Extracellular vesicles of Clonorchis sinensis promote the malignant phenotypes of cholangiocarcinoma via NF-κB/EMT axis

Xiaowen Pan; Qing He; Yingxuan Yin; Anyuan Xu; Aoxun Wu; Xueqing Yi; Zifeng Zhong; Yinjuan Wu; Xuerong Li

doi:10.1371/journal.pntd.0012545

Abstract

Clonorchis sinensis infection is an important risk factor for cholangiocarcinoma (CCA). It has been reported that extracellular vesicles (EVs) are involved in the parasite-host interaction, and EVs of C. sinensis (CsEVs) can contribute to biliary injuries and inflammation. However, uncertainty surrounds the function of CsEVs in the progression of CCA. In this study, differential ultracentrifugation was used to separate CsEVs from the culture supernatant of C. sinensis adult worms, and they were then identified by transmission electron microscopy, nanoparticle tracking analysis and proteome assays. CCK8, EdU-488 and colony formation assays were used to explore the effect of CsEVs on the proliferation of CCA cells in vitro. Wound healing assays, transwell assays and in vivo lung metastasis model were conducted to evaluate the migration and invasion abilities. Moreover, the involvement of EMT process, as well as NF-κB and ERK signaling pathway was assessed. Results showed that CsEVs were successfully isolated and could be taken up by CCA cells, which promoted proliferation by accelerating cell cycle progression. In addition, CsEVs could facilitate cell metastasis by triggering the epithelial-mesenchymal transition (EMT). Mechanistically, activation of NF-κB signaling pathway was involved in the CsEVs-mediated EMT, which could be reversed partly by BAY 11–7082 (an inhibitor of NF-κB). In conclusion, these findings suggested that CsEVs could induce the aberrant proliferation and metastasis of CCA cells by stimulating the NF-κB/EMT axis, providing a novel theoretical explanation for liver fluke-associated CCA.

Author summary

C. sinensis infection is closely related to the occurrence of CCA, and the International Agency for Research on Cancer (IARC) has classified C. sinensis as a class I carcinogen. The exact mechanism of C. sinensis causing CCA remains to be elucidated, which may be the result of the synergistic effect of multiple factors such as mechanical damage caused by the migration of C. sinensis adult worms, secondary bacterial infection and inflammatory response, toxicity of the excretory-secretory products, abnormal host immune response and gene mutations. Recently, extracellular vesicles have emerged as a potential mechanism participating in parasite-host interaction, and CsEVs may play an important role in the pathogenesis and carcinogenesis of C. sinensis. In this study, it was discovered that 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. Furthermore, the protein expression profile of CsEVs is revealed by this study, which provides a new perspective for the study of liver fluke-associated CCA and may therefore contribute to the development of novel therapeutic approaches to treat CCA caused by C. sinensis infection.

Citation: Pan X, He Q, Yin Y, Xu A, Wu A, Yi X, et al. (2024) Extracellular vesicles of Clonorchis sinensis promote the malignant phenotypes of cholangiocarcinoma via NF-κB/EMT axis. PLoS Negl Trop Dis 18(10): e0012545. https://doi.org/10.1371/journal.pntd.0012545

Editor: Javier Sotillo, Instituto de Salud Carlos III, SPAIN

Received: April 1, 2024; Accepted: September 17, 2024; Published: October 28, 2024

Copyright: © 2024 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting information files.

Funding: This work was supported by the National Key Research and Development Program of China (grant no. 2021YFC2300800, 2021YFC2300801), Natural Science Foundation of Guangdong Province (grant no. 2023A1515010955), Guangzhou Municipal Science and Technology Project (grant no. 202201011723) to XL, Natural Science Foundation of Guangdong Province (grant no. 2022A1515012560) to YW, and the Sun Yat-sen Creative Project (grant no. 202310706) to XY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors declare that they have no conflict of interest.

Introduction

Clonorchis sinensis, also known as liver fluke, is a neglected food-borne zoonotic parasite that causes clonorchiasis. Eating raw or undercooked freshwater fish or shrimp harboring live metacercariae frequently triggers infection in humans and other mammals [1]. It is speculated that 15 million people are infected around the world, with the majority of cases occurring in China, South Korea, and Vietnam, and more than 200 million individuals are thought to be at risk of infection [1–3]. C. sinensis adults, which inhabit and lay eggs in the bile duct, can inflame the liver and bile duct system and result in biliary epithelial hyperplasia, periductal fibrosis, hepatic fibrosis, cirrhosis and even cholangiocarcinoma (CCA) [4]. The International Agency for Research on Cancer (IARC) has classified C. sinensis as a Class I carcinogen due to its close association with CCA [5]. However, the underlying mechanism of CCA induced by C. sinensis has not been fully elucidated.

