INDUCTION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR EXPRESSION BY HYPOXIAAND BY GLUCOSE DEFICIENCY IN MULTICELL SPHEROIDS - IMPLICATIONS FOR TUMOR ANGIOGENESIS

Perfusion insufficiency, and the resultant hypoxia, often induces a co mpensatory neovascularization to satisfy the needs of the tissue. We h ave used multicellular tumor spheroids, simulating avascular microenvi ronments within a clonal population of glioma tumor cells, in conjunct ion with in situ analysis of gene expression, to study stress inducibi lity of candidate angiogenic factors. We show that expression of vascu lar endothelial growth factor (VEGF) is upregulated in chronically hyp oxic niches (inner layers) of the spheroid and that expression is reve rsed when hypoxia is relieved by hyperoxygenation, Acute glucose depri vation-another consequence of vascular insufficiency-also activates VE GF expression. Notably, glioma cells in two distinct regions of the sp heroid upregulated VEGF expression in response to hypoxia and to gluco se starvation. Experiments carried out in cell monolayers established that VEGF is independently induced by these two deficiencies. Upon imp lantation in nude mice, spheroids were efficiently neovascularized. Co ncomitant with invasion of blood vessels and restoration of normoxia t o the spheroid core, VEGF expression was gradually downregulated to a constitutive low level of expression, representing the output of nonst ressed glioma cells. These findings show that stress-induced VEGF acti vity may compound angiogenic activities generated through the tumor '' angiogenic switch'' and suggest that stress-induced VEGF should be tak en into account in any attempt to target tumor angiogenesis.