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.