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. 2023 Aug 22;12(8):12359. doi: 10.1002/jev2.12359

Clathrin light chain A‐enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis

Yi Xu 1,2,✉, Yue Yao 1,3, Liang Yu 1,2, Xiaoxin Zhang 1,4, Xiaowen Mao 1,5, Sze Keong Tey 6, Samuel Wan Ki Wong 1, Cherlie Lot Sum Yeung 1, Tung Him Ng 1, Melody YM Wong 7, Chi‐Ming Che 7,8, Terence Kin Wah Lee 9, Yi Gao 10, Yunfu Cui 2, Judy Wai Ping Yam 1,5,✉
PMCID: PMC10443339  PMID: 37606345

Abstract

Small extracellular vesicles (sEVs) play a key role in exchanging cargoes between cells in tumour microenvironment. This study aimed to elucidate the functions and mechanisms of hepatocellular carcinoma (HCC) derived sEV‐clathrin light chain A (CLTA) in remodelling microvascular niche. CLTA level in the circulating sEVs of HCC patients was analysed by enzyme‐linked immunosorbent assay (ELISA). The functions of sEV‐CLTA in affecting HCC cancerous properties were examined by multiple functional assays. Mass spectrometry was used to identify downstream effectors of sEV‐CLTA in human umbilical vein endothelial cells (HUVECs). Tube formation, sprouting, trans‐endothelial invasion and vascular leakiness assays were performed to determine the functions of sEV‐CLTA and its effector, basigin (BSG) in HUVECs. BSG inhibitor, SP‐8356, was tested in a mouse model of patient‐derived xenografts (PDXs). Circulating sEVs of HCC patients had markedly enhanced CLTA levels than control individuals and were reduced in patients after surgery. HCC derived sEV‐CLTA enhanced HCC cancerous properties, disrupted endothelial integrity and induced angiogenesis. Mechanistically, CLTA remodels microvascular niche by stabilizing and upregulating BSG. Last, SP‐8356 alone or in combination with sorafenib attenuated PDXs growth. The study reveals the role of HCC derived sEV‐CLTA in microvascular niche formation. Inhibition of CLTA and its mediated pathway may illuminate a new therapeutic strategy for HCC patients.

Keywords: clathrin light chain A, hepatocellular carcinoma, intercellular communication, premetastatic niche, small extracellular vesicles, vascular permeability

1. INTRODUCTION

Liver cancer is a major threat to human health worldwide, especially for East and Southeast Asian people. The latest study revealed that liver cancer ranks as the fifth leading cause of cancer death in males and seventh in females (Siegel et al., 2021). Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for over 80% of all cases (Chen, 2015). Compelling evidence reveals the concerted roles of the complex cellular and noncellular components within the tumour microenvironment during the progression of HCC (Tahmasebi Birgani et al., 2017). The bidirectional communication among different cell types confined to the local tumour microenvironment or over a distance at metastatic sites plays a defining role in tumour development and metastasis.

Small extracellular vesicles (sEVs), also called exosomes, are one of the most important nanosized communicators within the complex tumour microenvironment. sEVs are now considered as an additional mechanism for intercellular communication by delivering various types of active biomolecules (van Niel et al., 2018). The sEVs released by various types of cells have been demonstrated to be key mediators during the process of endothelial formation and vascular leakiness, which has attracted special attention (Gai et al., 2016). Knowledge of the cellular processes that govern sEV biology is indispensable for clarifying the physiological and pathological functions of these vesicles as well as for creating clinical applications involving their use and analysis (Gimona et al., 2017). The remodelling of microvascular niche is essential for tumour cells to disseminate to distant organs (Östman & Corvigno, 2018). sEVs have grown in popularity in cancer research due to their emerging role in promoting dynamic intercellular communication and remodelling the premetastatic niche (Fong et al., 2015).

Clathrin light chain A (CLTA) is one of the three subunits of the light chain of clathrin. Together with heavy chains, clathrin light chains form clathrin as a structural component of cytoplasmic coated pits, which are involved in receptor‐mediated endocytosis. Under membrane tension, CLTA stimulates selective myosin VI recruitment to clathrin‐coated pits (Biancospino et al., 2019). Apart from endocytosis, CLTA regulates synaptic vesicle formation (Redlingshofer et al., 2020), cell spreading and migration (Tsygankova et al., 2019). It has been found to be increased in lung tissues bearing macrometastases of mice implanted with breast cancer cells (Kurpinska et al., 2019). In the present study, we investigated the sEVs‐enriched proteins that may be responsible for HCC metastasis. The sEVs database indicated that CLTA extensively exists in 16 types of cancer cell derived sEVs. However, the functions of CLTA as a cargo of sEVs has not been investigated. In this study, CLTA was found to be present in sEVs of HCC cells and exerted oncogenic capacity in promoting angiogenesis and disrupting vascular endothelial barrier integrity by stabilizing basigin (BSG). Additionally, BSG inhibitor SP‐8356 alone or in combination with sorafenib could inhibit patient‐derived xenografts (PDXs) tumour progression. Moreover, the level of CLTA in circulating sEVs was significantly upregulated in HCC patients and decreased significantly after surgery.

2. MATERIAL AND METHODS

2.1. Human specimens

Clinical samples were employed in the current study. Control individuals with nonhepatic disease were recruited for serum collection. Patients diagnosed with chronic hepatitis B virus (HBV) infection, cirrhosis, and early‐ and advanced‐stage HCC who had not received any treatment were enrolled to collect serum. Patient donated serum specimens were recruited from Queen Mary Hospital, Hong Kong and Zhujiang Hospital, Guangzhou, China. The blood samples were drawn and handled according to updated Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines (Thery et al., 2018).

