Angiopoietin-2 Induces Angiogenesis via Exosomes in Human Hepatocellular Carcinoma

Background: 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. The expression of ANGPT2 in tumor-derived exosomes remains unknown. Methods: We detected the ANGPT2 expression in HCC-derived exosomes by the immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by the confocal laser scanning microscopy. 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 the Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. Findings: 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. Interpretation: 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. Funding Statement: National Natural Science Foundation of China (81672420, 81702406, 81673433); National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province, 2017B090903004); Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Tissue and serum samples were collected from patients after obtaining informed consent in accordance with a protocol approved by the Ethics Committee of Sun Yat-sen Memorial Hospital (Guangzhou, China). All experimental procedures involving animals were performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85-23, revised 2011) and in accordance with the institutional ethical guidelines for animal experiments.