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Inappropriate growth and survival signaling, which leads to the aberrant growth of cancer cells, is a driving force behind the development of tumors. Much current cancer research focuses on the kinase enzymes whose mutations are responsible for such disregulated signaling, and many successful molecularly targeted anti-cancer therapeutics are directed at inhibiting kinase activity.
Now, a team of investigators from the University of California, Los Angles (UCLA) has developed an in vitro method for assessing kinase activity in minute tissue samples from patients. The method involves an integrated microfluidics and imaging platform that can reproducibly measure kinase enzymatic activity from as few as 3,000 cells. In a paper published in the journal Cancer Research, the UCLA researchers describe several new technological advances in microfluidics and imaging detection they co-developed to measure kinase activity in small-input samples. The team applied their microfluidic kinase assay to human leukemia patient samples.
"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 Thomas Graeber, who along with Hsian-Rong Tseng and Arion Chatziioannou, led the research team. "For example, the stem cell properties of leukemia can be directly studied from patient samples." Drs. Graeber and Tseng are member of the Nanosystems Biology Cancer Center at UCLA, one of nine Centers of Cancer Nanotechnology Excellence funded by the National Cancer Institute's Alliance for Nanotechnology in Cancer.
To improve radio-signal detection, the team used a novel solid-state beta camera detector that can sensitively detect and spatially resolve radioactive signal directly from a microfluidic chip. The beta camera provides a picture of the activity on the chip, allowing real-time, quantitative 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. This mutation is targeted by the clinically successful kinase inhibitor Gleevec. "We are not aware of other work demonstrating solid-state integrated radioactive imaging from a microfluidic platform," said Dr. Chatziioannou.
The resulting microfluidic in vitro kinase radioassay improves reaction efficiency, compared with standard assays, and can be processed in much less time. This greater efficiency, coupled with the high sensitivity of the beta camera, reduces the amount of sample cell input by two to three orders of magnitude, compared with conventional and 96-well assays. The assay includes a kinase immunocapture step to increase specificity towards the kinase of interest. "To get the kinase assay to work in a microfluidic environment, we needed to develop new protocols and reagents for efficiently manipulating solid-support kinase capture beads using microfluidic trap-and-release valves," said Dr. Tseng.
Integration of the solid-state beta camera allowed the investigators to monitor the assay in real time, which proved useful during protocol development and testing. With the integration of the compact camera, the microfluidic format assay has the potential to be developed into inexpensive bench-top, stand-alone units. 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.
About The National Cancer Institute (NCI)
The NCI Alliance for Nanotechnology in Cancer is engaged in efforts to harness the power of nanotechnology to radically change the way we diagnose, treat and prevent cancer.
The NCI Alliance for Nanotechnology in Cancer is a comprehensive, systematized initiative encompassing the public and private sectors, designed to accelerate the application of the best capabilities of nanotechnology to cancer.
Currently, scientists are limited in their ability to turn promising molecular discoveries into benefits for cancer patients. Nanotechnology can provide the technical power and tools that will enable those developing new diagnostics, therapeutics, and preventives to keep pace with today’s explosion in knowledge.
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