Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs).In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis. ' 2007 Wiley-Liss, Inc.Key words: HUVEC; glioblastoma multiforme; telomerase; tumor xenograft Glioblastoma multiforme (GBM) is the most aggressive brain cancer. In this tumor, angiogenesis is so prominent to represent a landmark for histological diagnosis.1 Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is expressed by endothelial cells (ECs) of the tumor vasculature with increasing incidence and relative quantity during the progression of astrocytic tumors toward malignancy. We first observed telomerase involvement in the angiogenesis of astrocytic tumors on human tissue sections.2 Expression of hTERT by vascular ECs is a phenomenon strictly related to tumor environment, since it is absent in the ECs of nonneoplastic granulation tissues as well as in normal brain.2 Moreover expression of hTERT is induced in ECs at an early stage of tumor progression and it is positively correlated with the histological grade of the tumor. Using in vitro models, we demonstrated that GBM is able to activate hTERT transcription as well as telomerase activity in human umbilical vascular endothelial cells (HUVECs) and that such effects are likely mediated by diffusible factor/s.3 Taken together, our data suggest that telomerase reactivation might improve the replicative potential of ECs, and that this phenomenon might be induced by the tumor itself. Telomerase expressing ECs are supposed to exhibit survival advantages, i.e. resisting the apoptotic cell death triggered by replication under the cell stressing conditions of the tumor environment. 4 Here, we inhibited telomerase activity in human ECs, either by RNA interference (RNAi) tar...