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

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.

Actin-related protein 2/3 complex subunit 2-enriched extracellular vesicles drive liver cancer metastasis.

BACKGROUND: Extracellular vesicles (EVs) play pivotal roles in tumor growth, cancer metastasis and angiogenesis. Here, we aimed to identify proteins that contribute to the functionality of EVs derived from metastatic hepatocellular carcinoma (HCC) cells. METHODS: Proteins of EVs derived from metastatic HCC cells and normal liver cells were analyzed by mass spectrometry. Proteomic profiling identified actin-related protein 2/3 complex subunit 2 (ARPC2) to be highly expressed in EVs of metastatic HCC cells. The expression of ARPC2 in EVs and HCC tissues was examined using immunoblotting and TCGA database, respectively. The functional roles of EV-ARPC2 were investigated by knockout approach and various in vitro and in vivo assays. RESULTS: ARPC2 was highly expressed in EVs of metastatic cells but barely detected in non-metastatic HCC cells and normal liver cells. Immunogold labeling showed the presence of APRC2 on the surface of EVs. Analysis of TCGA database of liver cancer revealed ARPC2 overexpression was correlated with poor prognosis of patients. ARPC2 was knockout in metastatic HCC cells. EVs derived from knockout cells displayed compromised activity in enhancing cell growth, motility and metastasis compared to EVs of control cells. Pimozide, an inhibitor of APRC2, also inhibited the promoting effect of EVs of metastatic cells in lung colonization of tumor cells in mice. CONCLUSION: This study reveals previously unreported expression and function of ARPC2 in EVs. EVs with highly expressed ARPC2 enhance cancer cell growth and metastasis. ARPC2 may provide a prospective target for the novel treatment of HCC patients.

Therapy-induced modulation of extracellular vesicles in hepatocellular carcinoma.

Despite rapid development of anti-tumorigenic treatments, the clinical outcome for hepatocellular carcinoma (HCC) is still far from satisfactory. With a deeper understanding about tumor microenvironment (TME), the critical role of extracellular vesicles (EVs) as intercellular liaison has come into spotlight. The dynamic functionality of these nanoparticles revealed cancer cells can employ both tumor and non-tumorous components for their own benefit, so as to mediate cell-to-cell communication and interchange of oncogenic biomolecules. Increasing studies on HCC-derived EVs have identified various irregulated biomolecules, that may serve as biomarkers or therapeutic targets. In this review, we first introduce the current knowledge about EVs and how they operate to maintain a healthy liver microenvironment. We then summarize some of the aberrant observations reported on HCC-derived EVs and how they contribute to HCC pathogenesis. Finally, we describe how current treatments for HCC alter behavior of EVs, which may shed light for potential prognostic markers and therapeutic strategies.

Patient pIgR-enriched extracellular vesicles drive cancer stemness, tumorigenesis and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: Extracellular vesicles (EVs) play a pivotal role in connecting tumor cells with their local and distant microenvironments. Herein, we aimed to understand the role (on a molecular basis) patient-derived EVs play in modulating cancer stemness and tumorigenesis in the context of hepatocellular carcinoma (HCC). METHODS: EVs from patient sera were isolated, quantified and characterized. The EVs were vigorously tested, both in vitro and in vivo, through various functional assays. Proteomic analysis was performed to identify the functional components of EVs. The presence and level of polymeric immunoglobulin receptor (pIgR) in circulating EVs and tumor and non-tumorous tissues of patients with HCC were determined by ELISA, immunoblotting, immunohistochemistry and quantitative PCR. The functional role and underlying mechanism of EVs with enhanced pIgR expression were elucidated. Blockade of EV-pIgR with neutralizing antibody was performed in nude mice implanted with patient-derived tumor xenografts (PDTXs). RESULTS: Circulating EVs from patients with late-stage HCC (L-HCC) had significantly elevated pIgR expression compared to the EVs released by control individuals. The augmenting effect of L-HCC-EVs on cancer stemness and tumorigenesis was hindered by an anti-pIgR antibody. EVs enriched with pIgR consistently promoted cancer stemness and cancerous phenotypes in recipient cells. Mechanistically, EV-pIgR-induced cancer aggressiveness was abrogated by Akt and ß-catenin inhibitors, confirming that the role of EV-pIgR depends on the activation of the PDK1/Akt/GSK3ß/ß-catenin signaling axis. Furthermore, an anti-pIgR neutralizing antibody attenuated tumor growth in mice implanted with PDTXs. CONCLUSIONS: This study illustrates a previously unknown role of EV-pIgR in regulating cancer stemness and aggressiveness: EV-pIgR activates PDK1/Akt/GSK3ß/ß-catenin signaling cascades. The blockade of the intercellular communication mediated by EV-pIgR in the tumor microenvironment may provide a new therapeutic strategy for patients with cancer. LAY SUMMARY: The World Health Organization estimates that more than 1 million patients will die from liver cancer, mostly hepatocellular carcinoma (HCC), in 2030. Understanding the underlying mechanism by which HCC acquires aggressive attributes is crucial to improving the diagnosis and treatment of patients. Herein, we demonstrated that nanometer-sized extracellular vesicles released by tumors promote cancer stemness and tumorigenesis. Within these oncogenic vesicles, we identified a key component that functions as a potent modulator of cancer aggressiveness. By inhibiting this functional component of EVs using a neutralizing antibody, tumor growth was profoundly attenuated in mice. This hints at a potentially effective therapeutic alternative for patients with cancer.

Liver cancer cells with nuclear MET overexpression release translation regulatory protein-enriched extracellular vesicles exhibit metastasis promoting activity.

