Phenotypic and functional characterization of tumor-derived endothelial cells isolated from primary human hepatocellular carcinoma.

AIMS

Tumor endothelial cells (TECs) have been investigated using human tumor xenografts in mice models. In order to provide pure human TECs for the updating of clinical anti-angiogenic cancer therapy, in the present study we established a protocol of purification of TECs derived from clinical hepatocellular carcinoma (HCC) and revealed the TEC features by in vitro and in vivo assays.

METHODS

We isolated TECs from fresh surgical resections of HCC by magnetic-activated cell sorting and purified by flow cytometry sorting upon CD31 expression, referred to as ECDHCCs. Next, we identified cultured ECDHCCs by morphology, phenotype, genotype, and functional assays.

RESULTS

The ECDHCCs appeared as Weibel-Palade bodies under electron microscopy. They expressed endothelial markers, such as CD31, CD105, and vascular endothelial growth factor receptor 2, and expressed the genes that are associated with pro-angiogenesis, especially vascular endothelial growth factor, epiregulin, and programmed cell death 10. Functionally, ECDHCCs were capable of tube formation, wound healing, and Transwell migration in vitro. These in vitro behaviors were validated by in vivo Matrigel plug assay in mice. Finally, comparison of ECDHCC with the Hep-G2 liver cancer cell line showed there was no similarity of phenotype or function between these two types of cells.

CONCLUSIONS

Tumor endothelial cells derived from human HCC can be isolated and purified from clinical samples by flow cytometer. They have the endothelial phenotype and morphologic features and are capable of tube formation and migration. This study provides a useful model for researchers to study tumor angiogenesis and screening of candidate targets.