Epithelial-mesenchymal transition (EMT) is a crucial process in embryonic development, chronic inflammation, tissue fibrosis and cancer metastasis [6]. At inflammation and wound healing sites, EMT is greatly in favor of reestablishing epithelial and endothelial integrity. However, during the pathological process of tumor progression and metastasis, abnormal reactivation of EMT is related to the malignant properties of tumor cells, including enhancing the invasiveness and migration capability, increasing tumor stemness, and strengthening resistance to chemotherapy and immunotherapy [6–8]. The participation of EMT in the progression of CCA has been proposed to be strongly supported by mounting molecular and cellular evidence [9,10]. However, the relation between EMT and liver fluke-associated CCA is still poorly known and needs further research. It has been demonstrated that excretory-secretory products of C. sinensis (CsESPs) facilitate the migration and invasion of CCA cells [11]. Furthermore, our previous study has proved that C. sinensis granulin (CsGRN), a component of CsESPs, can promote the metastasis of CCA and hepatocellular carcinoma via EMT process [12–14].

Extracellular vesicles (EVs) are vesicular particles that are released by a wide range of living cells, containing many significant bioactive substances, such as proteins, lipids, and nucleic acids, which are involved in mediating intercellular communication, immunological regulation, as well as other physiological and pathological processes [15]. Increasing evidence points to the possibility that helminths can utilize EVs to facilitate parasite-host interaction and promote their survival and infectivity [16]. Yan et al. [17] discovered that CsEVs had a typical saucer-like structure with a diameter of 30~150 nm, and could promote bile duct injury and the activation of M1-type inflammatory macrophages by delivering microRNA Csi-let-7a-5p targeting at the Clec7a and SOCS1 mediated NF-κB signaling pathway. Another microRNA Csi-miR-96-5p, delivered by CsEVs, can promote tumor proliferation and migration through ferroptosis by regulating the expression of the PTEN/SLC7A11/GPX4 axis [18]. Moreover, CsEVs can promote bile duct epithelial cells to secrete IL-6 and TNF-α by activating Toll-like receptor (TLR) mediated ERK signaling pathway [19]. These results suggest that CsEVs may play a crucial role in the pathogenesis of C. sinensis, and the proteins and nucleic acids carried by CsEVs may be the key pathogenic molecules. However, the role of CsEVs in promoting the malignant phenotypes of liver fluke-associated CCA remains poorly understood, and the protein composition of CsEVs has not been reported.

In this study, we aimed to elucidate the impact of CsEVs on the malignant progression of CCA and reveal the protein profile of CsEVs to better understand the molecular mechanisms. It was verified that CsEVs could facilitate the aberrant proliferation and metastasis of CCA cells by activating NF-κB and ERK signaling pathway, where EMT played a prominent role. Our findings provide a new perspective for the study of liver fluke-associated CCA and may therefore contribute to the development of novel therapeutic approaches to treat CCA caused by C. sinensis infection.

Materials and methods

Ethics statement

All experimental procedures were approved by the Research Ethics Board of Zhongshan School of Medicine. The animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (SYSU-IACUC-2024-000343). All animal studies have followed the ARRIVE guidelines.

Cell culture and transfection

Human CCA cell lines (RBE and HuCCT1) used in our study were acquired from the Center of Hepato-Pancreato-Biliary Surgery, the First Affiliated Hospital of Sun Yat-sen University. RBE and HuCCT1 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (P/S, Hyclone, USA), and maintained at 37°C in an incubator with 5% CO₂.

Slug-specific small-interfering RNA (siSlug) and scramble negative control of small-interfering RNA (siCon) were designed and synthesized by HanyiBio (Guangzhou, China) (S1 Table). A total of 5×10⁵ CsEVs pretreated RBE and HuCCT1 were transfected using Lipofectamine 3000 (Invitrogen, California, USA) at a final concentration of 100 nM according to the manufacturer’s recommendations. After 48 h of transfection, the cells were collected, and Slug expression was detected by RT-qPCR.

C. sinensis and CsEVs preparation

To prepare CsEVs, adult worms of C. sinensis were extracted from the liver of infective cats purchased from the slaughter house. After being washed five times by sterilized phosphate buffer saline (PBS) containing 5% P/S, the adult worms were transferred to DMEM medium (Gibco, Carlsbad, USA) supplemented with 1% P/S for culture at 37°C with 5% CO₂ (20~30 worms/2 ml medium in a well). After that, the culture supernatant was collected for CsEVs isolation via differential ultracentrifugation as previously described with some minor adjustments [17,20].

Briefly, the culture supernatant was first centrifuged at 1 500g for 30 min at 4°C, and then the resulting supernatant was centrifuged at 3 500g for another 30 min at 4°C to eliminate worm eggs and excreting waste. Next, the supernatant was gathered and centrifuged at 12 000g for 30 min at 4°C, followed by centrifugation at 20 000g for 1 h at 4°C. Then, the obtained supernatant was filtered through a 0.22 μm PES membrane (Merck Millipore, USA). After that, the supernatant was transferred to the thick wall centrifuge tubes (Beckman Coulter, USA) and then centrifuged at 120 000g for 70 min at 4°C twice in an Optima XE-100 tabletop ultracentrifuge (Swinging bucket rotor, model SW32 Ti, Beckman Coulter, USA). The resultant precipitate containing CsEVs was resuspended in 100 μL PBS and the protein concentration was assessed with a BCA Protein Assay Kit (Thermo Scientific, USA).