MET receptor tyrosine kinase is a cell surface receptor that plays important role in embryonic development and tissue regeneration. Aberrant MET activation has been widely reported in different human cancers, making MET an attractive therapeutic target. The presence of truncated MET within the nucleus (nMET) with potential novel functions poses a great challenge to the current therapeutic strategies against MET surface receptor. Previous work has demonstrated the promoting effect of nMET in aggressive properties of hepatocellular carcinoma (HCC) cells by activating TAK1/NF-κB signalling pathway. Herein, we report the role of nMET in modulating tumour microenvironment and tumour metastasis mediated by extracellular vesicles (EVs). EVs released by nMET overexpressing cells enhanced cell motility and provoked metastasis. Proteomic profiling revealed the enrichment of translational regulatory proteins in EVs derived from nMET overexpressing cells. These proteins include eukaryotic initiation factor (EIF), ribosomal protein small subunit (RPS) and ribosomal protein larger subunit (RPL) gene families. Knockdown of EIF3I, RPS3A and RPL10 diminished the promoting effect of EVs in cell migration invasiveness and metastasis. In conclusion, the findings reveal an unrecognized capacity of nMET to augment HCC through the release of EVs with oncogenic effect. Targeting these translation-related proteins may serve as an alternative treatment for patients with nMET overexpression.

TPI1-reduced extracellular vesicles mediated by Rab20 downregulation promotes aerobic glycolysis to drive hepatocarcinogenesis.

Rab GTPases are major mediators that ensure the proper spatiotemporal regulation of intracellular trafficking. Functional impairment and altered expression of Rab proteins have been revealed in various human cancers. There is an emerging evidence about the role of Rab proteins in the biogenesis of extracellular vesicles (EVs). In hepatocellular carcinoma (HCC), using RNA sequencing comparing expression profiles of adjacent non-tumorous tissues and HCC, Rab20 is identified to be the most frequently downregulated Rab member in HCC. Functionally, restoration of Rab20 in metastatic HCC cells results in the release of EVs with a diminished activity to promote cell growth, motility and metastasis. Conversely, EVs released from normal liver cells with Rab20 knockdown loses suppressive effect on HCC cell growth and motility. Proteomic profiling revealed the level of triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, in EVs to be positively associated with Rab20 expression of the releasing cells. TPI1 targeted to be expressed in EVs released by Rab20 knockdown cells compromises the oncogenic activity of EVs. Besides, EVs released by TPI1 knockdown cells recapitulates the promoting effect of EVs derived from HCC cells with Rab20 underexpression. Aerobic glycolysis is beneficial to the survival and proliferation of tumour cells. Here, we observed that the enhanced cell growth and motility are driven by the enhanced aerobic glycolysis induced by EVs with reduced TPI1. The addition of glycolytic inhibitor blocks the promoting effect of EVs with reduced TPI1. Taken together, our study provides a mechanistic link among tumour cell-derived EVs and glucose metabolism in HCC with Rab20 deregulation.

Nidogen 1-Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1.

In hepatocellular carcinoma (HCC) patients with extrahepatic metastasis, the lung is the most frequent site of metastasis. However, how the lung microenvironment favors disseminated cells remains unclear. Here, it is found that nidogen 1 (NID1) in metastatic HCC cell-derived extracellular vesicles (EVs) promotes pre-metastatic niche formation in the lung by enhancing angiogenesis and pulmonary endothelial permeability to facilitate colonization of tumor cells and extrahepatic metastasis. EV-NID1 also activates fibroblasts, which secrete tumor necrosis factor receptor 1 (TNFR1), facilitate lung colonization of tumor cells, and augment HCC cell growth and motility. Administration of anti-TNFR1 antibody effectively diminishes lung metastasis induced by the metastatic HCC cell-derived EVs in mice. In the clinical perspective, analysis of serum EV-NID1 and TNFR1 in HCC patients reveals their positive correlation and association with tumor stages suggesting the potential of these molecules as noninvasive biomarkers for the early detection of HCC. In conclusion, these results demonstrate the interplay of HCC EVs and activated fibroblasts in pre-metastatic niche formation and how blockage of their functions inhibits distant metastasis to the lungs. This study offers promise for the new direction of HCC treatment by targeting oncogenic EV components and their mediated pathways.

Tumour extracellular vesicle-derived Complement Factor H promotes tumorigenesis and metastasis by inhibiting complement-dependent cytotoxicity of tumour cells.

The complement system is involved in the immunosurveillance of pathogens and tumour cells. Proteomic profiling revealed that extracellular vesicles (EVs) released by metastatic hepatocellular carcinoma (HCC) cells contained a significant number of complement proteins. Complement Factor H (CFH), an abundant soluble serum protein that inhibits the alternative complement pathway, was found to be highly expressed in EVs of metastatic HCC cell lines. Here, we investigated the functional role of EV-CFH and explored the therapeutic efficacy of targeting EV-CFH with an anti-CFH antibody in HCC. The results showed that EVs that are enriched in CFH promoted HCC cell growth, migration, invasiveness and enhanced liver tumour formation in mice. EV-CFH also promoted metastasis, which was significantly abrogated when treated with an anti-CFH antibody. These findings demonstrate an unexplored function of EV-CFH in protecting HCC cells by evading complement attack, thereby facilitating tumorigenesis and metastasis. Lastly, we demonstrated the therapeutic efficacy of an anti-CFH antibody in suppressing tumour formation in a syngeneic mouse model. This study suggests a new therapeutic strategy for HCC, by inhibiting EV-CFH with a tumour specific anti-CFH antibody.