypoxia/normoxia pairs of EV samples; each of these replicates for either hypoxia or normoxia was analyzed in duplicates. Obtained data were compared with proteomics analysis of corresponding cell culture media supernatants (SN) after EV removal with 100 000 g ultracentrifugation. We found that exposure of tumor cells to hypoxia (1% oxygen) induced EV secretion, altered EV sizes, and led to notable changes in the EV protein cargo in comparison to normoxia.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cortex Of Kidney, Kidney

DISEASE(S): Renal Cell Carcinoma

SUBMITTER: Anatoliy Samoylenko

LAB HEAD: Seppo Vainio

PROVIDER: PXD023546 | Pride | 2022-02-16

REPOSITORIES: Pride

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Publications

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles.

Samoylenko Anatoliy A Kögler Martin M Zhyvolozhnyi Artem A Makieieva Olha O Bart Geneviève G Andoh Sampson S SS Roussey Matthieu M Vainio Seppo J SJ Hiltunen Jussi J

Scientific reports 20211001 1

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell-cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics a ...[more]

PMID: 34599227

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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Project description:Extracellular vesicles (EVs) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. We have previously demonstrated that ultracentrifuged serum is depleted of EVs and, when used in cell culture media, is associated with declines in growth and viability of numerous cultured cell types. Although EVs had been reported to enhance or interfere with HIV-1 infection, depending on the setting, the effects of EVs on HIV-1 production and infectivity of released virions were unknown. In this study, we examined the effects of EV-depleted serum on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Cell culture media were prepared with EV-replete fetal bovine serum (FBS) or serum depleted of EVs via ultracentrifugation or a proprietary method (ThermoFisher/Gibco). T-cell and myeloid-lineage cell lines, including ACH-2 and U1 HIV-1 latency models, and primary cells were grown in 10% FBS-based culture media. Cell counts, viability, and proliferation were assessed throughout. HIV-1 production and infectivity were measured by p24 ELISA and luciferase reporter cell lines, respectively. Flow cytometry, Seahorse assays, and miRNA and mRNA expression arrays were done to assess cellular responses to EV-depleted conditions. Significant increases in HIV-1 production were observed in EV-depleted conditions, along with, in some cases, morphology changes and decreased cell viability. Add-back of pelleted EVs reduced HIV-1 production almost to baseline. Primary cells appeared to be less susceptible to EV depletion. ACH-2 and U1 latency models also produced more HIV-1 under EV-depleted conditions. Virus produced under EV-depleted conditions was more infectious. Changes in cellular metabolism and gene expression were associated with EV-depleted culture. The EV environment of HIV-1 infected cells appears to have a significant effect on virus production and infectivity. In cell lines of HIV-latency, significantly higher concentrations of p24 were observed in those cells cultured in EVD conditions. EV-dependence of cell cultures should be examined carefully prior to examining additional experimental variables. However, we also sound a cautionary note that direct actions of EVs may be accompanied by the effects of other, closely associated factors.

Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

Dataset Information

Renal Cell Carcinoma (Renca) Extracellular Vesicles under Hypoxia and Normoxia

Publications

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles.

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