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, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-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.

Figure 1

Uptake of SKOV3 exosomes by SKOV3 cells. (A) SKOV3 cells were incubated with Exos-CFSE (20 μg protein; green) for 4 h and were visualized in bright-field merged with fluorescence microscopy. Scale bar = 20 μm. (B) Detection of Exos-CFSE (green) and alpha-tubulin (red) by confocal immunofluorescence microscopy. Scale bar = 10 μm.

Figure 2

Path of internalization of exosomes in SKOV3 cells. (A) Exos-CFSE (20 μg protein; green) were incubated with SKOV3 cells at 37 or 4°C and uptake was monitored by flow cytometry analysis of cell fluorescence intensity. Solid and dashed lines represent Exos-CFSE uptake at 37 and 4°C, respectively. (B) Colocalization of Exos-CFSE (20 μg protein; green) with EEA1 (red), LAMP1 (red) and caveolin-1 (red). Secondary antibody was donkey anti-mouse IgG AlexaFluor 594. Colocalization is indicated in yellow. Images in the left bottom represent 4 × magnifications of selected areas. Scale bars = 10 μm. (C) Effect of chlorpromazine, cytochalasin D, EIPA and methyl-beta-cyclodextrin (30 min pre-incubation) on Exos-CFSE uptake (4, 4, 4 and 2 h, respectively) monitored by flow cytometry analysis (dashed lines). Controls consist of SKOV3 cells with no treatment for chlorpromazine and methyl-beta-cyclodextrin, or treated with DMSO for cytochalasin D and EIPA (solid lines). Unlabelled SKOV3 cells (grey) were used as control for cell autofluorescence. The results shown are representative of three independent experiments.

Figure 3

Effect of proteinase K treatment in SKOV3 exosomes uptake. (A) Exos-CFSE (20 μg protein; green) or (B) SKOV3 cells were treated with 100 μg/ml proteinase K for 30 min. Uptake was determined after 4 h of incubation by flow cytometry analysis and compared with uptake of Exos-CFSE with no treatment (solid lines). Unlabelled SKOV3 cells (grey) were used as control for cell autofluorescence. Dashed lines represent Exos-CFSE uptake after proteinase K digestion. The results shown are representative of three independent experiments performed in duplicate.

Figure 4

Western blot and lectin detection of glycoproteins from SKOV3 cellular extracts (Ext) and secreted vesicles (Ves). (A) Con A, SNA and MAL lectin detection in SKOV3 cellular extract and vesicles. Three μg total protein were applied per lane. As positive controls, 200 ng ribonuclease B (RNase B) [41], human plasma transferrin (TFR) [42] and erythropoietin (EPO_BRP) [43] were used. (B) SNA and MAL lectin analysis of desialylated SKOV3 cellular extracts and vesicles. Total proteins (3 μg) were digested with neuraminidases from V. cholerae (Vb), A. urefaciens (Au) and S. pneumonia (Sp). Input consisted of cellular extracts and exosomes without treatment. As loading control L1 was detected. (C) Vesicles from 1.5 × 10⁷ SKOV3 cells were fractionated in a sucrose gradient. Cellular extracts (Ext), secreted vesicles from 100,000 × g pellet (Ves), pooled fractions 2-5 (F2-5) (3 μg total protein), 20% of F1, F6-7, F8-11 and F12 were analysed. As positive control for exosomes, CD9 was detected. Detection was performed using the chemiluminescent method.

Figure 5

Con A, SNA and MAL lectin detection of glycoproteins from HEK293 and H4 cellular extract (Ext) and secreted vesicles (Ves). For the analysis, 3 μg of total protein were applied per lane. Detection was performed using the chemiluminescent method.

Figure 6

Effects of neuraminidase and sugars on SKOV3 exosomes uptake. (A) Exos-CFSE (20 μg protein) were incubated with 15 mU V. cholerae neuraminidase or corresponding buffer for 2 h before Exos-CFSE uptake. (B) Effect of 150 mM D-glucose (Glc), α-L-fucose (Fuc), α-D-mannose (Man), β-lactose (Lac), D-galactose (Gal) and D-N-acetylglucosamine (GlcNAc) (30 min pre-incubation) on Exos-CFSE uptake (4 h) monitored by flow cytometry analysis. Uptake efficiency was calculated relatively to the uptake without treatment, considered as 100%. Results are displayed in relative percentages ± S.D. The results shown are representative of three independent experiments performed in duplicate.