Fig 1.
Purification and identification of EVs derived from C. sinensis.
(A) CsEVs isolated from the liver of infected cats. Scale bar: 1 cm. (B) Procedure for purification of CsEVs using differential ultracentrifugation. (C) Morphological identification of CsEVs by negative-staining TEM, and typical vesicular structures can be observed. Scale bar: 200 nm. (D) Nanoparticle tracking analysis of purified CsEVs. The concentration and size distribution were plotted with peaks labeled with nm size.
Fig 2.
Bioinformatics analysis of the proteins contained in CsEVs.
(A) Subcellular location of the CsEVs proteins. Bar graph representing the abundance of CsEVs proteins at different subcellular locations. (B) Gene ontology (GO) annotation of the CsEVs proteins. Enrichment analyses by biological process, cell component, and molecular function, respectively. (C) Bubble map of KEGG pathway of the CsEVs proteins (Top 20). X-axis indicates the rich factor; Y-axis indicates the terms of KEGG pathway. The dot size means the protein number and dot color indicates the p-value. (D) Distribution of the enriched KEGG pathways (Top 20). Bar chart showing the abundance of CsEVs proteins at different pathways.
Fig 3.
CsEVs can be internalized by CCA cells.
CsEVs were stained with PKH26 and then incubated with RBE and HuCCT1 cells for 12 h. Laser scanning confocal microscope was used to detect the CsEVs internalization. Bright field showed the size and ratio of nuclei to cytoplasm in RBE and HuCCT1 cells. Scale bar: 100 μm. Red fluorescence representative PKH26-labeled CsEVs, and the blue are cell nuclei. As shown in the images, CsEVs were localized in the cytoplasm surrounding the nucleus. Scale bar: 50 μm.
Fig 4.
CsEVs induce the malignant proliferation of CCA cells.
(A) CCK8 assay was employed to detect cell proliferation. RBE and HuCCT1 cells were treated with different concentrations of CsEVs, respectively, and detected at 0, 24, 48 and 72 h after treatment. (B) EdU-488 assay and (C) colony formation assay were conducted to determine the proliferation capacity of RBE and HuCCT1 cells after treatment with or without 10 μg/ml of CsEVs for 48 h, respectively. Representative images were shown and the summary results were from three independent experiments. Scale bar: 100 μm. (D) Cell cycle distribution was analyzed after treatment with or without 10 μg/ml of CsEVs for 48 h by flow cytometry. The percentage of cell cycle phase and the proliferation index (PI) were calculated and shown. PI = (S+G2/M)/(G0/G1+S+G2/M).
Fig 5.
CsEVs are involved in regulating the expression of cell cycle-related molecules.
(A, C) The relative mRNA expression of cyclinD1, cyclinD3, CDK4, CDK6, and CDK2 were detected in RBE and HuCCT1 cells after treatment with or without 10 μg/ml of CsEVs for 48 h using RT-qPCR. Equal volume PBS-treated group was used as negative control, and the mRNA fold change was normalized to β-actin. (B, D) The relative protein levels of cyclinD1, cyclinD3, CDK6, and CDK2 were detected in RBE and HuCCT1 cells after treatment with or without 10 μg/ml of CsEVs for 48 h using western blot. Representative images were shown and the quantification of protein expression was measured by Image J and normalized to GAPDH. All experiments were conducted three times.
Fig 6.
CsEVs promote the migration and invasion of CCA cells.
(A) Wound healing assay for RBE and HuCCT1 cells showed motility changes after treatment with or without 10 μg/ml of CsEVs for 24 h. Images were taken at 0 h, 24 h, and 48 h after scratching, and representative pictures were shown. Scale Bar: 400 μm. (B) Transwell assay was conducted to determine the migration and invasion abilities of RBE and HuCCT1 cells after treatment with or without 10 μg/ml of CsEVs for 24 h. Representative images of matrigel-free (for migration) and matrigel-coated (for invasion) transwell assay were shown. Three independent experiments were implemented for each group. Scale Bar: 200 μm. (C) The in vivo lung metastasis model. Representative images of specimens and H&E staining images of lung metastasis for the indicated RBE cells were shown. Table shows incidences of lung metastasis in the different groups. Histogram shows number of metastatic nodules in the different groups. Scale Bar: 200 μm.
Fig 7.
CsEVs can induce EMT in CCA cells, and Slug is essential for CsEVs -mediated EMT and CCA metastasis.
(A) The expression of E-cadherin, N-cadherin, vimentin, and slug were detected in RBE and HuCCT1 cells after treatment with or without 10 μg/ml of CsEVs for 48 h by western blot. Representative images were shown and the quantification of protein expression was measured by Image J and normalized to GAPDH. (B) RBE and HuCCT1 cells were pretreated with 10 μg/ml of CsEVs for 24 h, followed by transfection with Slug siRNA for 48 h. siCon was used as a transcriptional negative control. Western blot showed that Slug knockdown reversed the levels of the EMT-related proteins affected by CsEVs treatment. (C) The relative mRNA expression of Slug was measured in transfected CCA cells using RT-qPCR, and the mRNA fold change was normalized to β-actin. (D) After the indicated treatment, representative images of transwell assays demonstrated that downregulated Slug reversed the number of migrated and invasive CCA cells pretreated with CsEVs. All experiments were repeated three times. Scale Bar: 200 μm.
Fig 8.
The NF-κB and ERK signaling pathways were involved in CsEVs- mediated EMT and malignant phenotypes.
(A) The expression of key proteins in NF-κB pathway were detected in RBE and HuCCT1 cells after treatment with or without 10 μg/ml of CsEVs for 48 h by western blot. Representative images were shown and the quantification of protein expression was measured by Image J. (B) The phosphorylation of ERK was determined by western blot after the indicated treatment. (C, D) RBE and HuCCT1 cells were pretreated with 10 μM BAY 11–7082 (a NF-κB inhibitor) for 1 h, followed by stimulation with 10 μg/ml of CsEVs for 24 h. Then, western blot assay was conducted to evaluate the expression levels of p-p65/p65, N-cadherin and vimentin. (E) EdU-488 assay and (F) Transwell assay were used to determine the proliferation and migration ability of RBE and HuCCT1 cells after treatment with NF-κB inhibitor. All assays were executed for three times.
Fig 9.
Schematic diagram of CsEVs in promoting malignant proliferation and metastasis of CCA cells.
EVs isolated from the culture supernatant of C. sinensis adult worms could facilitate the abnormal proliferation of CCA cells by regulating cell cycle progression, and promote the migration, invasion and EMT process through activating the NF-κB signaling pathway.