(4bf) Development of Hydrogel-Based Kinase Assay for Monitoring Cancer and Developing Patient-Specific Treatments

Protein array/microarray systems have received considerable attention because of their potential high throughput and cost-effective characterization of protein concentration and activity. However, the major drawbacks associated with these systems are low surface concentration of substrates and non specific binding of sample components resulting in low signal to noise ratios. Moreover, dehydration and denaturation of protein substrates immobilized onto solid surfaces reduce protein activity. We have developed hydrogel based array where the substrate is embedded within the polyacrylamide hydrogel by copolymerization. Immobilization of peptides or fusion protein constructs within the hydrogel network helps maintain the native form of the protein by providing a hydrated environment for the immobilized substrate. Moreover, the characteristic three dimensional network of the hydrogel increases the substrate capacity to a great extent. These characteristics provide a probe with high binding capacity and sensitivity, ultimately improving signal-to-noise ratio.

Epidermal growth factor receptor (EGFR) signaling plays important role in cancer progression and therapeutics directed against the tyrosine kinase domain of EGFR have been developed. However, therapeutics targeted against EGFR have been only partly successful to reproduce promising preclinical model results in clinical settings due to incomplete assessment of EGFR status in cancer, inappropriate drug dosage and /or development of drug resistance during treatment. Hence, a diagnostic tool capable of quantifying the activity and inhibition of EGFR activity will be beneficial in identifying the optimal therapy for individual cancer patients and monitoring for treatment progression. We have developed a hydrogel based assay for quantitative and reproducible determination of the activity of EGFR directly from cellular extracts as well as for determining the sensitivity of different inhibitors to EGFR activity. The protein array has been able to identify EGFR upregulation in a mixture containing 7% EGFR-overexpressing cell lysate diluted in lysate from a cell line expressing low levels of EGFR. Moreover, this system is also capable of evaluating the efficacy of different inhibitors and screening for the most promising therapeutics to overcome the acquired drug resistance.

In future, this assay can be adapted to not only quantify activities of other kinases dysregulated in different cancers but can also be extended into a multiplexed assay with multiple substrates for molecular monitoring of cancers as well as other diseases affected by dysregulated kinases. Moreover, this hydrogel based array can also be used to determine the effect of inhibitors of different kinases on stem cell biology. This is particularly important as cancer stem cells as been implicated in cancer relapse and eradicating cancer stem cells is of profound importance in long term cancer eradication.