(316a) Hydrogel-Based Protein Array for Quantification of EGFR Activity in Cancer Cellular Extracts | AIChE

(316a) Hydrogel-Based Protein Array for Quantification of EGFR Activity in Cancer Cellular Extracts


Ghosh, G. - Presenter, University of Wisconsin-Madison
G, A. - Presenter, University of Wisconsin-Madison
P, S. - Presenter, University of Wisconsin-Madison

Epidermal growth factor receptor (EGFR) signaling pathways have been implicated in playing an important role in cancer development and progression. This led to the development of therapeutics directed against the tyrosine kinase domain of EGFR. However, therapeutics targeted against EGFR either have failed to reproduce promising preclinical model results in clinical settings or have only been successful in a cohort of cancer patients bearing somatic mutation in EGFR kinase domain. The failure can be partly attributed to incomplete assessment of EGFR status in cancer, inappropriate drug dosage and /or development of drug resistance during treatment. One way to address this problem is to develop a customized targeted treatment. A patient-customized, predictive diagnostic that quantifies the effects of specific anti-EGFR therapies may improve outcomes in cancers where EGFR plays a mechanistic role. Nonetheless, major challenges in tailoring patient-specific treatment regimens involve predicting the most effective inhibitors and monitoring for treatment progression. We believe any system capable of directly measuring the EGFR activity will provide a more accurate report of the disease state than the conventional methods which involve quantification of EGFR at DNA, RNA, or protein levels. However, technical challenges in developing such a tool involved specific quantification of EGFR activity in the background of multiple tyrosine kinases present in cancer cellular extracts and obtaining accurate measurements from small biopsies. Here we report the development of a novel hydrogel based protein array for quantifying the activity of EGFR in cancer cellular extracts. Acryl-labeled EGFR substrate (glutathione S-transferase ? Eps15 (GST-Eps15) fusion protein) is polymerized within a polyacrylamide hydrogel that is immobilized to a surface. Incorporation of the substrate within the hydrogel network helps maintain the native form of the protein by providing a hydrated environment and increases the accessibility of the substrate to kinases in the cell lysate. These characteristics provide a probe with high binding capacity and sensitivity, ultimately improving signal-to-noise ratio. The extent of substrate phosphorylation upon incubation with lysates of cancer cells corresponded to the difference in activation status of EGFR in different cancer cell lines. Significant EGFR upregulation was detected in a mixture containing 7% EGFR-overexpressing (breast cancer cell line, MDA-MB-468) cell lysate diluted in lysate from a (breast cancer cell line, MDA-MB-453) cell line expressing low levels of EGFR. Approximately, 2000 cells are required for quantification of EGFR activity. Additionally, the ability of the protein array to quantify the extent of inhibition of EGFR activity by different tyrosine kinase inhibitors establishes the potential of this protein-acrylamide copolymer hydrogel array to screen for the most promising therapeutics for individual patients and monitor treatment progression.