Malignant gliomas (astrocytomas) are deadly, invasive brain tumors with high levels of expression for epidermal growth factor receptor (EGFR). Extension of tumor cell pseudopodia into interstitial spaces initiates invasion. The U87 glioma cell line cultured in minimal essential media with 10% fetal bovine serum (FBS) migrated spontaneously in phosphate buffered saline with 1% FBS, 0.1% bovine serum albumin (BSA), and miminal chemical additives (calcium, magnesium, etc.) through 8 μm porous filters at 5 hr. The cytoplasmic strands extended by U87 glioma cells through 3 μm pores of gelatin-coated filters were harvested at 5 hr (before their nuclei traversed the pores). Pseudopodia were solubilized in urea lysate buffer in sufficient quantity for examination with specific antibodies on immunoblots to identify proteins suspected as mediators of cell migration and invasion. An antibody with broad specificity for phosphorylated (activated) growth factor receptors, including Met (145 kD β subunit), platelet derived growth factor receptor, PDGFR (175 kD), EGFR (170 kD), fibroblast growth factor receptors (122 kD or less), and insulin receptor (132 kD or less) demonstrated reactivity with a single band in pseudopodial lysates migrating in the 170-188 kD range. Compared to whole cell lysates prepared similarly, the pseudopodial reactivity was increased 2.82-fold. Specific antibodies for phosphorylated EGFR, Tyr845, Tyr992, Tyr1045, and Tyr1068, demonstrated reactivity for all these isoforms within the pseudopodial lysate. All of the bands reactive with the phosphorylated EGFR antibodies migrated the same distance as the band reactive with the broadly specific growth factor receptor antibody. An antibody for phosphorylated PDGFR β (Tyr751) reacted predominantly with two bands migrating at 50 - 60 kD with no reactivity at or near 175 kD. Activated EGFR is a candidate for mediating pseudopodial formation in glioma cells in coordination with other constituents of the pseudopodial proteome described recently (Beckner ME et al., Lab Invest. in press). Potential ligands for the EGFR receptor were limited to those made by the U87 cells, captured by cells from 10% FBS in culture media prior to the assay, or bound by the pseudopodia from 1% FBS or 0.1% BSA during the assay. Harvested pseudopodia provide a subcellular compartment for analyzing activated EGFR’s role in the biology of brain tumors. Supported by The Nick Eric Wichman Foundation, Ellicott City, MD, and The Pittsburgh Foundation’s Walter L. Copeland Fund for Cranial Research.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]