Transmission electron microscopy (TEM)

The morphology of CsEVs was observed by negative-staining TEM. After the ultrafast centrifugation, 20 μL of CsEVs solution was immediately applied to a copper grid and left to rest for 1 min at room temperature (RT). The excess liquid was absorbed by filter paper, and then the grid was negatively stained with 3% phosphotungstic acid aqueous solutions for 1 min and dried at RT. Following, the grid containing CsEVs was imaged using TEM (FEI Tecnai G2 Sprit Twin TEM at 80 kV, USA).

Nanoparticle tracking analysis (NTA)

The size distribution and concentration of CsEVs were detected by NTA (NanoSight NS300, Malvern Instruments, UK). In brief, CsEVs were diluted 100-fold with ddH₂O to reach the detection range of the instrument, and then CsEVs diluent was drawn with a 1ml syringe and slowly injected into the sample chamber after it had been cleaned with filtered ddH₂O. Each sample was measured in triplicate with a 488 nm laser (Blue) and a high-sensitivity sCMOS camera at a camera level of 16 with an acquisition duration of 40 s and a detection threshold setting of 6. The data was analyzed by NTA 3.3 Dev Build 3.3.301 software.

Proteome assays

The proteome analysis of CsEVs was carried out as previously mentioned [21,22]. In brief, protein sample was extracted from CsEVs using an ultrasonic processor in the presence of lysis buffer containing protease inhibitors, followed by digestion with trypsin (Promega, USA) to obtain tryptic peptides. After desalted on a ZipTip C18 column and vacuum-dried, the peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an EASY nLC system coupled to a Q Exactive HF mass spectrometer (Thermo Scientific, USA). The raw data was searched against a C. sinensis protein database (uniprot-Proteome ID_UP000286415_20221115.fasta) downloaded from UniProt database using the PEAKS software (Bioinformatics Solutions Inc., Canada). Trypsin was chosen as the enzyme, and at most, 3 missed cleavages per peptide were permitted. Carbamidomethylation was specified as static modifications while oxidation (M) and acetylation (N-term) was set as dynamic modifications. 15 ppm of precursor mass tolerance and 0.03 Da of fragment mass tolerance was used. The peptides were considered when the false discovery rate (FDR) ≤ 0.05.

CsEVs uptake experiment

To determine whether CsEVs could be incorporated into human CCA cell lines, a PKH26 Red Fluorescent Cell Labeling Kit (Umibio, Shanghai, China) was used to trace CsEVs. In detail, the isolated CsEVs described above were resuspended in 100 μL of PBS, then 200 μL of Diluent C mixed with 1 μL of PKH26 dye were added. The mixture was incubated for 5 min at RT in darkness, and 200 μL of 1% BSA (PBS) was added to terminate staining. The PKH26-labeled CsEVs were washed with PBS by ultracentrifugation at 120 000g for 70 min at 4°C twice to remove excess dye, and resuspended in 200 μL of PBS. PKH26-labeled PBS were used as negative control. Subsequently, the PKH26-labeled PBS or CsEVs were incubated with RBE and HuCCT1 cells previously prepared on coverslips in 6-well plates at 37°C for 12 h. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde at RT for 15 min and permeabilized with 0.3% Triton X-100 for 10 min. After the slides dried naturally, add 2 drops of fluorescent mounting medium with DAPI (ZSGB, Beijing, China) to stain nuclei, and the cells were observed using a laser scanning confocal microscope (LSCM, Zeiss, Germany).

CCK8 assays

Cell Counting Kit 8 (CCK8, Dojindo Laboratories, Kumamoto, Japan) was used to detect cell proliferation. Briefly, RBE and HuCCT1 cells were seeded into 96-well plates at approximately 2×10³ cells/well and incubated overnight. Cells were treated with 0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml CsEVs, respectively. After 0 h, 24 h, 48 h and 72 h of culture, the medium was removed and 100 μL of serum-free RPMI-1640 medium supplemented with 10 μL CCK8 reagent was replaced in each well, and then the cells were incubated in the dark at 37°C for 1 to 4 h. The absorbance value at 450 nm wavelength was detected by a microplate reader (Molecular Devices, CA).

EdU-488 assays

EdU-488 incorporation assay kit (C0071S; Beyotime, China) was employed to estimate cell proliferation. Briefly, RBE and HuCCT1 cells with or without CsEVs treatment were incubated with EdU medium diluent for 2 h according to the product manual. After being fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100, cells were labeled with EdU-488 through Click reaction, and the cell nuclei were stained with Hoechst 33342. The stained cells were observed under a fluorescence microscope (Leica DMI8, Wetzlar, Germany) and EdU positive cells were analyzed by ImageJ software (National Institutes of Health, USA).

