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UW Madison Group Wins NCI Funding for Microfluidic Device to Aid CTC Analysis

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NEW YORK (GenomeWeb) — A group at the University of Wisconsin, Madison recently received a five-year grant totaling approximately $1.5 million from the National Cancer Institute to develop a microfluidic chip that will combine circulating tumor cell purification, DNA and RNA extraction, and protein analysis.

The integrated platform, the result of a collaboration between biomedical engineers and oncologists at UW-Madison's Wisconsin Institutes for Medical Research, is intended for clinical use, and could generate a "comprehensive snapshot" of CTC function at a molecular level.

In addition to mRNA for RT-PCR and DNA for SNP and sequencing readouts, the chip is being developed to analyze subcellular protein localization and quantify total protein levels. The resulting platform will then be tested in a prospective study measuring many manifestations of androgen receptor status in 40 prostate cancer patients.

Joshua Lang, an oncologist at UW-Madison's Carbone Cancer Center and project leader on the new grant, told PCR Insider the new device is based on a method to capture mRNA and DNA called selective nucleic acid removal via exclusion, or SNARE. That method was described in Analytical Chemistry last September.

In SNARE, simultaneous DNA and RNA extraction from single cells is accomplished using "pinned oil interfaces." In their Analytical Chemistry paper, the researchers showed that the microfluidic device isolated as much or more mRNA and DNA as the Qiagen Allprep DNA/RNA Micro Kit from ten or fewer cells, as demonstrated by qPCR. The resulting nucleic acids were also a suitable template for sequencing.

The current funding is to develop SNARE into a new platform, called VERSA, for vertical exclusion-based rare sample analysis. This chip will perform single-cell CTC purification and RNA and DNA extraction, as well as protein analysis.

Lang said the motivation for creating a CTC purification device that can also perform extraction came from challenges he sees in his clinical practice. "We have many therapeutic options, but limited tools to assess and identify patients most likely to respond to different treatments, [and] even fewer tools to evaluate them over the course of treatment, let alone identify what is the next therapy we could recommend," Lang said.

Lang's collaborators on the project specialize in microfluidics. The layout of his UW-Madison workplace encouraged such collaboration, he said. "I was fortunate that when I became interested in CTCs, [the bioengineers] were just one floor down. ...We have clinicians, biologists, engineers, all within the same building, so we can run back and forth and ask questions as they come up. That was actually how we started this entire project a couple of years ago," he said.

The new device uses antibody-coated magnetic particles to capture CTCs based on cell surface phenotype. The chip also integrates cell capture with extracellular staining for counting and classing tumor cells.

Although the duo of antibody and magnetic capture of CTCs is a component in other purification devices in development, Lang said his group's device uses a novel fluidics mechanism that takes advantage of a unique principle of microfluidics.

"At the microscale, surface tension is a dominant force over gravity," he explained. The chip consists of microscale chambers positioned in contact with each other, containing alternating aqueous and oil solutions. "That barrier between oil and water is essentially a filterless filter. We can bring anything that we've captured with the magnetic particle up to that oil and water interface, pull our magnetic particles across that, and because of surface tension, anything that is not bound to those particles remains behind."

This technique enables cell staining as well, and eliminates centrifuging and pipetting steps. "For example, we capture cells in our input well, pull them through the first oil barrier into another aqueous solution that has antibodies for extracellular staining," Lang said. "[Then] we pull our captured cells through another oil barrier, and that washes off any excess antibody and we can do our imaging and enumeration. After that, we can add a lysis buffer with magnetic beads to bind RNA and pull those through an oil barrier to purify mRNA in a single step. The tumor nuclei are still retained in the first well, so then we can add a high salt buffer to lyse [them] and add silica beads to bind DNA and pull those through a different pathway."

This oil/water method thus enables "paired transcriptomic and genomic analyses from the same cell population," Lang said. The device is also entirely closed. "Once we put the cells into the chip we never take them out. It really simplifies our workflow," he said.

This device will aid in obtaining material for multiple downstream analyses, like qPCR or sequencing, within a single patient sample. While that should prove useful to diagnostics and treatment monitoring, it may also help answer some nagging questions in cancer biology. Single-cell profiles of heterogenous CTCs could help characterize which ones have metastatic or proliferative potential, Lang said. "We really don't have that information right now with the current technology."

There are currently a number of devices in development for CTC purification. For example, a recent article in The Scientist profiled three popular methods of purifying CTCs, based on size, imaging, or passive capture. Lang's antibody-coated magnetic particle-based device would fit the passive capture category. Thus, it could ultimately be competing with another device highlighted in that article, designed at Harvard, licensed to Johnson & Johnson, and slated for commercialization in 2015.

However, Lang said he believes there is plenty of room in the market for different devices for CTCs. True, there are many different technologies, but there are also many contexts for use.

"When we look at the broad landscape of oncology, across different tumor types, disease settings, stages of disease, and therapeutic contexts, there are just so many different needs out there," he said. "I don't think there will be one technology or one assay that is suitable for all of those applications." His group's approach has been to "engineer as much flexibility into the platform and to integrate all of our capture methodologies with downstream assays."

Studies aimed at detecting emerging therapy resistance using the prototype VERSA are ongoing. Much of the group's current work is on prostate cancer and has been supported by Movember fundraising proceeds from the Prostate Cancer Foundation, Lang said. The current studies specifically examine the androgen receptor, which can be altered both at the genomic and transcriptomic levels, and show protein changes in individual cells. "It's a good model for this kind of comprehensive analysis," he said.

"From a PCR-related perspective, we're looking at doing qPCR for various targets in prostate cancer, such as splice variants in the androgen receptor, and looking at genomic alterations using PCR assays as well as other downstream DNA and RNA sequencing assays" from patient samples, Lang said. This work is currently being submitted for publication.

In terms of commercialization of the device, the group is in early stage discussions with a few different entities and patent applications have been filed on some aspects. The project is currently "focused on developing tools that have the potential to be CLIA certified and integrated into clinical care and prospective clinical trials," Lang said.

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