NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma

Background:Small extracellular vesicles(sEVs)mediate intercellular commu-nication that contributes to hepatocellular carcinoma(HCC)progression via multifaceted pathways.The success of cell entry determines the effect of sEV on recipient cells.Here,we aimed to delineate the mechanisms underlying the uptake of sEV in HCC.Abbreviations:AF,Alexa Fluor;ANOVA,analysis of variance;ATP9A,ATPase Phospholipid Transporting 9A;BCECF-AM,2’,7’-bis-(2-barboxyethyl)-5-(and-6)-carboxyfluorescein,acetoxymethyl ester;BSA,bovine serum albumin;CCMR,Centre for Comparative Medicine Research;CRISPR,clustered regularly interspaced short palindromic repeats;CTL,ctrl;CXCR4,C-X-C Chemokine Receptor Type 4;DAPI,4′,6-diamidino-2-phenylindole;DFS,disease-free survival;DMEM,Dulbecco’s Modified Eagle Medium;DMSO,dimethyl sulfoxide;Dox,doxycycline;EEA1,early endosome antigen 1;EIPA,5-(N-ethyl-N-isopropyl)-amiloride;FBS,fetal bovine serum;FITC,fluorescein isothiocyanate;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;GM130,Golgi matrix protein 130;HCC,hepatocellular carcinoma;HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;HPRT1,hypoxanthine phosphoribosyltransferase 1;H-score,histoscore;IAA,indole-3-acetic acid;KD,knockdown;KO,knockout;mAID,mini-auxin-inducible degron;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;NHE7,Na(+)/H(+)exchanger 7;ns,non-significant;OD,optical density;OS,overall survival;PBS,phosphate-buffered saline;PCR,polymerase chain reaction;pHe,endosomal pH;pHi,intracellular pH;PKH67,Paul Karl Horan 67;Rab21,Ras-associated binding protein 21;RIPA,radioimmunoprecipitation assay;SAM,synergistic activation mediator;SEMs,standard error of the means;sEVs,small extracellular vesicles;sgRNA,single-guide RNA;shRNA,short-hairpin RNA;SLC9,solute carrier gene 9;SLiCE,Seamless Ligation Cloning Extract;TCGA,The Cancer Genome Atlas;TCL,total cell lysates;TGN,trans-Golgi network;TMA,tissue microarray;TMR,tetramethyl rhodamine;TSG101,tumor susceptibility gene 101.Yue Yao and Yi Xu contributed equally to this work.Methods:Macropinocytosis was examined by the ability of cells to internalize dextran and sEV.Macropinocytosis was analyzed in Na(+)/H(+)exchanger 7(NHE7)-knockdown and-overexpressing cells.The properties of cells were stud-ied using functional assays.pH biosensor was used to evaluate the intracellular and endosomal pH.The expression of NHE7 in patients’liver tissues was exam-ined by immunofluorescent staining.Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.Results:The data revealed that macropinocytosis controlled the internaliza-tion of sEVs and their oncogenic effect on recipient cells.It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells.Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride(EIPA)limited the entry of sEVs and compromised cell aggressiveness.Mechanistically,we delineated that high level of NHE7,a sodium-hydrogen exchanger,alkalized intracellular pH and acidized endosomal pH,leading to the maturation of macropinosomes.Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis.Clinically,NHE7 expression was upregulated and linked to dismal prognosis of HCC.Conclusions:This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells.Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

Ferroptosis:From Basic Research to Clinical Therapeutics in Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common and highly heterogeneous malignancies worldwide.Despite the rapid development of multidisciplinary treatment and personalized precision medicine strategies,the overall survival of HCC patients remains poor.The limited survival benefit may be attributed to difficulty in early diagnosis,the high recurrence rate and high tumor heterogeneity.Ferroptosis,a novel mode of cell death driven by iron-dependent lipid peroxidation,has been implicated in the development and therapeutic response of various tumors,including HCC.In this review,we discuss the regulatory network of ferroptosis,describe the crosstalk between ferroptosis and HCCrelated signaling pathways,and elucidate the potential role of ferroptosis in various treatment modalities for HCC,such as systemic therapy,radiotherapy,immunotherapy,interventional therapy and nanotherapy,and applications in the diagnosis and prognosis of HCC,to provide a theoretical basis for the diagnosis and treatment of HCC to effectively improve the survival of HCC patients.

Roles and Molecular Mechanisms of Biomarkers in Hepatocellular Carcinoma with Microvascular Invasion: A Review

Hepatocellular carcinoma(HCC)being a leading cause of cancer-related death,has high associated mortality and recurrence rates.It has been of great necessity and urgency to find effective HCC diagnosis and treatment measures.Studies have shown that microvascular invasion(MVI)is an independent risk factor for poor prognosis after hepatectomy.The abnormal expression of biomacromolecules such as circ-RNAs,lncRNAs,STIP1,and PD-L1 in HCC patients is strongly correlated with MVI.Deregulation of several markers mentioned in this review affects the proliferation,invasion,metastasis,EMT,and anti-apoptotic processes of HCC cells through multiple complex mechanisms.Therefore,these biomarkers may have an important clinical role and serve as promising interventional targets for HCC.In this review,we provide a comprehensive overview on the functions and regulatory mechanisms of MVI-related biomarkers in HCC.

Role of Small Extracellular Vesicles in Liver Diseases:Pathogenesis,Diagnosis,and Treatment

Extracellular vesicles(EVs)are vesicular bodies that bud off from the cell membrane or are secreted virtually by all cell types.Small EVs(sEVs or exosomes)are key mediators of cell-cell communication by delivering their cargo,including proteins,lipids,or RNAs,to the recipient cells where they induce changes in signaling pathways and phenotypic prop-erties.Tangible findings have revealed the pivotal involve-ment of sEVs in the pathogenesis of various diseases.On the bright side,they are rich sources of biomarkers for diag-nosis,prognosis,treatment response,and disease monitor-ing.sEVs have high stability,biocompatibility,targetability,low toxicity,and are immunogenic in nature.Their intrinsic properties make sEVs an ideal delivery vehicle to be loaded with cargo for therapeutic interventions.Liver diseases are a major global health problem.This review aims to focus on the roles and mechanisms of sEVs in the pathogenesis of liver diseases,liver injury,liver failure,and liver can-cer.sEVs are released not only by hepatocytes but also by stromal and immune cells in the microenvironment.Early detection of liver disease determines the chance for cura-tive treatment and high survival of patients.This review focuses on the potential of circulating sEV cargo as specific and sensitive noninvasive biomarkers for the early detection and prognosis of liver diseases.In addition,the therapeutic use of sEVs derived from various cell types is discussed.Al-though sEVs hold promise for clinical applications,there are still challenges to be overcome by further research to bring utilization of sEVs into clinical practice.