Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown. We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC. Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy.

中文翻译：

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血管生成素2通过外来体诱导人肝细胞癌中的血管生成。

肝细胞癌（HCC）是最常见的原发性肝癌，是高度血管化的实体瘤。血管生成素2（ANGPT2）被描述为抗血管生成治疗的诱人靶标。外泌体是大多数细胞类型分泌的小细胞外囊泡，通过将功能性货物递送至受体细胞而有助于细胞间的通讯。ANGPT2在肿瘤来源的外泌体中的表达仍然未知。我们通过免疫印迹，酶联免疫吸附测定和免疫金标记检测了HCC来源的外泌体中的ANGPT2表达，然后通过共聚焦激光扫描显微镜，免疫共沉淀和免疫印迹观察了外泌体ANGPT2的内在化和再循环。我们使用了两种HCC细胞系（Hep3B和MHCC97H）通过慢病毒感染过表达ANGPT2或通过CRISPR / Cas系统敲除ANGPT2，然后分离外泌体，与人脐静脉内皮细胞（HUVEC）共培养，并通过Matrigel微管形成测定，穿孔迁移测定，伤口愈合测定，细胞计数kit-8测定，免疫印迹和体内肿瘤发生测定观察血管生成。我们发现，HCC衍生的外泌体携带ANGPT2，并通过外泌体内吞作用将其递送到HUVEC中，这种递送通过独立于Tie2的途径导致血管生成显着增加。同时，我们观察到HCC细胞分泌的外泌体ANGPT2被受体HUVEC回收利用，并可能被重复使用。此外，CRISPR-Cas系统敲除ANGPT2可以显着抑制HCC细胞分泌的外泌体ANGPT2诱导的血管生成，并明显抑制HCC的上皮-间质转化激活。在一起 这些结果揭示了由HCC细胞分泌的外泌体ANGPT2诱导的肿瘤血管生成的新途径，不同于经典的ANGPT2 / Tie2途径。这种方式可能是抗血管生成治疗的潜在治疗靶标。