2.2. Study approval

This study was authorized by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB), Zhujiang Hospital of Southern Medical University. Informed consent was obtained from each participant. All study procedures involving human specimens were handled according to the relevant ethical regulations.

2.3. Statistical analysis

All data presented in the study are the mean ± standard error of the mean (SEM). The procedures were repeated in triplicate and analysed by Student's t test or one‐way ANOVA using GraphPad Prism 8.30 (GraphPad, Inc., La Jolla, CA, USA). p < 0.05 was considered statistically significant.

Further details of the experimental methods are depicted in the Supplementary Materials and Methods section.

3. RESULTS

3.1. CLTA is overexpressed in sEVs of HCC

In the present study, we investigated the sEVs‐enriched proteins that may be responsible for HCC metastasis using the cancer genome atlas (TCGA) database. A total of 6362 candidates are significantly upregulated in tumour tissues than their normal counterparts. Furthermore, TCGA database indicates that 1456 genes potentially predict worse overall survival for HCC patients (p < 0.001). Among these, 276 gene products are related to vascular invasiveness. CD63 mainly resides in multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) of cells. It is reported that CD63 is approximately seven times enriched in the ILVs as compared to the endosomal limiting membrane (Escola et al., 1998), which allow it as a specific biomarker for sEVs. Therefore, we further investigated the target genes that positively correlated with CD63 expression in HCC. We assume the target genes co‐expressed with CD63 might be involved in sEV assembling, endocytosis, trafficking or enriched in sEV cargoes. Among the 276 candidates, 12 genes are well co‐expressed with CD63 by Spearman correlation analysis (R > 0.6, p < 0.05, Figure 1a). Taken together, CLTA, HOMER3, PTDSS2, NAP1L1, CLIC1, PA2G4, JPT1, RALY, SNRPD1, COPZ1, TPD52L2 and SNRPA were predicted to be significantly upregulated in HCC tissues, related to vascular invasiveness, worse overall survival, and positively correlated with CD63 expression. As shown in Figure 1(b), CLTA was found to be most powerful to predict distant metastasis for HCC patients (AUC = 0.694). In addition, Vesiclepedia database (Hina Kalra et al., 2016) indicated that CLTA extensively exists in 16 types of cancer cell derived sEVs, which is more than other genes. To the best of our knowledge, the function as well as mechanism of CLTA as a cargo of sEVs has not been investigated. We questioned whether CLTA could be expressed in sEVs of normal liver and HCC cell lines (Figure S1a). The isolated sEVs were validated by the detection of CD63 using immunogold labelling (Figure S1b), their size range (Figure S1c) and expression of sEV markers (Figure 1c). CLTA was significantly upregulated in PLC/PRF/5, MHCC97H, MHCC97L, and MHCCLM3 cell lysates than normal liver cells, MIHA. Similarly, CLTA was expressed in sEVs of HCC cells but barely detected in sEVs of MIHA (Figure 1c). To validate the localization of CLTA, proteinase K was used to degrade outer membrane‐localized proteins such as CD63 but not the intravesicular TSG101. The results showed that CLTA was expressed on the surface of sEVs (Figure S2). It was encouraging to observe a remarkable elevation of CLTA in circulating sEVs of HCC patients compared to control individuals with and without chronic hepatitis B virus infection and cirrhosis (Figure 1d). The level of sEV‐CLTA was decreased in 15 out of 19 patients after surgery (Figure 1e).

FIGURE 1.

FIGURE 1

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CLTA is overexpressed in sEVs of HCC. (a) Venn diagram showing the increased expression of mRNAs related to vascular invasion, worse overall survival, and CD63 expression in HCC. (b) The distant metastasis prediction potential of 12 candidates and their existence in cancerous sEVs. (c) Analysis of CLTA, positive sEVs markers (Alix, CD63, and CD9) and negative sEVs markers (GM130 and p62) expression in 20 μg of total cell lysates (TCL) and 10 μg of sEVs by western blot analysis. (d) Level of CLTA in circulating sEVs from control individuals (Normal, n = 23) and patients with HBV (HBV, n = 20), HBV‐related cirrhosis (Cirrhosis, n = 8), early‐stage HCC (I‐II, n = 40) or late‐stage HCC (III‐IV, n = 23) was determined by ELISA. (e) The CLTA level in circulating sEVs from HCC patients before and after surgery was measured (n = 19). Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.

3.2. HCC derived sEV‐CLTA promotes tumour progression in vitro and in vivo

sEVs derived from metastatic HCC cells have been shown to facilitate premetastatic niche formation and promote tumorigenesis and metastasis (Mao et al., 2020). To answer whether CLTA is a functional component in sEVs of HCC cells, sEVs were obtained from vector control (XPack), CLTA overexpressing (XP‐CLTA), non‐target control (CTL‐KD) and CLTA knockdown (CLTA‐KD1 and CLTA‐KD2) clones established in MHCCLM3 and MHCC97L cells (Xu et al., 2023) (Figure S3a–d, Figure 2a). The colony formation ability of HLE and Huh7 was significantly facilitated by both MHCCLM3 and MHCC97L XPack‐sEV compared to PBS treated cells. Moreover, XP‐CLTA‐sEV either from MHCCLM3 or MHCC97L further enhanced the number of colonies. Conversely, CLTA‐KD1‐sEV and CLTA‐KD2‐sEV both abrogated the enhanced colony formation of HLE and Huh7 cells induced by CTL‐KD‐sEV (Figure 2b, Figure S4). In line with the effect of MHCCLM3 and MHCC97L sEV‐CLTA on the colony forming ability of HCC cells, a similar effect on cell migration and invasiveness was observed (Figure 2b, Figure S4). In the subcutaneous injection model, coinjection with MHCCLM3 XPack‐sEV accelerated tumour development and formed larger CLTA knockdown tumours than cells without sEV injection. In addition, tumour development was further promoted by XP‐CLTA‐sEV (Figure 2c, d).

