INHIBITION OF EPIDERMAL GROWTH-FACTOR RECEPTOR-ASSOCIATED TYROSINE KINASE BLOCKS GLIOBLASTOMA INVASION OF THE BRAIN

OBJECTIVE: Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, i n part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor g rowth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro. METHODS: Cocultures of human gliobla stoma spheroids (derived from three separate patients) and fetal rat b rain aggregates were examined for infiltration using confocal microsco py, in the presence of 0 to 100 mu mol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mu mol/L tyrphostin A25, a specific EGFR-TK inhi bitor. RESULTS: Infiltration (not attachment) was completely inhibited by genistein at 10 mu mol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mu mol/L (the IC20 for monolay ers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion -hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.0 65% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% in hibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in E GFR-associated tyrosine phosphorylation at those doses in monolayers b y P-32 immunolabeling, consistent with the known effects of low concen trations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equa lly well inhibited by a monoclonal antibody to the high-affinity ligan d binding domain of EGFR and not by antibody to an inactive domain. CO NCLUSION: Our observations support the role of EGFR activation as a de terminant by which glioblastoma invades normal brain tissue, and we sh ow that invasion can be effectively inhibited at much fewer concentrat ions of TK inhibitors than are necessary for growth suppression.