All errors were calculated as the standard error of the mean (SEM). of Ang1 by blocking its cognate receptor Tie2, and we found that the formation of glomeruloid bodies was inhibited. Collectively, these results support our hypothesis that Ang1 is a key molecular regulator of pathological vascularization characteristic of malignant astrocytomas. Full-length human Ang1 cDNA was a gift from K. Alitalo (Helsinki, Finland) and was subcloned into the mammalian Prostaglandin E2 expression vector pSECTagB/Myc-HIS (Invitrogen, Mississauga, Ontario, Canada). The Ang-Myc/HIS sequence was subcloned into the pCAGG vector, containing a Cytomegalovirus promoter with a chicken -actin enhancer. Stable cell lines were Prostaglandin E2 generated by transfection of this vector into the U87-MG cells using Lipofectamine-2000 (Gibco/BRL, Burlington, Ontario, Canada), as per the manufacturer’s instructions. Stable clones were selected with 1 mg/ml Zeocin (Invitrogen), and cells were expanded. Twenty stable clones were examined for Ang1 expression levels using immunoprecipitation and Western blot analysis, as described below. Two of the highest-producing Ang1 clones (A1-1 and A1-2), plus one pooled clone (A1-p), were selected for studies (Figure 2growth effect of Ang1 on subcutaneous and intracranial xenograft models of GBMs. (A) The level of Ang1 expressed above baseline control by the corresponding stable U87-MG:Ang1 clones (A1-1, A1-2, and one pooled clone A1-p) is demonstrated at the bottom of the growth curve. (B) U87-MG:Ang1 astrocytoma stable clones grown as subcutaneous xenograft models in NOD-SCID mice demonstrate a faster growth rate and a final tumor size comparable to that of control empty vector-transfected clones. (C) Varying levels of Ang1 expression by addition of Dox (bottom) did not result Prostaglandin E2 in a statistically significant difference in survival, which were all decreased compared to control. (D) Intracranial xenografts of Tet-Off-regulated U87-MG:Ang1 tumors demonstrate decreased survival concordant with a faster growth rate, compared to U87-MG:Control (vector transfectants). As described previously, stable Tet-Off U87-MG cells have been established [31,32]. Briefly, U87-MG cell lines were transfected with the pTet-Off (Clontech, Palo Alto, CA) vector, and stable clones were selected and maintained in 1 mg/ml and 500 g/ml G418, respectively. Thirty CANPml of the Tet-Off clones were assayed by transfecting with pTRE-LUC and, using a luciferase assay, the highest tetracycline-inducible clone was selected for generating double-stable Tet-Off cell lines (data not shown). Double-stable Tet-Off U87-MG cell lines overexpressing Ang1 were generated by cotransfecting U87-MG:Tet-Off stable cells with pTRE-Ang1 using the Prostaglandin E2 pTK-puromycin vector. Stable clones were selected in 3 g/ml puromycin, and 20 clones were tested for induction of Ang1 expression using immunoprecipitation followed by Western blot analysis, as described below. For control U87-MG:Tet-Off double-stable cell lines, pTRE-Red vector (Clontech) expressing the dsRed fluorescent protein was used. testing of tetracycline induction was determined using varying doses of doxycycline (Dox), with the most tightly regulated clones expressing Ang1 selected for experiments (Figure 2Tumor Models Subcutaneous xenografts were generated by growing U87-MG stable clones overexpressing Ang1 in the flanks of NOD-SCID mice. For each stable clone, seven mice were injected with 10E7 cells suspended in 300 l of PBS, with five mice injected with control empty vector transfectants. Tumor growth was measured biweekly, using calipers, by two observers in a blinded fashion. Tumor volume was calculated using the formula: (diameter2 x length)/2. As per animal protocol, mice were sacrificed by cervical dislocation after 100 mg/kg bromodeoxyuridine (BrDu) (Sigma-Aldrich, Toronto, Canada) injection. Tumors were cut in cross sections, with two cross sections kept in formaldehyde for paraffin blocks and immunohistochemical analysis and with the remaining tumor stored in liquid nitrogen. All tumor models were repeated in duplicate. For orthotopic xenograft models, Tet-Off-regulated human U87-MG:Ang1 cells (10E6) were stereotactically injected 3 mm deep.