FIGURE 2.

FIGURE 2

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HCC derived sEV‐CLTA promotes tumour progression in vitro and in vivo. (a) Immunoblotting of CLTA in sEVs derived from vector control (XPack), CLTA overexpressing (XP‐CLTA), non‐target control (CTL‐KD) and CLTA knockdown (CLTA‐KD1 and CLTA‐KD2) clones established in MHCCLM3 (upper) and MHCC97L (bottom) cells. (b) HLE cells treated with the indicated sEVs were evaluated by colony formation, migration and invasion assays. Scale bar, 200 μm. (c) Image of mice subjected to subcutaneous injection of MHCCLM3 CTL‐KD and CLTA‐KD1 cells with PBS or the indicated sEVs (right). The tumour size was measured regularly (left). (d) Images of tumours harvested from mice (left). The weight of the tumours was measured (right). (e) Schematic diagram of the sEV education model. (f) Bioluminescence imaging of animals at the end of the experiment (left). The intensity of the signals in whole mice imaging (middle). Image of the dissected livers (right). Liver tumours are indicated by dotted lines. (g) Ex vivo bioluminescence imaging of lung tissues (left). The intensity of the lung signal (middle). H&E staining of lung tissues (right). Tumour nodules are indicated by arrowheads. Scale bar, 200 μm. (h) Schematic diagram of the experimental lung metastasis assay. (i) Bioluminescence imaging of animals at the end of the experiment (left‐upper). H&E staining of lung tissues (left‐bottom). Tumour nodules are indicated by arrowheads. Scale bar, 200 μm. The intensity of the lung signal. (right). Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.

An sEV education model was performed to evaluate the influence of sEV‐CLTA on the premetastatic niche, which is crucial to HCC metastasis (Figure 2e). Smaller implanted tumours (Figure 2f) and fewer lung metastatic nodules in the lungs (Figure 2g) formed in mice implanted with CLTA knockdown cells compared to mice implanted with CTL‐KD cells. Mice educated with XPack‐sEV formed larger liver tumours (Figure 2f) and more intense lung metastasis (Figure 2g) than mice without sEV education. XP‐CLTA displayed a more potent promoting effect than XPack‐sEV, implicating the potential of sEV‐CLTA in facilitating a favourable microenvironment for the colonization and growth of disseminating cells (Figure 2f, g). In line with these observations, MHCCLM3 and MHCC97L derived sEV‐CLTA significantly facilitated the colonization of murine p53‐/‐;Myc‐transduced hepatoblasts in the lungs in an experimental metastasis assay (Figure 2h, i, Figure S5).

3.3. CLTA‐enriched sEVs induce angiogenesis, disrupt vascular endothelial barrier integrity and enhance pulmonary vessel leakage

The hypervascularized nature of HCC and lung as a common site of distant metastasis prompted us to investigate whether CLTA, carried by sEVs, affects endothelial cells. The results demonstrated that XPack‐sEVs strikingly enhanced the number of tubes (Figure 3a) and sprouts (Figure 3b) of HUVECs. CLTA‐enriched sEVs obtained from XP‐CLTA cells further increased the tube forming and sprouting abilities of HUVECs, and such an increase was not detected in cells treated with CLTA‐KD1‐sEVs (Figure 3a, b). In subcutaneous tumours derived from CLTA‐KD1 cells, lower microvascular density (MVD) was observed compared to tumours derived from CTL‐KD cells. CLTA expression and MVD were elevated in tumours derived from CLTA‐KD1 cells coinjected with XP‐CLTA‐sEVs (Figure 3c).

FIGURE 3.

FIGURE 3

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CLTA‐enriched sEVs induce angiogenesis, disrupt vascular endothelial barrier integrity and enhance pulmonary vessel leakage. HUVECs treated with the indicated sEVs were subjected to tube formation (a) and spheroid‐based sprouting (b) assays. The number of tubes and lengths of the sprouts were analysed. Scale bar, 50 μm (a); 200 μm (b). (c) Immunohistochemistry of CLTA and CD31 in subcutaneous tumours derived from MHCCLM3 CTL‐KD and CLTA‐KD1 cells injected with the indicated sEVs. Scale bar, 200 μm. (d) Schematic diagram of the TMR‐dextran leakiness assay (left). The amount of TMR‐dextran in the lower chamber was determined (right). (e) Schematic diagram of the trans‐endothelial invasion experiment (left). The number of MitoTracker‐stained HLE cells that passed through the HUVEC monolayer was determined (right). Scale bar, 50 μm. (f) Schematic diagram of the pulmonary vessel leakiness assay (left). The areas with Texas‐Red dextran were analysed (right). Arrowheads indicate diffused dextran. Scale bar, 50 μm. Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Furthermore, we examined whether sEV‐CLTA facilitates cancer cell extravasation and induces vascular permeability. The HUVEC monolayer showed increased leakiness to tetramethylrhodamine (TMR)‐dextran (Figure 3d) and green fluorescence‐labelled HLE cells (Figure 3e) after exposure to XPack‐sEVs compared to PBS. The HUVEC monolayer was more permeable to TMR‐dextran and HLE cells when pretreated with XP‐CLTA‐sEVs but not when pretreated with CLTA‐KD1‐sEVs (Figure 3d, e). To this end, we evaluated whether sEV‐CLTA could modulate vascular permeability in vivo. In accordance with the in vitro findings, mice injected with either MHCCLM3 or MHCC97L XP‐CLTA‐sEVs resulted in the most regions with diffuse Texas‐Red dextran, an indicator of pulmonary vessel leakiness, compared to mice injected with control sEVs and CLTA‐KD1‐sEVs (Figure 3f).