Colony formation assays

Colony formation assays were used to test the effects of CsEVs on the proliferation ability and tumorigenesis of RBE and HuCCT1 cells. In brief, RBE and HuCCT1 cells with or without CsEVs treatment were seeded into 6-well plates at about 1000 cells/well and cultured for 2 weeks with medium replacement every 3 days. After being washed twice with PBS, cell colonies were fixed with 4% paraformaldehyde and then stained with 1% crystal violet stain solution (Solarbio, Beijing, China) for 30 min. Wash off the excess dye and take photographs. The cell colonies were counted via ImageJ software (NIH).

Cell cycle analysis

Cell cycle staining kit [MultiSciences (Lianke) Biotech, China] was used to analyze cell cycle distribution. Firstly, 1×10⁶ cells with or without CsEVs treatment were collected and washed once with PBS. Then, Propidium iodide (PI) with RNase A and permeabilization solution were added and incubated at room temperature in darkness for 30 min to stain the cells. The samples were detected using flow cytometry (CytoFLEX S, Beckman Coulter, USA) and the data was analyzed with the ModFit LT 4.1 program (Verity Software House, Topsham, USA).

Wound healing assays

Wound healing assays were used to analyze the effects of CsEVs on the migration of RBE and HuCCT1 cells. Briefly, RBE and HuCCT1 cells were seeded into 6-well plates at about 5×10⁵ cells/well and incubated to 80% confluence, followed by treatment with or without CsEVs for 24 h. After that, scratches were created vertically with a 200-μL pipette tip and suspension cells were washed off with PBS. The cells were cultured in serum-free RPMI-1640 medium and photographed under a microscope (Leica DMI4000B, Wetzlar, Germany) at 0 h, 24 h and 48 h after scratching. Finally, the scratch area was measured with ImageJ software (NIH).

Transwell assays

The transwell chambers (Corning, NY, USA) were used to evaluate the migration and invasion ability of RBE and HuCCT1 cells. For the transwell migration assays, 1×10⁵ treated cells were seeded into the upper chamber in serum-free RPMI-1640 medium, while the lower chamber was inserted into 24-well plates supplemented with 600 μL of medium containing 10% FBS. After incubation for 24 h, the migrated cells were fixed with 4% paraformaldehyde for 15 min, and 1% crystal violet solution (Solarbio, Beijing, China) was used to stain. The cells on the upper membrane surface were wiped off, and then the lower membrane surface was randomly imaged with a microscope (Leica DMI4000B, Wetzlar, Germany). The migrated cells were calculated with ImageJ software (NIH). The procedure for the transwell invasion assays was the same as for the migration assays, but the upper chamber was precoated with Matrigel (Corning, NY, USA).

In vivo metastasis assays

Twenty athymic male BALB/c nude mice (5-week-old) were purchased from the Laboratory Animal Center of Sun Yat-sen University. The following experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (No. SYSU-IACUC-2024-000343). For the lung metastasis model, approximately 2 × 10⁶ control or CsEVs treated-RBE cells were resuspended in 200 μL PBS and injected into the nude mice through the tail vein (n = 10/per group). After about 4 weeks of inoculation, the mice were sacrificed by cervical dislocation after anesthetization via intraperitoneal injection with 1% sodium pentobarbital, and their lungs were separated for the subsequent histopathologic analyses with hematoxylin-eosin staining.

RT-qPCR

Total RNA was extracted from cells using the EZ-press RNA Purification Kit (EZBioscience, USA) according to the manufacturer’s instructions, and an Epoch Microplate Spectrophotometer (BioTek, USA) was used to evaluate the RNA quality by determining parameters of concentration and purity. Complementary DNA (cDNA) was synthesized using 5× Evo M-MLV RT Master Mix (AGBio, China), and quantitative analysis was performed using SYBR Green Premix Pro Taq HS qPCR Kit (AGBio, China) on a CFX96 Real-Time PCR system (Bio-Rad). The specific primers used in this study were synthesized by Sangon Biotech (Shanghai, China) and shown in S2 Table. Relative mRNA expression was calculated using the 2^-ΔΔCt formula as in a previous study [12]. All experiments were performed in triplicate.

Western blot

Western blot was carried out in accordance with a previous study [13]. Briefly, total protein was extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors and phosphatase inhibitors. Protein samples were quantified with a BCA Protein Assay kit (Thermo Fisher Scientific, USA) and denatured with the 5×SDS loading buffer at 100°C for 10 min. A total of 30 μg protein from each sample was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, USA). After blocking with 5% skim milk for 2 h, the membranes were incubated with the corresponding primary antibodies (S3 Table) at 4°C overnight, including anti-E-cadherin (1:2,000), anti-N-cadherin (1:1,000), anti-vimentin (1:1,000), anti-slug (1:1,000), anti-CDK2 (1:1,000), anti-CDK6 (1:2,000), anti-CyclinD1 (1:1,000), anti-CyclinD3 (1:2,000), anti-p-ERK (1:2,000), anti-ERK (1:1,000), anti-p-IKKα/β (1:1,000), anti-IKKα (1:1,000), anti-IKKβ (1:1,000), anti-p-IKBα (1:1,000), anti-IKBα (1:1,000), anti-p-p65 (1:1,000), anti-p65 (1:1,000), and anti-GAPDH (1:1,000). Subsequently, the membranes were supplemented with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; Proteintech, Wuhan, China) for 2 h incubation at RT. The enhanced chemiluminescence reagent (ECL; EpiZyme, Shanghai, China) was used for development and ImageJ software (NIH) was used for quantification.

