Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates.
Overactive and overexpressed kinases have been implicated in the cause and progression of many cancers. Kinase inhibitors offer a targeted approach for treating cancers associated with increased or deregulated kinase activity. Often, however, cancer cells exhibit initial resistance to these inhibitors or evolve to develop resistance during treatment. Additionally, cancers of any one tissue type are typically heterogeneous in their oncogenesis mechanisms, and thus diagnosis of a particular type of cancer does not necessarily provide insight into what kinase therapies may be effective. For example, while some lung cancer cells that overexpress the epidermal growth factor receptor (EFGR) respond to treatment with EGFR kinase inhibitors, overexpression or hyperactivity of Met kinase correlates with resistance to EGFR kinase inhibitors. Here we describe a microfluidic-based assay for quantifying Met kinase activity in cancer cell lysates with the eventual goals of predicting cancer cell responsiveness to kinase inhibitors and monitoring development of resistance to these inhibitors. In this assay, we immobilized a phosphorylation substrate for Met kinase into macroporous hydrogel micropillars. We then exposed the micropillars to a cancer cell lysate and detected substrate phosphorylation using a fluorescently conjugated antibody. This assay is able to quantify Met kinase activity in whole cell lysate from as few as 150 cancer cells. It can also detect cells expressing overactive Met kinase in a background of up to 75% non-cancerous cells. Additionally, the assay can quantify kinase inhibition by the Met-specific kinase inhibitors SU11274 and PHA665752, suggesting predictive capability for cellular response to kinase inhibitors.
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Alicia D
AD
Powers
Bi
B
Liu
Andrew G
AG
Lee
Sean P
SP
Palecek
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry; D046210:Microfluidic Analytical Techniques; D010766:Phosphorylation; D010879:Piperazines / Q000737:chemistry; D016062:Porosity; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D019859:Proto-Oncogene Proteins c-met / Q000032:analysis* / Q000037:antagonists & inhibitors; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry; D013450:Sulfones / Q000737:chemistry
C478479:((3Z)-N-(3-chlorophenyl)-3-((3,5-dimethyl-4-((4-methylpiperazin-1-yl)carbonyl)-1H-pyrrol-2-yl)methylene)-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide); C480541:5-((2,6-dichlorobenzyl)sulfonyl)-3-((3,5-dimethyl-4-((2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)carbonyl)-1H-pyrrol-2-yl)methylene)-1,3-dihydro-2H-indol-2-one; D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D005456:Fluorescent Dyes; C098587:GAB1 protein, human; D007211:Indoles; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D013450:Sulfones; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D019859:Proto-Oncogene Proteins c-met