3.4. sEV‐CLTA interacts with and stabilizes BSG in HUVECs

The proteomic profiles of HUVECs treated with MHCCLM3 CTL‐KD‐sEVs and CLTA‐KD1‐sEVs were compared. Twenty‐one proteins were found to be downregulated by at least 2‐fold and with p value < 0.05 in CLTA‐KD1‐sEV‐treated cells compared to CTL‐KD‐sEV. When comparing the proteome of HUVECs treated with XP‐CLTA‐sEV and XPack‐sEV, 23 proteins were found to be significantly upregulated in cells treated with XP‐CLTA‐sEV. BSG was among the top‐ranked differentially deregulated proteins (Figure 4a). Gene Ontology (GO) analysis using the database for annotation, visualization and integrated discovery (DAVID) platform revealed a marked enrichment of proteins involved in endomembrane system organization (Figure 4b), implicating BSG, a member of the endomembrane component, as a promising target regulated by CLTA. The expression of BSG was upregulated when HUVECs were treated with XP‐CLTA‐sEVs, while CLTA‐KD1‐sEVs failed to elevate BSG levels in HUVECs (Figure 4c, Figure S6a). It was noted that the transcriptional level of BSG remained unchanged in cells treated with sEV‐CLTA (Figure S6b). To understand whether sEV‐CLTA could stabilize BSG protein, the kinetics of BSG degradation were analysed in sEV‐treated HUVECs with the addition of cycloheximide (CHX). The results showed that BSG was stabilized in cells treated with either MHCC97L or MHCCLM3 XPack‐sEV. The degradation of BSG was further compromised in cells treated with XP‐CLTA‐sEV derived from MHCC97L or MHCCLM3 (Figure 4d, Figure S6c). Coimmunoprecipitation demonstrated the interaction between CLTA and BSG in HUVECs (Figure 4e). CLTA and BSG were shown to be colocalized in cells. The causal relationship between CLTA and BSG was revealed when their expression levels were both significantly elevated in cells treated with CLTA‐enriched sEVs (Figure 4f, Figure S6d). It is reported that F‐Box Protein 22 (FBXO22) mediated polyubiquitination and degradation of BSG by interacting with it (Wu et al., 2017). We postulated whether sEV‐CLTA interferes the interaction between BSG and FBXO22. Thus, we used PBS, XPack‐sEV and XP‐CLTA‐sEV to treat HUVECs and evaluated the interaction between BSG and FBXO22. The result showed that XPack‐sEV treatment reduced the BSG‐FBXO22 interaction compared to PBS treatment. XP‐CLTA‐sEV further decreased the interaction between BSG and FBXO22 (Figure S6e). Taken together, HCC cells derived sEV‐CLTA binds to BSG, thereby alleviating the interaction between BSG and FBXO22 and preventing BSG from polyubiquitination and degradation induced by FBXO22.

FIGURE 4.

FIGURE 4

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sEV‐CLTA upregulates and stabilizes BSG in endothelial cells. (a) HUVECs treated with CTL‐KD‐sEVs and CLTA‐KD1‐sEVs were subjected to mass spectrometry (left). Volcano plots show the differentially expressed proteins. Proteins that were modulated by at least 2‐fold and with significance are coloured. A similar analysis was performed on HUVECs treated with XPack‐sEVs and XP‐CLTA‐sEVs (right). (b) GO enrichment analysis was performed by DAVID in terms of biological process and cellular components. (c) Immunoblotting of BSG in HUVECs treated with the indicated sEVs. (d) Immunoblotting of BSG in CHX‐ and sEV‐treated HUVECs (left). The band intensity of BSG normalized to that of β‐actin was plotted (right). (e) Coimmunoprecipitation was performed on HUVECs using anti‐IgG or anti‐CLTA antibodies, followed by immunoblotting. (f) Immunofluorescence of CLTA (green) and BSG (red) in HUVECs treated with XPack‐sEV and XP‐CLTA‐sEV (left). Signals of CLTA (middle) and BSG (right) were determined. Scale bar, 20 μm. Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

3.5. HCC derived sEV‐CLTA remodels the tumour microvascular niche via BSG

To ascertain whether the effect of sEV‐CLTA in modulating endothelial cells is mediated by BSG, we examined whether SP‐8356, an inhibitor of BSG, could hinder the effect of XP‐CLTA‐sEVs in HUVECs. The enhancing role of sEV‐CLTA in tube forming and sprouting abilities of HUVECs was abrogated by the addition of SP‐8356 (Figure 5a). The vascular leakiness to TMR‐dextran (Figure 5b) and trans‐endothelial activity of HLE cells (Figure 5c) induced by sEV‐CLTA were both compromised by SP‐8356. The in vivo effect of MHCCLM3 and MHCC97L derived sEV‐CLTA in inducing pulmonary vascular leakiness was reduced in mice administered with SP‐8356 compared to mice treated with vehicle (Figure 5d, e). Taken together, these data support the contributing role of BSG in sEV‐CLTA‐induced endothelial modulation.

FIGURE 5.

