NEW YORK ─ A dearth of methods to rapidly crank out microfluidic tools has hindered researchers looking to develop liquid-biopsy assays that detect extracellular vesicles as markers of disease. University of Kansas (KU) researchers believe they found a way around that using inkjet printing.
In a proof-of-concept study published recently in Science Translational Medicine, the group printed a microfluidic chip using nanopatterned glass as part of a test that detected MMP14, a biomarker of breast tumor metastasis.
The microfluidic tool detected the extracellular vesicles (EVs) ─ particles released from cells that are delimited by a lipid bilayer and cannot replicate ─ in patient samples.
"Among the key advances [of the STM study] are the nanofabrication capabilities to enable scalability of the platform, which can become key bottlenecks with other technologies," Cesar Castro, director of the cancer program at Massachusetts General Hospital Center for Systems Biology, said in an interview.
The need for innovative diagnostic platforms and multi-disciplinary approaches are still paramount in the exosome diagnostics field, said Castro, who is not involved in the KU project.
In the KU study, the EVs were bound to MMP14, proteins of the matrix metalloproteinase (MMP) family involved in disease processes such as metastasis. Using cancer cell lines and mouse models, the researchers leveraged the microfluidic tool to conduct an integrative analysis of the expression and proteolytic activity of MMP14 on EVs to detect in vitro cell invasiveness and monitor in vivo tumor metastasis, they said.
To detect EVs associated with disease, the KU researchers developed a specific fabrication method that they call high-resolution colloidal inkjet printing to enable scalable manufacturing of three-dimensional (3D) patterned devices on nanochips. The approach replaces more time consuming and laborious fabrication methods, according to the researchers.
The microfluidic system when used to analyze clinical plasma specimens demonstrated its potential to detect cancer biomarkers by classifying samples from control subjects and patients with in situ ductal carcinoma, invasive ductal carcinoma, and localized metastatic breast cancer, the researchers said.
Larger studies consisting of greater volumes of patient samples are needed to prove the clinical utility of the tool, and further work needs to be done to develop the device as a cartridge and instrument pairing, Yong Zeng, a study author and bioengineer in the department of chemistry at the University of Kansas, said in an interview.
However, with further clinical validation, the technology could prove useful as part of a liquid biopsy platform to improve cancer diagnostic testing and enable real-time surveillance of tumors as they evolve in patients to inform treatments, he added.
The design of the microfluidic system's 3D structure is among the most important features of the study, Zeng said. The tool consists of nanoparticles coated with antibodies that recognize and capture cancer-specific proteins carried by the vesicles. As plasma flows through the chip, antibodies immobilized on its surface capture the vesicles of interest, and the device washes away unwanted proteins, leaving behind the exosomes derived from tumors. Reagents introduced to the system interact with target enzymes on the surface of the vesicles. The system uses electronics to control valves and pumps that manage the flow of reagents and a microscope to detect the intensity of fluorescence signals associated with the concentration and activity of the tumor vesicles.
Such a microfluidic chip has the potential to support studies designed to validate EV-based diagnostic tests, said Andrew Godwin, deputy director of the University of Kansas Cancer Center. Godwin supplied samples for the STM study and is collaborating with the KU researchers to translate their research into clinical applications.
Like others doing research in this field, the KU group has been relying on time-consuming fabrication methods, involving molding and stamping, to build microfluidic prototypes, Godwin said. However, the scale of the studies needed to validate microfluidic tools and develop them for commercial use requires high-throughput fabrication enabled by inkjet printing, Godwin added.
One such study for which Godwin and the KU microfluidics researchers are seeking funding involves evaluation of the clinical utility of the microfluidic tool and biomarkers to detect early-stage ovarian cancers. The study would require an analysis of about 800 plasma samples over three years and involve refining and validating current biomarkers.
In a training cohort for the STM breast cancer study, the microfluidic test had achieved 96.7 percent accuracy using 30 samples, and in an independent validation cohort, it achieved 92.9 percent accuracy using 70 samples.
For ovarian cancer screening, Godwin said, the group will need to push for higher levels of specificity and sensitivity and test out many biomarker candidates. However, if it is successful in the screening study, the group anticipates launching a laboratory-developed screening test to further explore the platform's clinical utility.
Godwin participated in a study that the microfluidic nanochip developers published in Nature last year, describing a microfluidic chip that used self-assembled three-dimensional herringbone nanopatterns to detect low levels of cancer-based exosomes in plasma. The device detected approximately 200 vesicles per 20 μl of a spiked sample, a level undetectable by standard microfluidic systems for biosensing, the researchers said.
The current prototype is more advanced because its fabrication method enables large-scale manufacturing.
In the future, the group will also look to engage in a collaboration with a company that has expertise in developing instrument platforms for its microfluidic cartridges. "We have a proof-of-concept assay but building a combined prototype cartridge and instrument is a long-term research project," Zeng said.
Being able to launch a diagnostic test based on its platform depends on a lot of things, including demonstrating high performance and clinical utility in clinical trials and obtaining funding to continue developing the platform. However, Zeng said he is encouraged that companies are showing how microfluidics can be integrated with instruments for diagnostic testing, and research to identify exosome-based disease biomarkers and develop tests "is taking off rapidly."
Castro, for example, is developing an exosome-based nanoplasmonic assay to fit into clinical workflows as a high-throughput detection tool for pancreatic ductal adenocarcinoma.
Meanwhile, Bio-Techne's Exosome Diagnostics sells a urine-based assay, the ExoDx Prostate IntelliScore (EPI) prostate cancer test, which tests for three exosomal RNA biomarkers, and the firm has been engaged in efforts to demonstrate clinical utility and drive health plan coverage.
Castro, whose team has licensed some of its technology to Exosome Diagnostics, noted that the overall field of liquid biopsy testing "leverages the various advantages that cancer-derived exosomes confer over other circulating biomarkers, such as circulating tumor cells or cell-free DNA. Exosomes display longer half-lives and are highly abundant with cargo loads directly from parental cells, thus offering true peripheral windows into living tumors."
The KU researchers could further improve upon their prototypes by augmenting their sample processing and multiplexing capabilities, Castro said. "Since [the researchers] invoke clinical implications, technology design should evolve to align with the high throughput and rapid turnaround times of clinical workflows in order to promote end-user adoption," he added.
Tony Jun Huang, a professor of mechanical engineering and materials science at Duke University and a developer of exosome-based microfluidic diagnostic tests, said that the work described in the STM study has broad potential. It could be used to "improve health monitoring and diagnosis of a number of human diseases, including cancers, neurodegenerative diseases, and cardiovascular disease," he said.