Researchers have developed an in vitro method involving an integrated microfluidics and imaging platform that can reproducibly measure kinase enzymatic activity from as few as 3,000 cells in human leukemia patient samples in a study published Nov. 1 in Cancer Research.
Many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, Thomas Graeber, PhD, assistant professor of molecular and medical pharmacology at Crump Institute for Molecular Imaging in Los Angeles and his team developed an immunocapture-based in vitro kinase assay on an integrated microfluidics platform that adopted the radiometric 32P-ATP–labeled phosphate transfer assay.
"Because the device requires only a very small tissue sample to give results, this method creates new potential for direct kinase experimentation and diagnostics on patient blood, bone marrow and needle biopsy samples," said Graeber.
To improve radio-signal detection, the team used a novel imaging detector, in the form of a solid-state beta camera, which can sensitively detect and spatially resolve radioactive signal directly from a microfluidic chip. The beta camera provided a picture of the activity on the chip, allowing real-time monitoring of the assay performance and outcome.
In their first application of the device, the team measured the activity of the mutated kinase responsible for chronic myelogenous leukemia. The resulting microfluidic in vitro kinase radioassay improved reaction efficiency, compared with standard assays, and can be processed in much less time, according to the researchers.
"Integration of the solid-state beta camera allows researchers to monitor the assay in real time, which proved useful during our protocol development and testing," said Cong Fang, PhD, from University of California, Los Angeles. "The integrated microfluidic and imaging platform opens new possibilities and makes miniaturization of many common radioactivity-based bioassays to the microfluidic realm possible."
"Taken together, the reduced sample input required, the decreased assay time, and the digitally controlled reproducibility of the team's microfluidic kinase radioassay facilitates direct experimentation on clinical samples that are either precious or perishable," said Yanju Wang, PhD, postdoctoral fellow at University of California, Los Angeles.
Future experiments will develop reproducible sample collection and measurement conditions for primary patient samples. Other applications could include profiling of patient and animal model samples for their kinase-inhibitor drug sensitivity, or measurement of kinase activity from stem cells, cancer stem cells and other rare immune cells, according to the researchers.