FIGURE 5

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HCC‐derived sEV‐CLTA remodels the tumour microvascular niche through BSG. (a) HUVECs treated with XPack‐sEV and XP‐CLTA‐sEV in the presence of vehicle or SP‐8356 were subjected to tube formation (left‐upper) and spheroid‐based sprouting (left‐bottom) assays. The number of tubes and length of sprouts were analysed (right). Scale bar, 200 μm. (b) A TMR‐dextran leakiness assay was performed on HUVECs treated with XPack‐sEV and XP‐CLTA‐sEV in the presence of vehicle or SP‐8356. The amount of TMR‐dextran in the lower chamber was determined. (c) Trans‐endothelial invasion determined the number of MitoTracker‐stained HLE cells that passed through HUVECs treated with the indicated sEVs with or without SP‐8356. Scale bar, 50 μm. A pulmonary vessel leakiness assay was performed in mice injected with Texas‐Red dextran, Alexa Fluor 488 concanavalin A, MHCCLM3‐derived sEVs (d) or MHCC97L‐derived sEVs (e) and SP‐8356. Arrowheads indicate diffused dextran (left). Scale bar, 50 μm. Areas with Texas‐Red dextran were analysed (right). Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.

3.6. SP‐8356 reverses the tumour progression promoting effect caused by sEV‐CLTA

We further studied whether the blockade of BSG by SP‐8356 would suppress tumour growth induced by sEV‐CLTA (Figure 6a). In a subcutaneous mouse model, coinjection of HCC cells with MHCCLM3 XP‐CLTA‐sEVs accelerated tumour development and resulted in the formation of larger tumours compared to HCC cells coinjected with or without XPack‐sEVs. Tumour development was delayed, and the tumour size was significantly reduced, in mice injected with SP‐8356 (Figure 6b–d). The dissected tumours showed enhanced CLTA and CD31 expressions when XPack‐sEVs and XP‐CLTA‐sEVs were administered. The MVD in tumours was markedly reduced in tumours obtained from mice treated with SP‐8356 (Figure 6e). Similar trends were also identified in lung colonization study. SP‐8356 strikingly prohibited the tumour cell colonization to lungs induced by MHCC97L sEV‐CLTA (Figure 6f, g).

FIGURE 6.

FIGURE 6

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SP‐8356 reverses the tumour progression promoting effect caused by sEV‐CLTA. (a) Schematic diagram of the treatment regimen applied to mice subcutaneously coinjected with MHCC97L and the indicated sEVs. (b) Image of mice at the end of the experiment. Subcutaneous tumours are indicated by arrows. (c) Tumour size was measured regularly and plotted. (d) Tumours harvested from mice (left). The volume (middle) and weight (right) of tumours were analysed. (e) Immunohistochemistry of CLTA and CD31 in dissected tumours (left). Scale bar, 200 μm. The intensities of CLTA (middle) and microvessels (right) were quantified. (f) Bioluminescence imaging of animals at the end of the lung metastasis assay (left). The intensity of the lung signal. (right). (g) H&E staining of lung tissues. Tumour nodules are indicated by arrowheads. Scale bar, 200 μm. Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.

3.7. Blockade of BSG using inhibitor suppresses the development of HCC patient‐derived xenografts

In this study, a mouse model of a subcutaneous HCC PDXs was employed to test the therapeutic efficacy of pharmacological inhibition of BSG using SP‐8356. Sorafenib, the first‐line treatment for advanced unresectable HCC, alone or in combination with SP‐8356, were administered to the mice implanted with PDXs (Figure 7a). Both SP‐8356 and sorafenib significantly inhibited tumour development and resulted in smaller tumours compared to those in vehicle group. Combined treatment of sorafenib and SP‐8356 showed an enhanced inhibitory effect compared to treatment using a single agent (Figure 7b, c). Immunohistochemistry (IHC) indicated decreased expression of Ki67 and CD31 in tumours formed from mice either treated with SP‐8356 or sorafenib. Furthermore, for the mice treated with SP‐8356 and sorafenib simultaneously, Ki67 and CD31 expression levels were further decreased (Figure 7d).

FIGURE 7.

FIGURE 7

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Blockade of BSG using inhibitor suppresses the development of HCC patient‐derived xenografts. (a) The diagram illustrates the treatment regimen of sorafenib and SP‐8356 administered to mice subcutaneously implanted with PDXs. (b) Image of mice at the end of the experiment (left). Tumour size was measured regularly and plotted (right). (c) The tumours were harvested at the end of the experiment (left). The volume (middle) and weight (right) of the tumours were determined. (d) IHC of Ki67 and CD31 in dissected tumours (left). Scale bar, 200 μm. The intensities of Ki67 (middle) and microvessels (right) were quantified. Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.

4. DISCUSSION

Using the public TCGA dataset, 12 candidates including CLTA, HOMER3, PTDSS2, NAP1L1, CLIC1, PA2G4, JPT1, RALY, SNRPD1, COPZ1, TPD52L2 and SNRPA were predicted to be significantly upregulated in HCC tissues and correlated to vascular invasiveness, worse overall survival, and CD63 expression. Among these candidates, CLTA was found to be most powerful to predict distant metastasis for HCC patients. In addition, vesiclepedia database (Hina Kalra et al., 2016) indicated that CLTA extensively exists in 16 types of cancer cell derived sEVs, which is more than other candidates.

Clathrin acts as the main structural component of the lattice‐type cytoplasmic face of coated pits to facilitate sEVs internalization (Lampe et al., 2016). Clathrin has three highly conserved light chain subunits, including CLTA, CLTB, and CLTC. Unlike most of the studies focused on the endocytosis function of CLTA (Grimm et al., 2022; Huang et al., 2022), CLTA was detected for the first time in sEVs with functional capacity. Functional assays indicated the enrichment of CLTA in HCC derived sEVs and revealed its oncogenic role in promoting HCC progression and aggressiveness.

