University of California, Los Angeles, researchers have built an integrated chip-based microfluidic and imaging platform capable of measuring kinase activity in samples containing as few as 3,000 cells.
The platform, which the scientists used to measure ABL kinase activity – the target of the kinase inhibitor Gleevec – in BCR-ABL-positive leukemia patient samples, could potentially be expanded into a multiplex version for use in high-throughput kinase studies, said Thomas Graeber, assistant professor of molecular and medical pharmacology at UCLA and one of the platform's developers.
Graeber's team is also behind a chip-based microfluidic platform and accompanying brain tumor biomarker assay that biotech startup CytoScale Diagnostics licensed in August for commercial development (PM 08/06/2010). The new kinase platform is a "more sophisticated version" of that microfluidic device, Graeber told ProteoMonitor, with pneumatic valves that can be controlled via computer, allowing researchers to manipulate samples directly in the device.
In the ABL kinase study, which was detailed in a paper published in this month's edition of Cancer Research, the scientists adapted the standard radiometric 32P-ATP-labeled phosphate transfer assay for the microfluidic platform. For detection they used a solid-state beta-particle camera embedded directly below the device, allowing real-time, position-sensitive monitoring of the assay.
Given the importance of protein signaling networks in a wide range of diseases including cancer, kinases have become a major area of focus for researchers and drugmakers. The UCLA microfluidic assay allows the direct measurement of a kinase's activity, making it a potentially useful complement to approaches to protein signaling research like reverse phase protein microarrays or mass spec-based phosphoproteomics.
"My lab does a lot of phosphoproteomics, quantitating how much of a phosphorylated protein is around," Graeber said. "That's all very rich information, but it's a surrogate for [kinase] activity; it's not directly measuring the kinase activity. Sometimes it's helpful to see the other substrates and products and monitor those, but sometimes it's helpful just to measure the direct amount" of kinase activity.
"If you think about a living system, whether something is phosphorylated or not will be the combined effect of the kinases that phosphorylate it and the phosphatases that desphosphorylate it," he added. "So you could have a highly active kinase that's offset by a highly active phosphatase."
Leukemia offered a good first test of the assay, Graeber said, because the samples could be collected through a blood draw, making them relatively easy to obtain. Ultimately, though, his team hopes to apply the technique to samples like patient biopsies, as well. He named melanoma as one of the candidates they are considering for study.
"Our main interest is to be able to use this device directly on patient samples. So the first goal was to get the amount of sample needed low. Being able to go down a couple orders of magnitude in sample input opens up new possibilities. That moves you into being able to look at small patient biopsies, rare populations of cells, anything where your source is limiting," he said.
In the Cancer Research study, the scientists were able to perform the microfluidic assay using roughly two to three orders of magnitude less sample than is required for a conventional 96-well assay.
In addition to enabling studies involving limited sample sources, the lower input required by the microfluidic assay also has the advantage of using less radioactive 32P, Graeber noted.
"Radioactivity has its advantages," he said. "Radioactive phosphate looks essentially like regular phosphate and doesn't really change the properties of the reaction at all. It's not like you're adding a big fluorescent molecule or tag of some sort. One of the disadvantages, of course, is the safety concern. But by miniaturizing it, the amount of radioactivity goes down, so it becomes safer in that regard."
Currently the platform consists of two microfluidic modules that can be operated in parallel, but, Graeber said, the researchers plan to expand the number of modules to enable its use in "high-throughput, systems-biology" research.
"We have just two units here, but if we could expand that to 20 or more units then it would actually become something in the lab that we can use to measure many kinases, or many different time points, or many different drugs in a screening assay," he said.
"I don't think there are any real hurdles that aren't conquerable. It's more an engineering question and just something that needs the time and effort put towards it," he added.
The researchers have patented the platform through UCLA and are currently working with the university to explore commercialization opportunities. CytoScale is a possible commercial partner, Graeber said, but "that's not something that's been settled at this point."
CytoScale CEO David Franklin told ProteoMonitor that the new platform has "come across our desk, and we're interested in it, but nothing has moved forward."
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