Statistical analysis

Statistical analysis was performed by GraphPad Prism 8.0 (GraphPad, San Diego, USA). In the case of comparison of two groups, the differences were evaluated using a two-tailed Student’s t-test, and One-way ANOVA analysis was used for comparison of differences among more than two groups. All results were from three independent experiments and the data were expressed as Mean ± SD. The differences were statistically significant under the following circumstances: *P < 0.05, **P < 0.01, ***P< 0.001, ****P<0.0001.

Results

Purification and identification of CsEVs

To obtain CsEVs, we first collected the adult worms of C. sinensis from the liver bile duct of the infected cats, and then cultured them in DMEM medium after washing procedures (Fig 1A). Next, CsEVs were separated from the culture supernatant via differential ultracentrifugation (Fig 1B). Negative-staining TEM was used to determine the morphological properties of CsEVs, which showed the typical cup-shaped or rounded structures of extracellular vesicles with a double-layer membrane and the diameter was between 30~150 nm (Fig 1C). Furthermore, the size distribution detected by NTA revealed a consistent result with a peak at 67.1 to 92.5 nm in diameter, and the concentration of CsEVs in our preparation was 70.6 particles per frame, which was equal to a concentration of 1.5×10¹¹ particles/mL before 100-fold dilution with ddH₂O (Fig 1D). These results indicated that CsEVs were successfully isolated from the supernatant.

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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.

https://doi.org/10.1371/journal.pntd.0012545.g001

Proteomic analysis of CsEVs

To elucidate the protein composition of isolated CsEVs, LC-MS/MS assays were performed. A total of 114 proteins present within the CsEVs fraction were identified, of which 11 were uncharacterized proteins (S4 Table). Among the characterized 103 proteins, some of the most frequently identified proteins in EVs, such as tetraspanin, annexin, tubulin, actin and heat shock protein (HSP) 90, which were implicated in EVs biogenesis, secretion and cell targeting, were also found to exist in CsEVs [23]. Additionally, some proteins, such as myoferlin, leucine aminopeptidase 2 and cathepsin B, matched with those previously identified in CsESPs, and may be related to the pathogenesis and oncogenicity of C. sinensis [22].

Subcellular localization of the CsEVs proteins was predicted using WoLF PSORT software [24]. The majority of the proteins were localized in the cytoplasm (45), with the remaining proteins sequentially deriving from the nucleus (20), plasma membrane (19), extracellular space (13), cytoskeleton (10), and mitochondrion (9) (Fig 2A). Then, the identified CsEVs proteins were annotated using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) and classified based on three categories. In the case of biological processes, CsEVs proteins were primarily enriched in the metabolic processes. GO enrichment of cellular components revealed that most of the CsEVs proteins were found to be cytoskeletal and nucleosome proteins. When it comes to molecular function, CsEVs proteins were considerably enriched in binding functions (Fig 2B). The KEGG analysis showed that CsEVs proteins were mainly abundant in gap junction, collecting duct acid secretion, bile secretion, arachidonic acid metabolism, etc., and most were predicted to be associated with the metabolic pathways (Fig 2C and 2D).

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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.

https://doi.org/10.1371/journal.pntd.0012545.g002

CsEVs can be internalized by CCA cell lines

To clarify the effects of CsEVs on the biological function of CCA cells, we first examined whether CsEVs could be captured by RBE and HuCCT1 cells. PKH26, a lipophilic red fluorescent dye, was used to label CsEVs specifically. Then these PKH26-labeled CsEVs were incubated with RBE and HuCCT1 cells for 12 h. PKH26-labeled PBS treated cells were used as negative control. As shown in Fig 3, red fluorescence signals were clearly observed in RBE and HuCCT1 cells around the nucleus (blue) by using a laser scanning confocal microscope (LSCM), suggesting that CsEVs can be internally absorbed by CCA cells.

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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.

https://doi.org/10.1371/journal.pntd.0012545.g003

CsEVs induce the proliferation of CCA cells in vitro

Next, we explored whether CsEVs could affect the growth of CCA cells. As demonstrated by CCK8 assays, the proliferation ability of RBE and HuCCT1 cells treated with 10 μg/mL of CsEVs was significantly improved compared with other groups (p < 0.0001), while the effect was lost at 5 μg/mL and substantially dampened at 20 μg/mL. (Fig 4A). Similarly, CsEVs treatment increased the colony formation numbers and resulted in more EdU positive cells relative to the control (Fig 4B and 4C), indicating that CsEVs facilitated the proliferation of CCA cells. Moreover, to determine how the proliferation of CCA cells was induced by CsEVs, we further conducted the cell cycle analysis via flow cytometry, which showed that there was elevated cell aggregation in both S and G2/M phase with an increased proliferation index after CsEVs treatment (Fig 4D). These results demonstrate that CsEVs can promote cell division and proliferation by accelerating cell cycle progression.