Considering the prediction effect of CLTA in vascular invasiveness and distant metastasis in HCC, we wonder whether CLTA could be delivered to HUVECs, where it augments the ability of cells to form tubes and sprouts and the formation of microvessels in tumours. CLTA‐enriched sEVs also increased endothelial leakiness, which is crucial to metastasis (Fong et al., 2015; Sleeman et al., 2015; Xu et al., 2021). In an animal model injected with CLTA‐enriched sEVs, accelerated tumour development and provoked metastasis were observed. With the evident role of sEV‐CLTA in modulating endothelial cells, the enhanced tumour growth and metastasis could be ascribed to angiogenesis induced by sEV‐CLTA that facilitates spreading of disseminated cancer cells and the induced pulmonary vasculature leakiness and tumour‐endothelial cell adhesion that potentially assists the extravasation of tumour cells during metastasis.

Mass spectrometry identified BSG as regulated by sEV‐CLTA upon internalization by HUVECs. This study demonstrated that CLTA interacted and colocalized with BSG in HUVECs. CLTA was found to upregulate BSG via protein stabilization rather than mRNA regulation. The ubiquitination of BSG has been reported before (Luo et al., 2016). Furthermore, it was found that FBXO22 could interact with BSG to mediate it polyubiquitination and degradation (Wu et al., 2017). In this study, we further advanced the knowledge that HCC derived sEV‐CLTA could bind to BSG, thereby alleviating the extent of FBXO22‐BSG interaction. sEV‐CLTA could protect BSG from polyubiquitination and degradation induced by FBXO22. BSG, also known as CD147, is reported to be a coreceptor for vascular endothelial growth factor receptor 2 (KDR/VEGFR2) in endothelial cells, enhancing its VEGFA‐mediated activation and downstream signalling (Khayati et al., 2015; Yin et al., 2020). Additionally, evidence indicated the elevation of BSG in activated HUVECs could enhance the angiogenesis by regulating PI3K/Akt pathway (Chen et al., 2009). Here, we advanced the understanding of functional outcomes due to aberrant BSG expression by sEV‐CLTA. We showed that the effect of sEV‐CLTA on angiogenesis and endothelial monolayer permeability of HUVECs is partly attributed to BSG stability, thereby facilitating tumour metastasis. The current study provides evidence about the interplay of CLTA, carried by sEVs, and BSG in forming a microvascular niche that is pivotal for metastasis, especially for HCC, which is hypervascular. The role of BSG in HCC was corroborated by its expression and correlation with CLTA in HCC. TCGA dataset of liver cancer revealed that BSG expression was upregulated in HCC tissue samples (Figure S7a) and markedly correlated with worse overall survival for patients with HCC (Figure S7b; Hazard ratio: 1.8, p = 0.0014). In addition, BSG upregulation in HCC tissues was significantly associated with vascular invasion (Figure S7c; Hazard ratio: 1.284, p = 0.0002). BSG and CLTA were found to be the most significantly upregulated in HCC and cholangiocarcinoma among all the cancer types (Figure S7d, e). Additionally, the strongest positive correlation of CLTA and BSG was found in HCC than other types of cancers (Figure S7f).

Recently, a prognostic model based on single‐cell RNA sequencing data indicated a 3‐gene signature (including CLTA, TALDO1 and CSTB) as a predictor of a poor outcome of HCC (Lu et al., 2022). However, the role of CLTA as a diagnostic biomarker has not been revealed. In the present study, sEV‐CLTA was found to be significantly elevated in circulating sEVs of HCC patients in both the early and advanced stages. Most of the HCC patients displayed a reduced level of serum sEV‐CLTA after surgery. These findings support the potential of circulating sEV‐CLTA to be applied as a noninvasive reliable biomarker to differentiate HCC patients and healthy individuals. Nevertheless, a larger cohort of patients should be included to further confirm the clinical value of sEV‐CLTA.

Sorafenib is the first‐line systemic therapeutic drug for patients with unresectable and metastatic HCC. Unfortunately, the effect of sorafenib on tumour shrinkage is modest, and some patients are rather refractory to sorafenib (Niu et al., 2017). The effects of other multikinase inhibitors, including lenvatinib, regorafenib and cabozantinib inhibitors, are not better than those of sorafenib considering their benefit in prolonging lifespan. HCC is a typical hypervascular tumour by which the extensive architecture of blood vessels facilitates the dissemination of cancer cells through the hematogenous route. Thus, the blockade of neoangiogenesis mediated by sEV‐CLTA could be a way to improve the current therapeutics for HCC treatment. Indeed, the blockade of the effector of CLTA, BSG, using SP‐8356 could effectively inhibit the growth of HCC PDXs in nude mice. When combined with sorafenib, the inhibitor could further increase the therapeutic efficacy in suppressing PDXs growth.

In summary, this study demonstrated the role of CLTA in remodelling the premetastatic microvascular niche by stabilizing BSG via its transfer to endothelial cells by sEVs. The findings point to the clinical relevance of the potential application of circulating sEV‐CLTA in liquid biopsy for the early detection of HCC. This study also provides insights into a new therapeutic strategy by inhibiting BSG and blocking its mediated effect on neoangiogenesis.

AUTHOR CONTRIBUTIONS

Yi Xu: Conceptualization; funding acquisition; investigation; methodology; writing—original draft. Yue Yao: Investigation. Liang Yu: Investigation. Xiaoxin Zhang: Investigation. Xiaowen Mao: Methodology. Sze Keong Tey: Methodology. Terence Kin Wah Lee: Resources. Yi Gao: Resources. Yunfu Cui: Resources. Judy Wai Ping Yam: Conceptualization; funding acquisition; methodology; project administration; Supervision; Writing—review & editing.

CONFLICT OF INTEREST STATEMENT

The authors have declared that no conflict of interest exists.

