Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, China.
Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, China.
Cell Commun Signal. 2020 Mar 17;18(1):46. doi: 10.1186/s12964-020-00535-8.
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. Video Abstract.
肝细胞癌（HCC）是最常见的原发性肝癌，是一种高度血管化的实体肿瘤。血管生成素-2（ANGPT2）已被描述为抗血管生成治疗的一个有吸引力的靶点。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到受体细胞来促进细胞间通讯。肿瘤来源的外泌体中 ANGPT2 的表达尚不清楚。
我们通过免疫印迹、酶联免疫吸附试验和免疫金标记检测 HCC 衍生的外泌体中的 ANGPT2 表达，然后通过共焦激光扫描显微镜、共免疫沉淀和免疫印迹观察外泌体 ANGPT2 的内化和再循环。我们使用两种 HCC 细胞系（Hep3B 和 MHCC97H）通过慢病毒感染过表达 ANGPT2 或通过 CRISPR/Cas 系统敲低 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 微管形成试验、Transwell 迁移试验、划痕愈合试验、细胞计数试剂盒-8 试验、免疫印迹和体内肿瘤发生试验观察血管生成。
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，这种递呈通过非 Tie2 依赖性途径显著增加了血管生成。同时，我们观察到 HCC 细胞分泌的外泌体 ANGPT2 被受体 HUVEC 再循环，并可能被再利用。此外，CRISPR-Cas 系统敲低 ANGPT2 显著抑制了 HCC 细胞分泌的外泌体 ANGPT2 诱导的血管生成，并明显抑制了 HCC 中的上皮间质转化激活。
总之，这些结果揭示了 HCC 细胞分泌的外泌体 ANGPT2 诱导肿瘤血管生成的一种新途径，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一个潜在治疗靶点。