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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).

https://doi.org/10.1371/journal.pntd.0012545.g004

CsEVs facilitate the proliferation and oncogenicity of CCA cells by regulating cell cycle-related molecules

Cell cycle regulation is crucial for cell proliferation and growth, which is orchestrated by an intricate and delicate molecular network with the participation of multiple proteins, enzymes, cytokines, and cell cycle signaling pathways [25,26]. To shed light on the potential molecular mechanism of CsEVs induced malignant proliferation in CCA cells, we examined the expression of various cell cycle-related regulators both at the transcriptional and translational levels. As shown in Fig 5, the relative mRNA and protein expression of cyclinD1 (RBE: RT-qPCR: p < 0.001, WB: p < 0.01; HuCCT1: RT-qPCR: p < 0.01, WB: p < 0.05), cyclinD3 (RBE: RT-qPCR: p < 0.01, WB: p < 0.0001; HuCCT1: RT-qPCR: p < 0.01, WB: p < 0.01), CDK4 (RBE: RT-qPCR: p < 0.001, WB: No data; HuCCT1: RT-qPCR: p < 0.001, WB: No data), CDK6 (RBE: RT-qPCR: p < 0.001, WB: p < 0.01; HuCCT1: RT-qPCR: p < 0.0001, WB: p < 0.01) and CDK2 (RBE: RT-qPCR: p < 0.001, WB: p < 0.001; HuCCT1: RT-qPCR: p < 0.01, WB: p < 0.05) were significantly increased in RBE and HuCCT1 cells following CsEVs treatment, implying that CsEVs can accelerate G1/S transition by regulating cell cycle-related molecules, thereby promoting the proliferation and oncogenicity of CCA cells.

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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.

https://doi.org/10.1371/journal.pntd.0012545.g005

CsEVs promote the migration, invasion, and lung metastasis of CCA cells

We then detected whether CsEVs could facilitate the migration and invasion of CCA cells in vitro and in vivo. Wound healing assays showed that CsEVs significantly increased the 24-hour and 48-hour migration rates of RBE and HuCCT1 cells in comparison to the control group (p < 0.0001) (Fig 6A). Moreover, results of Transwell assays were consistent with those of the wound healing assays. Both matrigel-free (for migration) and matrigel-coated (for invasion) Transwell assays showed that the number of cells passing through the chamber within 24 h after CsEVs treatment were markedly elevated (p < 0.0001) (Fig 6B). The lung metastasis model was established by injecting CCA cells into athymic nude mice through their tail veins to evaluate cell metastasis ability in vivo. We found that CsEVs-treated RBE cells were more likely to increase the incidence of lung metastasis and the number of metastatic nodules than control cells (p < 0.05) (Fig 6C). Taken together, these experiments provide evidence that CsEVs are involved in the malignancy of CCA by promoting cell metastasis.

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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.

https://doi.org/10.1371/journal.pntd.0012545.g006

CsEVs can induce EMT, and Slug is essential for CsEVs-mediated EMT and CCA metastasis

EMT is a key process by which tumor cells acquire highly migratory and invasive phenotypic characteristics [27], so we tried to investigate the correlation between CsEVs and EMT in CCA. Western blot assays were performed to analyze the effect of CsEVs on EMT-related markers in RBE and HuCCT1 cells, and it was revealed that the expression of E-cadherin (an epithelial type marker) was decreased followed by CsEVs treatment, whereas the levels of N-cadherin and vimentin (mesenchymal type markers) was up-regulated. Concurrently, CsEVs overexpression in RBE and HuCCT1 cells significantly increased (RBE: p < 0.001, HuCCT1: p < 0.01) the expression of Slug, one of the most important EMT-associated transcription factors that can participate in the regulation of EMT markers [28] (Fig 7A). We hypothesized that Slug might play an important role in CsEVs-mediated EMT progression, and therefore, siRNA was used to specifically knock down Slug. The results indicated that the expression of Slug was decreased at both transcriptional and translational levels after transfection with Slug siRNA (Fig 7B and 7C), while the expression of E-cadherin was restored and N-cadherin as well as vimentin had an inverse expression pattern in the transfected RBE and HuCCT1 cells (Fig 7B). Consistent conclusions were obtained by transwell assays. Knockdown of Slug suppressed the migration and invasion of CsEVs treated RBE and HuCCT1 cells (Figs 7D and S1). Together, these results indicate that CsEVs can induce EMT process, and Slug is crucial for CsEVs-mediated EMT and metastasis in CCA.

