Human Hepatocellular Carcinoma Tumor-Derived Endothelial Cells Present Increased Angiogenesis Capability And Drug Resistance Compared With Normal Endothelial Cells

Hepatocellular carcinoma(HCC)is one of the most prevalent cancers in Asia and Africa.Despite endeavors have been made to improve its prognosis,the overall survival,however,is still unsatisfied.The high rate of metastasis and recurrence after curative resection was responsible for the poor prognosis of resectable HCC.Anti-angiogenesis is an attractive treatment for HCC,because HCC is a typical hypervascular cancer,its growth,metastasis or even hepatocarcinogenesis depend on angiogenesis.As a major part of tumor vessels,tumor-drived endothelial cells(TEC) are heterogeneous in different organs,tissues and tumors.Alterations in growth factor receptors,cellular survival pathways,surface adhesion molecules,and passage-related changes in EC markers are all consistent with molecular perturbations in TEC,which may contribute to the function distinction between TEC and normal endothelial cells (NEC).Moreover,recent studies indicate that TEC from breast tumor,renal tumor and nasopharyngeal carcinoma,compared with NEC,were less sensitive to chemotherapeutic agents such as vincristine and doxorubicin in vitro.While whether acquired drug resistance to anti-angiogenic therapies might be a feature of the TEC has not yet been reported.The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma1.Isolation and culture TECs or NECs from HCC or adjacent normal liver tissueAs a main target of anti-angiogenesis therapy,TECs represent only a minor fraction of the total cell population within tumor tissues.Along with the fact that endothelial cells are enmeshed in a complex tissue consisting of a variety of cells,analysis of TEC has long been considered a tough task due to the difficulty of isolation and purification.In this study,a modified immunomagnetic methods using magnetic beads conjugated with anti-CD 105 antibody were used to isolate vascular endothelial cells from human HCC or adjacent normal liver tissue.The isolated NEC presented typical"cobblestone"appearance while TEC not.Negative expression of alpha fetoprotein (AFP)and CD68 in isolated ECs excludes the contamination of tumor cells and macrophages.The expression of CD105 and von Willebrand factor(vWF)was positive in more than 99%of isolated cells.Internalization of acetylated low-density lipoprotein(DiI-Ac-LDL)was positive in more than 95%of isolated cells.The isolated cells could form capillary-like tubes on BD Matrigel Matrix.Also,the isolated cells could express a panel of endothelial markers including CD105,CD31, CD34,VE-cadherin(CD144),vascular endothelial growth factor receptor-1 (VEGFR1),VEGFR2 with different ratio.Thus,TEC and NEC can be isolated from HCC and adjacent normal liver tissue by using modified immunomagnetic methods, and these cells can be serially subcultured and used for angiogenesis related researches in HCC.2.Functional characteristics of CD105~+ TECTo compare the function distinction between CD105~+ TEC and CD105~+ NEC or human umbilical vein endothelial cells(HUVEC),a series experiments have been done.Cell proliferation was measured over a period of 72 hrs in full medium. Proliferation of CD105+ TEC was significantly more active than CD105+ NEC and HUVEC when cultured in the serum supplemented medium for 24,48,and 72 hrs(P＜0.05).CD105+ TEC was more resistant to apoptosis in the serum-free medium compared with CD105+ NEC and HUVEC.Wound healing assay revealed increased migration ability of CD105+ TEC and endothelial cell-tumor cell interaction assay revealed increased ability to adhere more HCC cells of CD105+ TEC,with respect to CD105+ NEC and HUVEC.CD105+ TEC also could form capillary-like structures on Matrigel in the absence of serum while HUVEC not.Moreover,CD105+ TEC was presented increased drug resistance to cytotoxic therapy including adriamycin and 5-fluorouracil(5-Fu)than CD105+ NEC and HUVEC.Those data confirmed CD105+ TECs did not undergo cell senescence in serum-free medium and showed enhanced proliferation,motility,pro-angiogenesis properties,and resistance to cytotoxic drug treatment.3.The increased resistance of CD105+ TECs to sorafenib and its mechanism A handful of laboratories have reported successful isolation and subsequent culture of TEC and explored some characteristics of TEC,while whether acquired drug resistance to anti-angiogenic therapies might be a feature of the TEC has not yet been reported.In this study,we have compared the response of CD105+ TEC,CD105+ NEC,and HUVEC to the anti-angiogenic drug sorafenib.Cell proliferation revealed that HUVECs and CD105+ NECs were sensitive to sorafenib in a dose-dependent manner when the concentration was over 2μM,while CD105+ TEC were resistant to sorafenib at the higher dosage of 10μM.The tube formation experiment and EC-spheroid assay showed CD105+ TEC could form capillary-like tubes and produce sprouts at 5μM sorafenib while CD105+ NEC,and HUVEC not.These results demonstrated CD105+ TECs acquire more resistance to anti-angiogenic therapies than normal EC.Then we investigated the expression levels of p-STAT3,STAT3, p-Akt,Akt,p-MAPK and MAPK of different types of EC after sorafenib treatment, and found that the expression of p-STAT3 and p-Akt in CD105+ TECs before and after sorafenib treatment were higher than NECs.We also found that the baseline level of p-MAPK in CD105+ TEC was lower than NEC,and after sorafenib treatment for 24 hrs,in CD105+ TECs p-MAPK expression was significantly higher than untreated cells.These changes may contribute to the resistance of CD105+ TEC to sorafenib,and suggest a shorter dosing interval of sorafenib may of clinical benefit.Conclusions1.The modified immunomagnetic affinity purification can successfully isolate CD105+ endothelial cells from HCC and adjacent normal liver tissue,and these cells can be serially subcultured.2.Differential endothelial markers expression does exist between CD105+ TEC, CD105+ NEC,and HUVEC.3.Differential functional characteristics do exist between CD105+ TEC,CD105+ NEC,and HUVEC.CD105+ TEC acquire more pro-angiogenesis properties and resistance to cytotoxic drug treatment.4.CD105+ TEC acquire more resistance to sorafenib than normal ECs.The underlying mechanism may be related to the differential expression of p-STAT3, p-Akt and P-MAPK between CD105+ TEC and normal EC.The novelties of this work:1.Isolated and serially subcultured endothelial cells from HCC and adjacent normal liver tissue,and these cells can be used for angiogenesis related researches in HCC.2.For the first time,identified tumor endothelial cells acquired drug resistance to anti-angiogenic therapies,and explored the underlying mechanism.