Supporting information

Supporting Information

Click here for additional data file. (4.9MB, docx)

ACKNOWLEDGEMENTS

The authors would like to acknowledge the assistance of Centre for PanorOmic Sciences Imaging, Li Ka Shing Faculty of Medicine, The University of Hong Kong for providing equipment needed for animal imaging and confocal microscopy. We also thank Centre for Comparative Medicine Research for providing animals and facility for animal experimentation and the Electron Microscope Unit for providing service and support needed for experiments involving electron microscope. We also acknowledge the funding support from Laboratory for Synthetic Chemistry and Chemical Biology under the Health@InnoHK Program launched by Innovation and Technology Commission, The Government of Hong Kong Special Administrative Region of the People's Republic of China. This work was supported by National Natural Science Foundation of China (grant number 81872340, 82072626, and 81902431); Hong Kong Scholars Program (grant number XJ2020012 and 2020–036); Marshal Initiative Funding of Harbin Medical University (grant number HMUMIF‐22008); Natural Science Foundation of Heilongjiang Province (grant number LH2023H043).

Xu, Y. i. , Yao, Y. , Yu, L. , Zhang, X. , Mao, X. , Tey, S. K. , Wong, S W. K. , Yeung, C. L. S. , Ng, T. H. , Wong, M. Y. M. , Che, C.‐M. , Lee, T. K. W. , Gao, Y. , Cui, Y. , & Yam, J. W. P. (2023). Clathrin light chain A‐enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis. Journal of Extracellular Vesicles, 12, e12359. 10.1002/jev2.12359

Yi Xu, Yue Yao and Liang Yu Contributed equally.

Contributor Information

Yi Xu, Email: xuyihrb@pathology.hku.hk.

Judy Wai Ping Yam, Email: judyyam@pathology.hku.hk.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