Download:

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.

https://doi.org/10.1371/journal.pntd.0012545.g007

CsEVs promote EMT and malignant phenotypes of CCA cell lines through activating the NF-κB signaling pathway

To further explore the potential mechanism of CsEVs-induced alterations in CCA cells, we examined the NF-κB and ERK pathways using western blot analysis, which might be involved in the regulation of EMT and tumor metastasis [13,29]. As shown in Fig 8A, the phosphorylation of IκB kinase α/β (p-IKKα/β), IκB-α (p-IκB-α) and p65 (p-p65), some essential molecules in the activation of the NF-κB signaling pathway, were considerably elevated in RBE and HuCCT1 cells after CsEVs stimulation, especially p-p65. Also, the phosphorylation of ERK (p-ERK) was simultaneously upregulated (Fig 8B). These results demonstrated that CsEVs activated the NF-κB and ERK signaling pathways by promoting protein phosphorylation. Given that the NF-κB/EMT axis was found to participate in facilitating metastasis of cancer cells [30], we pondered whether the NF-κB pathway indeed regulated the malignant phenotypes of CCA via its impact on EMT mechanism. BAY 11–7082, a specific NF-κB inhibitor, was employed to pretreat RBE and HuCCT1 cells for 1 h, followed by CsEVs treatment. Results showed that NF-κB inhibitor could impede the phosphorylation of p65 and decrease the expression levels of N-cadherin and vimentin upregulated by CsEVs (Fig 8C and 8D). Furthermore, EdU-488 and Transwell assays revealed that NF-κB inhibitor markedly reversed the CsEVs-induced malignant proliferation and migration in both RBE and HuCCT1 cells (Fig 8E and 8F and S2 Fig). Taken together, these findings suggested that CsEVs accelerated malignant proliferation, migration and invasion of CCA cells through NF-κB/EMT axis.

Download:

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.

https://doi.org/10.1371/journal.pntd.0012545.g008

Discussion

In endemic areas, chronic liver fluke infection is thought to be a significant initiator of CCA [31]. Clinical statistics showed that the patients with CCA complicated by C. sinensis infection tend to be younger than those with CCA alone, and those under the age of 64 had a worse overall survival rate [32]. So far, the exact mechanism by which C. sinensis infection drives CCA to proceed to malignancy has not been established. Parasite-secreted EVs have been reported to affect the normal physiological function of recipient cells by transferring proteins, lipids, and nucleic acids in parasite-host interactions, leading to the pathogenesis of host [16]. For example, it has been shown that CsEVs can promote bile duct damage and inflammation, which are the important stages before carcinogenesis [17,19]. Numerous studies have shown a clear connection between EVs and tumor progression [33,34]. However, studies on the associations between CsEVs and CCA are limited. Therefore, further research into the specific mechanisms through which CsEVs affect the malignant phenotypes of CCA is required. In the present study, it was discovered that 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 (Fig 9).

Download:

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.

https://doi.org/10.1371/journal.pntd.0012545.g009

Different EVs separation techniques will generate high heterogeneity of their concentration and purity, which may yield discrepant research results [35]. Considering that helminth-derived EVs have been successfully separated using the gold standard approach of differential ultracentrifugation [17,20], we referred to these protocols with slight adjustments to enrich CsEVs. The isolated CsEVs appeared to have a cup-like or circular form with a peak size of 79 nm, which was compatible with the defining characteristics of EVs [23]. Furthermore, our findings provided the first insight into the protein expression profile in CsEVs. In comparison with the proteins enriched in EVs, there are extensive match [23]. Also, some proteins have been identified in CsESPs previously [22], which deserve further research. For instance, myoferlin, a membrane protein of ferlin family, has been reported to be involved in tumor progression by promoting angiogenesis, energy metabolism reprogramming, EMT and exosome modulation, which may be a potential target for tumor detection and therapy [36]. These results support that we have successfully obtained CsEVs.

PKH26 is a lipophilic fluorescent dye that can specifically label exosomes by interacting with the lipid bilayer of the membrane structure [37]. We used PKH26-labeled CsEVs to detect the uptake ability of CCA cells, and the results illustrated that CsEVs could be captured and functioned inside the cells. To discover the pathophysiologic significance and underlying mechanism of CsEVs in CCA, a series of experiments were performed, which showed that the proliferation, migration and invasion abilities of CCA cells were enhanced after treatment with CsEVs. To investigate the possible mechanism by which CsEVs promote proliferation, we examined the cell cycle distribution and expression levels of related regulators, as a disorder of the cell cycle can cause malignant proliferation and ultimately contribute to cancer initiation and progression [26]. The results suggested that CsEVs could accelerate G1/S transition to expedite cell proliferation by increasing the expression of positive cell cycle regulators. However, we did not delve into the detailed mechanisms of cell cycle dysregulation brought on by CsEVs, and we hope to explore this aspect in future studies.

Malignant tumor cells generated from epithelium go through a critical biological process called EMT to gain the capacity for invasion and migration, including CCA [9]. We hypothesized that CsEVs may promote CCA metastasis by inducing the EMT process, and we demonstrated that CsEVs could affect the expression of EMT-related markers at the transcriptional and translational levels. The decrease of E-cadherin can destroy the tight junction between cells, which is conducive to cell metastasis. Meanwhile, the up-regulation of N-cadherin and vimentin also mediate cell adhesion and migration. Most importantly, we discovered that Slug, one of the most significant EMT-associated transcription factors that can regulate EMT markers [28], was dramatically upregulated after stimulation with CsEVs. When being transfected with siRNA to knock down Slug, the EMT process was partially reversed, hinting that Slug may be an important target molecule in CsEVs-mediated EMT.

