Improved effect of an antiangiogenic tyrosine kinase inhibitor (SU5416) by combinations with fractionated radiotherapy or low molecular weight heparin.

The effect of combining SU5416 with fractionated radiotherapy or with low molecular weight (LMW) heparin (dalteparin) was studied in U87 human glioblastoma xenografts in nude mice. SU5416 is antiangiogenic by a specific inhibition of the vascular endothelial growth factor receptor 2 (VEGFR-2), and heparins are assumed to bind VEGF. Both SU5416 (100 mg/kg every second day in 5 days) and 3 Gyx5 produced moderate, yet significant, growth inhibition. Tumors treated with concomitant irradiation and short-term SU5416 maintained a lower growth rate during regrowth than the other treatment groups (P=.007). Dalteparin (1000 IE/kg subcutaneously once a day) had no growth-inhibitory effect on its own, but when this LMW heparin was added to the SU5416 schedule, a significantly enhanced growth inhibition was obtained. VEGF protein content in tumors was not significantly altered by SU5416, but a significant decrease in VEGF levels was found in tumors treated with concomitant dalteparin and SU5416 compared with controls (P=.03). We conclude that: 1) an additive growth-inhibitory effect is obtained by combining SU5416 and fractionated radiotherapy; and 2) LMW heparin (dalteparin), in combination with SU5416, decreases the level of VEGF in tumors and increases the growth-inhibitory effect of SU5416.

Angiopoietin-4 inhibits angiogenesis and reduces interstitial fluid pressure.

Angiopoietins (Ang) are involved in the remodeling, maturation, and stabilization of the vascular network. Ang-4 was discovered more recently; thus, its effect on angiogenesis and its interplay with other angiogenic factors have not been equivocally established. The role of Ang-4 in angiogenesis was tested in Matrigel chambers implanted into the subcutaneous space of nude mice. Ang-4 inhibited the angiogenic response of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and GLC19 tumor cells. In Matrigel chambers with Ang-4-transfected cells, the mean response was significantly lower than that of mock cells. Subcutaneous tumor interstitial fluid pressure (IFP) was significantly lower in Ang-4-transfected GLC19 tumors than in mock-transfected tumors. IFP reduction in Ang-4-transfected tumors was comparable to the reduction seen after bevacizumab treatment. In vitro, we examined the effect of recombinant Ang-4 on endothelial cell migration in Boyden chambers. Human umbilical vein endothelial cell (HUVEC) migration induced by bFGF and VEGF was inhibited by Ang-4 to control levels. In conclusion, we show that rhAng-4, as well as transfection with Ang-4, inhibits angiogenesis induced by GLC19 tumor cells and that Ang-4 expression reduces elevated tumor IFP. In addition, we demonstrate that rhAng-4 inhibits HUVEC migration and growth factor-induced angiogenesis.

How few cancer cells can be detected by positron emission tomography? A frequent question addressed by an in vitro study.

PURPOSE: Positron emission tomography (PET) has gained widespread use in cancer diagnosis and treatment, but how many malignant cells are required for a tumour to be detected by PET? METHODS: Three human cancer cell lines [glioblastoma and two subtypes of small cell lung cancer (SCLC)] in concentrations from 10(4) to 10(7) were seeded on six-well plates or plastic tubes and treated with [(18)F]fluorodeoxy-glucose (FDG) in vitro. FDG retention was measured in a PET/CT scanner and in a calibrated well counter. The clinical situation was simulated using a cylinder phantom with a background concentration of FDG. RESULTS: The theoretical detection limit was found to be around 10(5) malignant cells. In a cylinder phantom the detection limit was increased by a factor of 10. The FDG retention by the glioblastoma cell line was significantly higher than the activity of the SCLC cell line. FDG retention measured by PET and a gamma counter was closely correlated to the number of cells and a linear relationship was found. DISCUSSION: The detection limit of PET is in the magnitude of 10(5) to 10(6) malignant cells. The experimental set-up was robust and well suited as a platform for further investigations of factors influencing the detection limit of PET.

Angiogenic synergy of bFGF and VEGF is antagonized by Angiopoietin-2 in a modified in vivo Matrigel assay.

A murine modification of the Matrigel chamber assay originally developed for use on rats is presented. This modified assay permits improved quantification due to subcutaneous Matrigel implants of constant shape and volume. We have quantitatively assessed the angiogenic potential of the growth factors basic fibroblast growth factor (bFGF), VEGF, and Angiopoietin-2 (Ang-2) with special emphasis on their mutual interactions. A reproducible dose-response relationship for bFGF was established for doses between 150 and 1000 ng per chamber, whereas VEGF did not display angiogenic activity on its own in the tested dose of up to 200 ng per chamber. Conversely, we found a strong synergistic action of bFGF and VEGF when combined in a 3:1 ratio. Two other combinations (ratios) with greater VEGF doses were also tested, but the synergistic effect was only observed when 50 ng of VEGF was added to 150 ng per chamber of bFGF. This synergistic effect of bFGF and VEGF was significantly reduced by further addition of 100 ng Angiopoietin-2. Inhibition of the response to bFGF and VEGF was confirmed by in vitro EC migration experiments, which, together with our in vivo results, indicates that Ang-2 may target chemotactic responses to bFGF and VEGF in vivo.

Differential regulation of VEGF, HIF1alpha and angiopoietin-1, -2 and -4 by hypoxia and ionizing radiation in human glioblastoma.

We examined how ionizing radiation (IR) delivered under either severe hypoxia (< 0.1% O2) or normoxia affects the expression of hypoxia inducible factor 1alpha (HIF-1alpha) and the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins 1, 2 and 4 in U87 human glioblastoma cells. IR was delivered as single doses of 0, 2, 5, 10 and 20 Gy after 6-hr hypoxic incubation and in normoxic controls. Irradiation at any dose did not affect the cellular protein levels of any of the angiopoietins, whereas hypoxia led to increasing levels of both angiopoietin-4 and angiopoietin-2. Levels of angiopoietin-1 protein were unaltered throughout the observation period. A dose-dependent increase in levels of secreted VEGF in the medium occurred after IR at doses from 5-20 Gy. In hypoxic cells, 20 Gy IR induced an additional significant increase in VEGF relative to nonirradiated hypoxic control cells with elevated baseline VEGF levels induced by hypoxia. HIF-1alpha and glucose transporter-1 (Glut-1) were not correspondingly upregulated by IR. Blocking HIF-1alpha by antisense treatment induced a reduced baseline VEGF at normoxia, while the relative upregulation of VEGF by IR was unaffected. These data provide evidence that VEGF is upregulated by IR by mechanisms independent of HIF-1 transactivation.