Comparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysis

Abstract

Background: Extracellular vesicles (EVs) are nanoscale structures involved in intercellular communication and play a key role in cancer pathology. Intrahepatic cholangiocarcinoma (ICC) is a highly invasive malignancy marked by abnormal sialylated glycosylation. Analyzing proteins and glycans in EVs provides insights into ICC molecular subtyping and mechanisms. Optimizing EV isolation methods for ICC-derived EVs enables comprehensive proteomic and glycomic analysis.

Methods: We systematically evaluated five EV isolation methods-Ultracentrifugation (UC), exoEasy, Total Exosome Isolation (TEI), EVtrap, and ÄKTA-by analyzing the biophysical properties, proteomic profiles, and glycomic structures of EVs. Subsequently, we applied TMT-based quantitative proteome and light/heavy methylamine labeling for the quantification of sialylated N-glycan linkage isomers to investigate alterations in proteins and N-glycans within EVs secreted by HuCCT1 and HCCC-9810 cells with overexpressing ST6 β‑galactoside α2,6‑sialyltransferase 1 (ST6GAL1).

Results: By evaluating the biophysical properties, proteome, and N-glycome of EVs extracted using five different methods, UC was identified as the optimal approach for this study, as it offered a balance between operational complexity, cost-effectiveness, and the preservation of EVs activity. In this study, a total of 1,928 high-confidence proteins and over 84 high-confidence glycans were quantified. EVs secreted by HuCCT1 and HCCC-9810 cells overexpressing ST6GAL1 exhibited consistent upregulation of 16 proteins, consistent downregulation of 10 proteins, as well as consistent upregulation of 3 glycans and consistent downregulation of 3 glycans.

Conclusions: Quantitative proteomic and glycomic analysis of ICC-derived EVs revealed that ST6GAL1 overexpression led to significant alterations in proteins involved in cancer cell adhesion and glycosylation pathways, along with specific changes in N-glycan structures. Notably, these modifications extended beyond α2,6-sialylation, suggesting that interactions between glycosyltransferases and glycans may drive these alterations.

Keywords: Cell culture supernatants; Extracellular vesicles; N-glycome; Proteome.

Fig. 1

Schematic overview of the experimental evaluation workflow for EV isolation methods

Fig. 2

valuation of EV isolation methods. A Particle size distribution and representative TEM image of EVs (scale bar: 100 nm). B EV size distributions. C EV concentrations. D Particle count to protein ratio. E NanoFCM analysis of CD9, CD81, and CD63 tetraspanins. F Quantification of tetraspanin markers by NanoFCM. Data were analyzed by one-way ANOVA with Tukey’s HSD for multiple comparisons (n = 3) [16]. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Proteomic analysis of EVs isolated by different methods. A Protein identification comparison across methods. B Stacked bar chart of proteins identified in three technical replicates, color-coded by replicate count. C Venn diagram showing overlap of EV proteins identified by different methods. D Venn diagram comparing identified proteins with Vesiclepedia, ExoCarta, and top 100 proteins. E 3D-PCA of proteomes from EVs showing five clusters based on three principal components. F GO analysis of shared proteins by biological process, cellular component, and molecular function. G KEGG pathway analysis of shared proteins

Fig. 4

Glycomic analysis of EVs isolated by different methods. MALDI-MS spectra of glycans from EVs isolated by UC (A), exoEasy (B), TEI (C), EVtrap (D), and ÄKTA (E). Glycan peaks are marked with “*”. Red squares represent glycans identified from UC, blue squares from exoEasy, orange squares from TEI, green squares from EVtrap, purple squares from ÄKTA, and white squares indicate unidentified glycans

Fig. 5

Comparison of EVs internalization by HuCCT1 cells isolated by different methods. A Fluorescence images of EVs labeled with DAPI (blue, 0.5 μg/mL) and PKH67 (green, 100 μM) after incubation with HuCCT1 cells. B Mean fluorescence intensity of PKH67-labeled EVs after 6 h of incubation with HuCCT1 cells. **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Quantitative proteomics and bioinformatics analysis of EV proteins. A Volcano plot comparing the EV proteins between HuCCT1-ST6GAL1 and HuCCT1-Vector. B Volcano plot comparing the EV proteins between HCCC- 9810-ST6GAL1 and HCCC- 9810-Vector. C GO analysis of EV proteins significantly upregulated or downregulated in HuCCT1-ST6GAL1. D GO analysis of EV proteins significantly upregulated or downregulated in HCCC- 9810-ST6GAL1. E KEGG pathway analysis of EV proteins significantly upregulated or downregulated in HuCCT1-ST6GAL1. F KEGG pathway analysis of EV proteins significantly upregulated or downregulated in HCCC- 9810-ST6GAL1

Fig. 7

Quantitative glycomics analysis. A MALDI-TOF MS analysis of sialylated N-glycan isomers in EVs derived from HuCCT1-ST6GAL1 and HuCCT1-Vector, with an m/z range of 1000–4000. Selected N-glycan structures are highlighted with their corresponding MS peaks, where red indicates upregulated N-glycans and green indicates downregulated N-glycans. B The distribution of abundance proportions of different N-glycan types

Fig. 8

Differential and pathway analysis of all N-glycans in EVs from ICC cells. N-glycans with different numbers of monosaccharides are displayed in separate columns. The color scheme in the figure conveys specific meanings: dark blue represents N-glycans uniquely detected in EVs derived from ST6GAL1-overexpressing cells; light blue indicates N-glycans with higher expression levels in ST6GAL1-overexpressing EVs; dark red denotes components exclusively found in EVs from vector-transfected cells; and light red indicates N-glycans with lower expression levels in ST6GAL1-overexpressing EVs. Lines connecting different N-glycans signify monosaccharide additions: the red line indicates the addition of NeuAc (N-acetylneuraminic acid), the green line represents dHex (fucose), the blue line denotes HexNAc (N-acetylhexosamine), and the orange line corresponds to Hex (hexose)