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Abstract：
Small extracellular vesicles (sEVs) mediate intercellular communication 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.
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 studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients\' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.
The data revealed that macropinocytosis controlled the internalization 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.
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.
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 studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients\' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.
The data revealed that macropinocytosis controlled the internalization 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.
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.
摘要：
背景：小细胞外囊泡（sEV）介导细胞间通讯，通过多方面途径促进肝细胞癌（HCC）的进展。细胞进入的成功决定了sEV对受体细胞的影响。这里,我们的目的是描述肝癌中sEV摄取的潜在机制。
方法：通过细胞内化葡聚糖和sEV的能力来检查巨噬细胞胞吞作用。在Na(+)/H(+)交换体7(NHE7)-敲低和过表达细胞中分析巨噬细胞胞吞作用。使用功能测定研究细胞的性质。pH生物传感器用于评估细胞内和内体pH。免疫荧光染色检测患者肝组织中NHE7的表达。在已建立的肿瘤中进行NHE7的可诱导沉默以揭示靶向NHE7的治疗潜力。
结果：数据显示，巨噬细胞胞吞作用控制了sEV的内化及其对受体细胞的致癌作用。发现相对于正常肝细胞和非转移性HCC细胞，转移性HCC细胞表现出最高的sEV摄取效率。5-（N-乙基-N-异丙基）-阿米洛利（EIPA）对巨噬细胞活性的减弱限制了sEV的进入并损害了细胞侵袭性。机械上,我们描绘了高水平的NHE7，一种钠-氢交换剂，碱化的细胞内pH和酸化的内体pH，导致macropinosomes的成熟。在小鼠中形成的已建立的肿瘤中NHE7的可诱导抑制延迟了肿瘤发展并抑制了肺转移。临床上,NHE7表达上调，并与HCC预后不良有关。
结论：这项研究促进了以下认识：NHE7通过大细胞胞吞作用增强sEV摄取，以促进HCC细胞的恶性特性。通过大细胞胞吞作用抑制sEV摄取可被用作单独治疗或与HCC的常规治疗方法组合的治疗。
方法：通过细胞内化葡聚糖和sEV的能力来检查巨噬细胞胞吞作用。在Na(+)/H(+)交换体7(NHE7)-敲低和过表达细胞中分析巨噬细胞胞吞作用。使用功能测定研究细胞的性质。pH生物传感器用于评估细胞内和内体pH。免疫荧光染色检测患者肝组织中NHE7的表达。在已建立的肿瘤中进行NHE7的可诱导沉默以揭示靶向NHE7的治疗潜力。
结果：数据显示，巨噬细胞胞吞作用控制了sEV的内化及其对受体细胞的致癌作用。发现相对于正常肝细胞和非转移性HCC细胞，转移性HCC细胞表现出最高的sEV摄取效率。5-（N-乙基-N-异丙基）-阿米洛利（EIPA）对巨噬细胞活性的减弱限制了sEV的进入并损害了细胞侵袭性。机械上,我们描绘了高水平的NHE7，一种钠-氢交换剂，碱化的细胞内pH和酸化的内体pH，导致macropinosomes的成熟。在小鼠中形成的已建立的肿瘤中NHE7的可诱导抑制延迟了肿瘤发展并抑制了肺转移。临床上,NHE7表达上调，并与HCC预后不良有关。
结论：这项研究促进了以下认识：NHE7通过大细胞胞吞作用增强sEV摄取，以促进HCC细胞的恶性特性。通过大细胞胞吞作用抑制sEV摄取可被用作单独治疗或与HCC的常规治疗方法组合的治疗。
