Hydrogel-based protein array for quantifying epidermal growth factor receptor activity in cell lysates

Abstract

Epidermal growth factor receptor (EGFR) signaling plays an important role in a majority of solid tumors, and therapeutics targeted against EGFR has demonstrated promise in slowing growth of these tumors. However, many of these drugs either have failed to reproduce promising preclinical model results in clinical settings or have been successful in only a subgroup of cancer patients due partly to incomplete assessment of EGFR status in cancer. A patient-customized, predictive diagnostic for the effects of specific anti-EGFR therapies may improve outcomes. Here we report the development of a hydrogel-based protein array for quantitative and reproducible determination of the activity of EGFR directly from cellular extracts. In this study, we used glutathione S-transferase-fused Eps15 (GST–Eps15) fusion proteins immobilized within a polyacrylamide hydrogel as a substrate for quantifying EGFR kinase activity from the extracts of EGFR-expressing cell lines. Significant EGFR up-regulation was detected in a mixture containing 7% EGFR-overexpressing cell lysate diluted in lysate from a cell line expressing low levels of EGFR. In addition, the GST–Eps15 protein array was capable of detecting inhibition of EGFR activity when incubated with different tyrosine kinase inhibitors. These findings establish the potential of this protein–acrylamide copolymer hydrogel array not only to evaluate EGFR status in cancer cell lysates but also to screen for the most promising therapeutics for individual patients and monitor treatment progression.