After introducing a urine sample onto the device, extracellular vesicles are captured by a nanowire substrate via electrostatic forces. MicroRNAs can then be directly extracted from the substrate. [Takao Yasui]
Researchers in Japan, Thailand and China have developed a microfluidic device containing embedded nanowires that can capture minute quantities of microRNA (miRNA)-carrying extracellular vesicles (EVs) in urine, as a potential method for noninvasive cancer diagnosis or screening. In tests on human donor urine samples, the device allowed scientists to identify cancer-specific urinary EV-encapsulated miRNA expression patterns that could represent biomarkers for urologic and for non-urologic cancers.
“EVs are potentially useful as clinical markers,” explains Nagoya University’s Takao Yasui, who is lead author of the researchers’ published paper in Science Advances. “The ongoing challenge for physicians in any field is to find a noninvasive diagnostic tool that allows them to monitor their patients on a regular basis—for example, a simple urine test … The composition of the molecules contained in an EV may provide a diagnostic signature for certain diseases.” The published paper is entitled, “Unveiling Massive Numbers of Cancer-Related Urinary-microRNA Candidates via Nanowires.”
Circulating extracellular vesicles containing messenger molecules represent an important system for cell-cell communication. EVs that contain miRNAs are found in a variety of body fluids, including urine, and differences in EV-encapsulated miRNAs between healthy individuals and those with cancer could potentially represent “a warning sign for various disease scenarios,” the authors write. Unfortunately traditional methods for collecting EVs aren’t suitable for routine screening or diagnosis, primarily because urine contains only tiny numbers of these EVS. “The content of EVs in urine is extremely low, at less than 0.01% of the total fluid volume,” Yasui notes. “This is a major barrier to their diagnostic utility.”
Dr. Yasui’s team has now developed a microfluidic device comprising zinc oxide nanowires embedded in a specialized polymer, which use electrostatic forces to collect miRNA-containing EVs from as little as 1 ml of urine. In initial tests, the device collected EVs encapsulating about 1,000 different types of miRNA sequence, far higher than the 200–300 different miRNAs that have been extracted from urine using conventional ultracentrifugation methods. “Surprisingly, the device could extract a much larger variety of species of miRNAs than ultracentrifugation despite the fact that the device uses a smaller sample volume and shorter treatment time than the latter method,” the authors state. The combined processes of nanowire capture of EVs and miRNA extraction also takes just 40 minutes.
The researchers next used their microfluidic nanowire device to compare the microRNAs of EVs isolated from healthy patients, and those from patients with bladder and prostate cancers—which are urologic malignancies—but also from patients with non-urologic cancers including lung, pancreas and liver. The results showed very different expression levels of miRNAs between the cancer and noncancer donor urine samples, which suggests that it may be possible to identify specific combinations of down-regulated and overexpressed miRNAs as cancer biomarkers. This is “contrary to the sterotype thinking of depending on a single miRNA biomarker,” they point out.
“Finding a specific, reproducible marker to help confirm a cancer diagnosis is difficult,” notes coauthor Yoshinobu Baba. “This is especially true for microRNAs, which are a relatively new class of markers in the field. Sometimes finding just one reliable microRNA is considered a success. Using this approach, we were surprised to find that not just one, but whole combinations of microRNAs might be associated with different types of cancers. The findings are preliminary, of course, but we hope our device can help to lay the groundwork for easier ways to diagnose life-threatening diseases as early as possible.”
The authors acknowledge that further trials will be needed to test the technology for biomarker-based diagnostic or screening applications. Nevertheless, they write, “the present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer.”