NF-κB is an essential intracellular nuclear transcription factor, which participates in a variety of physiological and pathological processes such as inflammation, immune response, cell survival and tumorigenesis [29]. Moreover, the interaction between the NF-κB pathway and the EMT process has been reported in various cancers, showing that NF-κB can act as an upstream mediator to induce EMT for expediting tumor metastasis by collaborating with multiple other signaling molecules and pathways. NF-κB/EMT axis could be a critical way in triggering the malignant progression of tumor cells [30]. Thus, we concentrated on the interaction between NF-κB signaling pathway and CsEVs-mediated EMT. Here, it was demonstrated that the phosphorylation of key molecules in the NF-κB pathway was provoked by CsEVs, and its downstream effects were crucial for the occurrence of EMT and malignant metastasis in CCA. Previous studies have reported that ERK pathway is involved in inducing abnormal proliferation, invasive growth, and distant metastasis of tumors [38]. We currently found the phosphorylation of ERK, but further studies are requisite to verify the role of ERK pathway in CsEVs-induced CCA malignant phenotypes. In addition, we did not perform enough in vivo experiments to corroborate these findings. Therefore, future research will explore the role of CsEVs in the malignant phenotypes and tumor microenvironment of CCA by constructing a tumor-bearing mouse model. Moreover, elucidation of exactly which proteins in CsEVs are acting to activate such a broad range of signaling pathways is needed.

In conclusion, our study provides evidence that CsEVs can induce the malignant progression of CCA cells by promoting proliferation, migration and invasion via NF-κB/EMT axis. Meanwhile, the protein expression profile of CsEVs is revealed by this study, which provides a theoretical basis for understanding the function of CsEVs and proposes a novel mechanism to explain the liver fluke-associated CCA.

Supporting information

S1 Fig. The quantification of migrated and invasive CCA cells after CsEVs treatment and Slug knockdown.

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. Transwell assays demonstrated that downregulated Slug reversed the number of migrated (A) and invasive (B) CCA cells.

https://doi.org/10.1371/journal.pntd.0012545.s001

(DOCX)

S2 Fig. The quantification of EdU-488 assays and transwell assays after the employment of NF-κB inhibitor.

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. EdU-488 assays (A, B) and Transwell assays (C, D) revealed that NF-κB inhibitor markedly reversed the CsEVs-induced malignant proliferation and migration in both RBE and HuCCT1 cells.

https://doi.org/10.1371/journal.pntd.0012545.s002

(DOCX)

S1 Table. siRNA for transfection.

https://doi.org/10.1371/journal.pntd.0012545.s003

(DOCX)

S2 Table. Primer sequences of human genes used in RT-qPCR.

https://doi.org/10.1371/journal.pntd.0012545.s004

(DOCX)

S3 Table. Specifications of primary antibodies.

https://doi.org/10.1371/journal.pntd.0012545.s005

(DOCX)

S4 Table. The information of the identified CsEVs proteins.

https://doi.org/10.1371/journal.pntd.0012545.s006

(XLSX)

S1 File. Raw data and descriptive statistics.

https://doi.org/10.1371/journal.pntd.0012545.s007

(DOCX)

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Figures

Abstract

Author summary

Introduction

Materials and methods

Ethics statement

Cell culture and transfection

C. sinensis and CsEVs preparation

Transmission electron microscopy (TEM)

Nanoparticle tracking analysis (NTA)

Proteome assays

CsEVs uptake experiment

CCK8 assays

EdU-488 assays

Colony formation assays

Cell cycle analysis

Wound healing assays

Transwell assays

In vivo metastasis assays

RT-qPCR

Western blot

Statistical analysis

Results

Purification and identification of CsEVs

Proteomic analysis of CsEVs

CsEVs can be internalized by CCA cell lines

CsEVs induce the proliferation of CCA cells in vitro

CsEVs facilitate the proliferation and oncogenicity of CCA cells by regulating cell cycle-related molecules

CsEVs promote the migration, invasion, and lung metastasis of CCA cells

CsEVs can induce EMT, and Slug is essential for CsEVs-mediated EMT and CCA metastasis

CsEVs promote EMT and malignant phenotypes of CCA cell lines through activating the NF-κB signaling pathway

Discussion

Supporting information

S1 Fig. The quantification of migrated and invasive CCA cells after CsEVs treatment and Slug knockdown.

S2 Fig. The quantification of EdU-488 assays and transwell assays after the employment of NF-κB inhibitor.

S1 Table. siRNA for transfection.

S2 Table. Primer sequences of human genes used in RT-qPCR.

S3 Table. Specifications of primary antibodies.

S4 Table. The information of the identified CsEVs proteins.

S1 File. Raw data and descriptive statistics.

References