REFERENCES

  1. Biancospino, M. , Buel, G. R. , Nino, C. A. , Maspero, E. , Scotto di Perrotolo, R. , Raimondi, A. , Redlingshöfer, L. , Weber, J. , Brodsky, F. M. , Walters, K. J. , & Polo, S. (2019). Clathrin light chain A drives selective myosin VI recruitment to clathrin‐coated pits under membrane tension. Nature Communications, 10(1), 4974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen, W. (2015). Cancer statistics: Updated cancer burden in China. Chinese Journal of Cancer Research, 27, 1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen, Y. , Zhang, H. , Gou, X. , Horikawa, Y. , Xing, J. , & Chen, Z. (2009). Upregulation of HAb18G/CD147 in activated human umbilical vein endothelial cells enhances the angiogenesis. Cancer Letters, 278(1), 113–121. [DOI] [PubMed] [Google Scholar]
  4. Escola, J. M. , Kleijmeer, M. J. , Stoorvogel, W. , Griffith, J. M. , Yoshie, O. , & Geuze, H. J (1998). Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B‐lymphocytes. Journal of Biological Chemistry, 273(32), 20121–20127. [DOI] [PubMed] [Google Scholar]
  5. Fong, M. Y. , Zhou, W. , Liu, L. , Alontaga, A. Y. , Chandra, M. , Ashby, J. , Chow, A. , O'Connor, S. T. F. , Li, S. , Chin, A. R. , Somlo, G. , Palomares, M. , Li, Z. , Tremblay, J. R. , Tsuyada, A. , Sun, G. , Reid, M. A. , Wu, X. , Swiderski, P. , … Wang, S. E. (2015). Breast‐cancer‐secreted miR‐122 reprograms glucose metabolism in premetastatic niche to promote metastasis. Nature Cell Biology, 17(2), 183–194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gai, C. , Carpanetto, A. , Deregibus, M. C. , & Camussi, G. (2016). Extracellular vesicle‐mediated modulation of angiogenesis. Histology and Histopathology, 31, 379–391. [DOI] [PubMed] [Google Scholar]
  7. Gimona, M. , Pachler, K. , Laner‐Plamberger, S. , Schallmoser, K. , & Rohde, E. (2017). Manufacturing of human extracellular vesicle‐based therapeutics for clinical use. International Journal of Molecular Sciences, 18(6), 1190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grimm, E. , van der Hoeven, F. , Sardella, D. , Willig, K. I. , Engel, U. , Veits, N. , Engel, R. , Cavalcanti‐Adam, E. A. , Bestvater, F. , Bordoni, L. , Jennemann, R. , Schönig, K. , Schiessl, I. M. , & Sandhoff, R. (2022). A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis. PLoS ONE, 17(9), e0273660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huang, Z. , Luo, R. , Yang, L. , Chen, H. , Zhang, X. , Han, J. , Wang, H. , Zhou, Z. , Wang, Z. , & Shao, L. (2022). CPT1A‐mediated fatty acid oxidation promotes precursor osteoclast fusion in rheumatoid arthritis. Frontiers in immunology, 13, 838664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kalra, H. , Drummen, G. P. C. , & Mathivanan, S. (2016). Focus on extracellular vesicles: Introducing the next small big thing. International Journal of Molecular Sciences, 17(2), 170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khayati, F. , Perez‐Cano, L. , Maouche, K. , Sadoux, A. , Boutalbi, Z. , Podgorniak, M. P. , Maskos, U. , Setterblad, N. , Janin, A. , Calvo, F. , Lebbé, C. , Menashi, S. , Fernandez‐Recio, J. , & Mourah, S. (2015). EMMPRIN/CD147 is a novel coreceptor of VEGFR‐2 mediating its activation by VEGF. Oncotarget, 6, 9766–9780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kurpinska, A. , Suraj, J. , Bonar, E. , Zakrzewska, A. , Stojak, M. , Sternak, M. , Jasztal, A. , & Walczak, M. (2019). Proteomic characterization of early lung response to breast cancer metastasis in mice. Experimental and Molecular Pathology, 107, 129–140. [DOI] [PubMed] [Google Scholar]
  13. Lampe, M. , Vassilopoulos, S. , & Merrifield, C. (2016). Clathrin coated pits, plaques and adhesion. Journal of Structural Biology, 196, 48–56. [DOI] [PubMed] [Google Scholar]
  14. Lu, J. , Chen, Y. , Zhang, X. , Guo, J. , Xu, K. , & Li, L. (2022). A novel prognostic model based on single‐cell RNA sequencing data for hepatocellular carcinoma. Cancer Cell International, 22, 38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Luo, Z. , Zhang, X. , Zeng, W. , Su, J. , Yang, K. , Lu, L. , Lim, C. B. , Tang, W. , Wu, L. , Zhao, S. , Jia, X. , Peng, C. , & Chen, X. (2016). TRAF6 regulates melanoma invasion and metastasis through ubiquitination of Basigin. Oncotarget, 7(6), 7179–7192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mao, X. , Tey, S. K. , Yeung, C. L. S. , Kwong, E. M. L. , Fung, Y. M. E. , Chung, C. Y. S. , Mak, L. , Wong, D. K. H. , Yuen, M. , Ho, J. C. M. , Pang, H. , Wong, M. P. , Leung, C. O. , Lee, T. K. W. , Ma, V. , Cho, W. C. , Cao, P. , Xu, X. , Gao, Y. , … Yam, J. W. P. (2020). Nidogen 1‐enriched extracellular vesicles facilitate extrahepatic metastasis of liver cancer by activating pulmonary fibroblasts to secrete tumor necrosis factor receptor 1. Advanced Science (Weinh), 7(21), 2002157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Niu, L. , Liu, L. , Yang, S. , Ren, J. , Lai, P. B. S. , & Chen, G. G (2017). New insights into sorafenib resistance in hepatocellular carcinoma: Responsible mechanisms and promising strategies. Biochimica et Biophysica Acta: Reviews on Cancer, 1868, 564–570. [DOI] [PubMed] [Google Scholar]
  18. Östman, A. , & Corvigno, S. (2018). Microvascular mural cells in cancer. Trends in Cancer, 4(12), 838–848. [DOI] [PubMed] [Google Scholar]
  19. Redlingshofer, L. , McLeod, F. , Chen, Y. , Camus, M. D. , Burden, J. J. , Palomer, E. , Briant, K. , Dannhauser, P. N. , Salinas, P. C. , & Brodsky, F. M. (2020). Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo. PNAS, 117(38), 23527–23538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Siegel, R. L. , Miller, K. D. , Fuchs, H. E. , & Jemal, A. (2021). Cancer Statistics, 2021. CA: A Cancer Journal for Clinicians, 71, 7–33. [DOI] [PubMed] [Google Scholar]
  21. Sleeman, J. P (2015). The lymph node pre‐metastatic niche. Journal of Molecular Medicine (Berl), 93, 1173–1184. [DOI] [PubMed] [Google Scholar]
  22. Tahmasebi Birgani, M. , & Carloni, V. (2017). Tumor microenvironment, a paradigm in hepatocellular carcinoma progression and therapy. International Journal of Molecular Sciences, 18(2), 405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thery, C. , Witwer, K. W. , Aikawa, E. , Alcaraz, M. J. , Anderson, J. D. , Andriantsitohaina, R. , Antoniou, A. , Arab, T. , Archer, F. , Atkin‐Smith, G. K. , Ayre, D. C. , Bach, J.‐M. , Bachurski, D. , Baharvand, H. , Balaj, L. , Baldacchino, S. , Bauer, N. N. , Baxter, A. A. , Bebawy, M. , … Zuba‐Surma, E. K. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles, 7(1), 1535750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tsygankova, O. M. , & Keen, J. H (2019). A unique role for clathrin light chain A in cell spreading and migration. Journal of Cell Science, 132(10), jcs224030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. van Niel, G. , D'Angelo, G. , & Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews Molecular Cell Biology, 19, 213–228. [DOI] [PubMed] [Google Scholar]
  26. Wu, B. , Liu, Z. Y. , Cui, J. , Yang, X. M. , Jing, L. , Zhou, Y. , Chen, Z.‐N. , & Jiang, J.‐L. (2017). F‐box protein FBXO22 mediates polyubiquitination and degradation of CD147 to reverse cisplatin resistance of tumor cells. International Journal of Molecular Sciences, 18, 212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Xu, Y. , Leng, K. , Yao, Y. , Kang, P. , Liao, G. , Han, Y. , Shi, G. , Ji, D. , Huang, P. , Zheng, W. , Li, Z. , Li, J. , Huang, L. , Yu, L. , Zhou, Y. , Jiang, X. , Wang, H. , Li, C. , Su, Z. , … Cui, Y. (2021). A circular RNA, cholangiocarcinoma‐associated circular RNA 1, contributes to cholangiocarcinoma progression, induces angiogenesis, and disrupts vascular endothelial barriers. Hepatology, 73, 1419–1435. [DOI] [PubMed] [Google Scholar]
  28. Xu, Y. , Yao, Y. , Yu, L. , Fung, H. L. , Tang, A. H. N. , Ng, I. O. L. , Wong, M. Y. M. , Che, C.‐M. , Yun, J. P. , Cui, Y. , & Yam, J. W. P. (2023). Clathrin light chain A facilitates small extracellular vesicles uptake to promote hepatocellular carcinoma progression. Hepatology International, 10.1007/s12072-023-10562-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yin, Z. , Cai, H. , Wang, Z. , & Jiang, Y. (2020). Pseudolaric acid B inhibits proliferation, invasion, and angiogenesis in esophageal squamous cell carcinoma through regulating CD147. Drug Design, Development and Therapy, 14, 4561–4573. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

Click here for additional data file. (4.9MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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