Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates

Abstract

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.

Multiplexed tyrosine kinase activity detection in cancer cells using a hydrogel immobilized substrate

“…The previously described Escherichia coli BL21 strains containing the pGEX-4T1 vector with inserted amino acid sequences for Gab1 residues 431 to 561, Crkl residues 120 to 303, or EGFR pathway substrate 15 (Eps15) residues 758 to 881 were used to produce the fusion proteins GST-Gab1, GST-Crkl, and GST-Eps15 [12, 14, 22]. An additional BL21 strain containing the pGEX-4T1 vector with the inserted sequence for tensin residues 1392 to 1672 was used to produce GST-tensin.…”

“…Microchannels were assembled as previously described [12, 13] and as shown in Figure 1. Briefly, we piranha-cleaned glass microscope slides and acrylic functionalized them with 2% (v/v) (3-acryloxypropyl)-trimethoxysilane.…”

“…When EGFR kinase activity was measured, 10 µg/ml of EGF was also included in the reaction solution. After the reaction, the channels were washed, blocked, and incubated with 2 µg/mL 4G10 mouse α-phosphotyrosine antibody and 6 µg/mL rabbit α -GST antibody (Invitrogen) as previously described [12]. After washing again, the channels were incubated with a secondary antibody solution containing Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 donkey anti-rabbit (Invitrogen) at 10 µg/mL overnight at 4 °C.…”

“…Fluorescence signal intensity was analyzed for brightness of the hydrogel pillar using Matlab as previously described [12]. For the microchannel data, we normalized the data in each experiment.…”

“…Hydrogels offer a variety of advantages for biological diagnostics and have been used to detect mRNA and proteins, recognize glycoproteins, and measure kinase activity [12–18]. The hydrated, 3-D environment within hydrogels allows proteins to remain in a more native and active conformation, and tunable parameters such as stiffness and porosity may be modified to best fit the needs of the application [19].…”