The morphological abnormalities in tumor blood vessels compared to normal vessels raises questions as to whether there are phenotypical differences at the molecular and functional levels between tumor and normal endothelial cells. To address this question, tumor endothelial cells isolated from tumor tissue were required. However, there have been few reports about isolation of tumor endothelial cells until recently. In fact, for a long time, most studies on tumor angiogenesis were carried out using normal endothelial cells such as human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells, or bovine aortic endothelial cells. To isolate tumor endothelial cells for global analysis of gene expression has been difficult because endothelial cells are usually enmeshed in a complex tissue consisting of vessel wall components, stromal cells, and tumor cells, and only a small fraction of cells within these tissues are endothelial cells. Besides technical difficulties, there might have been concerns about trials to isolate tumor endothelial cells themselves, because they were sometimes considered to lose their specific phenotype soon after being isolated from tumor tissue. In the first report about tumor endothelial-specific markers, St Croix et al. succeeded in isolating endothelial cells from colon carcinoma and normal colonic mucosa and compared the gene expression profiles between tumor and normal endothelial cells in a relatively low number of cells. They identified the specific genes for tumor endothelial cells and designated them as tumor endothelial markers (TEMs) using serial analysis of gene expression (SAGE). SAGE revealed that there are 46 TEMs.(14) Some of them (TEM1, TEM5, TEM7, and TEM8) are transmembrane proteins and are also conserved in mice.(15,16) Very recently, they showed that these TEMs, except TEM8, are also overexpressed during physiological angiogenesis, as well as in tumor endothelial cells. Instead, they identified 13 novel cell surface proteins as TEMs.(17) Other studies about the gene profile of tumor endothelial cells using global analysis have been published recently (Table 1). Buckanovich et al. identified 12 ovarian tumor vascular markers from vascular cells captured by laser-capture microdissection and some tumor vascular markers correlated with the prognosis of patients. However, they commented that these markers are not strictly specific to tumor endothelial cells, because laser-capture microdissection-captured cells contain not only endothelial cells but also mural cells such as pericytes or smooth muscle cells.(18) Ovarian tumor endothelial cells were also isolated with magnetic beads and 23 TEMs were identified by DNA microarray.(19) Among the 23 markers, several genes are involved in the pro-angiogenic pathway. Colon carcinoma endothelial cell markers were also identified by SAGE.(17,20) These studies on the gene profiling of tumor endothelial cells are listed in Table 1. However, tumor endothelial cells were not cultured in these studies and the biological phenotype in tumor endothelial cells remains to be clarified. Another study, based on cultured tumor endothelial cells, found that human renal cell carcinoma endothelial cells did not undergo the senescence that is typical of normal endothelial cells, and were resistant to apoptotic stimuli such as serum-starvation and vincristine. They showed higher proliferation rates in low serum, enhanced Akt activation, and decreased expression of the tumor suppressor, PTEN.(21) Murine Lewis lung carcinoma was characterized by elongated morphology, and upregulated adhesion molecules such as CD31 or ICAM-1. They required a tumor-specific matrix to maintain their characteristics. Sca-1 expression was also elevated in these cells, suggesting the presence of circulating endothelial progenitors in their tumor endothelial cells.(22) We have also purified tumor endothelial cells in an attempt to better understand the effects of the tumor microenvironment on endothelial cell properties.(23) Human tumor xenograft models in nude mice were established as sources of mouse tumor endothelial cells. Murine tumor (melanoma and liposarcoma) endothelial cells and normal (skin and adipose) endothelial cell counterparts were isolated with high purity by magnetic bead cell sorting(24) (Fig. 3). As it is known that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a receptor of diphtheria toxin (DT) in human cells, but not mouse cells, and DT binds to human cells expressing HB-EGF-like growth factor and is toxic to them while mouse cells are resistant to DT,(25) we used DT in tumor endothelial cell isolation.(24) To remove any human tumor cell contamination which might have overgrown in the endothelial cell culture, DT was added to the tumor endothelial cell subculture to kill human cells and normal endothelial cells for technical consistency. The mouse tumor endothelial cells expressed typical endothelial cell markers such as CD31 and vascular endothelial growth factor (VEGF) receptors, and upregulated several tumor endothelial markers that have already been reported, such as TEMs(24) or aminopeptidase N (CD13) (Matsuda et al. unpublished data, 2007). From these data, tumor endothelial cells retain their specificity for tumor endothelial cells (at least some) even in culture. After this publication, we isolated two more tumor endothelial cells from oral carcinoma and renal carcinoma (data not shown). Microarray analysis showed several genes were overexpressed in four different tumor endothelial cells commonly compared to normal skin endothelial cells. There were approximately 50 genes that were expressed 10-fold higher in tumor endothelial cells than in normal endothelial cells (data not shown). These genes are now under investigation to identify novel tumor endothelial cell-specific markers. Our tumor endothelial cells retained endothelial cell properties at least up to 20 passages and the cells could be maintained in culture for at least 50 passages. Tumor endothelial cells grew faster, had a lower serum requirement, and were more responsive to angiogenic growth factors such as basic fibroblast growth factor (bFGF) and VEGF compared to normal counterpart endothelial cells.(23) Furthermore, Amin et al. have found that tumor endothelial cells express high levels of epidermal growth factor receptor (EGFR), not usually expressed in normal endothelial cells such as HUVEC.(26) EGF can induce phosphorylation of tumor endothelial cell EGFR and stimulate tumor endothelial cell proliferation. EGFR tyrosine kinase inhibitors inhibit EGF-induced EGFR activation and proliferation of tumor endothelial cells. Thus, it was suggested that EGFR kinase inhibitors might target not only tumor cells, but also tumor endothelial cell EGFR. This data has clinical significance. Anti-EGFR therapy could target tumor vasculature specifically. Moreover, this therapy can be applied to any cancer in which tumor cells do not express, or express a low level of, EGFR.