Isolation, Identification, And Comparison Of Differentsubtypes Of Tumor-derived Endothelial Cells From Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC)is the third most common cause of death from cancer worldwide,and the second cause of death from cancer in China. Rich and distorted vascular is an important characteristic of HCC and anti-angiogenesis therapy has become an important treatment for advanced HCC, of which, the major target is the surface receptors on tumor endothelial cells (TEC). In this study, we obtained and compareddifferent subtypes of TECusing magnetic activated cell sorting (MACS)assay, which is helpful for further study in themechanismsof tumor angiogenesis and the the study in antiangiogenic drugs.Materials and methodsPaired tumors with corresponding peritumoral liver tissues from HCC patients who underwent curative liver resection in the Department of Liver Surgery, ZhongshanHospital during October 2009 and March 2011 were enrolled. Inclusion criteria:hepatitis B infection, alpha fetoprotein (AFP) positive, and pathological confirmedHCC without any preoperativeanti-cancertherapy.CD105+or CD31+TEC and liver-derived endothelial cells (NEC) were isolated from ten pairs of tumor and peritumoral liver tissues using MACS assay.The tumor tissue and peripheral blood were collected, patients'clinical features and survival information were recorded. The endothelial cells were culturedin vitro and 3-5 generation were selected for the followingexperiments.The morphology was observed;the expression of CD 105, CD31, CD68 and alpha SMA were detected by flow cytometric (FCM) analysis, AFP expression was detected by RT-PCR with the purpose to exclude macrophages, fibroblasts, or tumor cells contamination.Usingthe acetylation low density lipoprotein absorb (Dil-Ac-LDL) experiment, tubular formation experiment and spheroid assay to verify whether positive sorting cells with endothelial cell function.The expression of the markers of endothelial cells, including VEGFR1 and VEGFR2 was detected by FCM. ResultsThe isolated CD105+TEC presented "fibrous bands" appearance, while CD31+TEC presented "fusiformis" appearance and NEC presented "cobblestone" appearance. Macrophages, fibrocytes, and tumor cells contamination were excluded. Surface CD68 and a-SMA expression were less than 5%; AFP expression was significantly lower than that of Hepatocarcinoma (P＜0.05), Internalization of DiI-Ac-LDL was positive in more than 95% of isolated cells. The optical density of CD105+TEC and CD31+TEC grown for 48 h were 0.45±0.04 and 0.35±0.02 (P 0.023), respectively. Proliferation of CD105+TEC was significantly more active than CD31+TEC. The isolated cells could form capillary-like tubes on Matrigel and CD31+TEC has more excellent capability of capillary-like tubes than CD105+TEC.FCM analysis technology was adopted to test the expression of VEGFR1 and VEGFR2 etc. in various subtypes of TEC and NEC (including the CD105+TEC/NEC and CD31+TEC/NEC).We found that the expression of VEGFR2 and VEGFR1 were different among differentsubtypes, although the difference was not significant.The expression of VEGFR1and VEGFR2 in NEC membrane reduced more significant than that of TEC after incubation with anti-angiogenesis drugs.Meanwhile, we also found the reduction of VEGFR1 and VEGFR2 expression was more significant in CD105+TEC than in CD31+TEC.ConclusionsMACS with magnetic beads conjugated with anti-CD 105 and anti-CD31 antibody could be successfully used to isolate TEC and NEC, namely CD31+TEC/NEC and CD105+TEC/NEC.Endothelial cells from different tissue(HCC or peritumoral liver tissue) or isolated with different markers (CD105 or CD31)were proved to be different subtypes, by morphologicaland biological studies. These differences should be studied by furtherexpriments.Potential applicationThemembrane receptor differed among different subtypes of TEC, which may resulted indifferent therapy response and resistance development among different patient.These findings may help to findnew molecular targetsof anti-angiogenesis therapy and to explore drug-resistant mechanism. NoveltyWe confirmdifferent subtypes of TECexist in human HCC.They are distinct in biological behavior or function and different in anti-angiogenesis drugs related to their membrane receptors.