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Interaction and uptake of exosomes by ovarian cancer cells.

ABSTRACT: BACKGROUND:Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells. METHODS:SKOV3 exosomes were labelled with carboxyfluorescein diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts. RESULTS:In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, ?-L-fucose, ?-D-mannose, D-N-acetylglucosamine and the disaccharide ?-lactose reduced exosomes uptake to a comparable extent as the control D-glucose. CONCLUSIONS:In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.

SUBMITTER: Escrevente C

PROVIDER: S-EPMC3072949 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Interaction and uptake of exosomes by ovarian cancer cells.

Escrevente Cristina C Keller Sascha S Altevogt Peter P Costa Júlia J

BMC cancer 20110327

<h4>Background</h4>Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells.<h4>Methods</h4>SKOV3 exosomes were labelled with carboxyfluorescein diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated ...[more]

PMID: 21439085

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Project description:BACKGROUND:How exosomal microRNAs (miRNAs) derived from macrophages contribute to the development of drug resistance in the context of the hypoxic tumor microenvironment in epithelial ovarian cancer (EOC) remains poorly understood. METHODS:The miRNA levels were detected by qRT-PCR. Protein levels of HIF-1α, CD163 and PTEN-PI3K/AKT pathway were assessed by Western blot (WB) and Immunohistochemistry (IHC). Exosomes were isolated, and then confirmed by Transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and WB. Internalization of macrophages-secreted exosomes in EOC cells was detected by Confocal microscope. Subsequently, Dual-luciferase reporter assay verified PTEN was the target of miR-223. Gain- and loss-of-function experiments, rescue experiments, and SKOV3 xenograft models were performed to uncover the underlying mechanisms of miR-223 and PTEN-PI3K/AKT pathway, as well as the exosomal miR-223 in inducing multidrug resistance in vitro and in vivo. RESULTS:Here, we showed hypoxic EOC cells triggered macrophages recruitment and induced macrophages into a tumor-associated macrophage (TAM)-like phenotype; exosomes derived from hypoxic macrophages enhanced the malignant phenotype of EOC cells, miR-223 was enriched in exosomes released from macrophages under hypoxia, which could be transferred to the co-cultivated EOC cells, accompanied by enhanced drug resistant of EOC cells. Besides, results from a functional assay revealed that exosomal miR-223 derived from macrophages promoted the drug resistance of EOC cells via the PTEN-PI3K/AKT pathway both in vivo and in vitro. Furthermore, patients with high HIF-1a expression had statistically higher CD163+ cell infiltration and intertumoral levels of miR-223. Finally, circulating exosomal miR-223 levels were closely related to the recurrence of EOC. CONCLUSIONS:These data indicate a unique role of exosomal miR-223 in the cross-talk between macrophages and EOC cells in chemotherapy resistance, through a novel exosomal miR-223/PTEN-PI3K/AKT signaling pathway.

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Interaction and uptake of exosomes by ovarian cancer cells.

Publications

Interaction and uptake of exosomes by ovarian cancer cells.

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