By Deborah Borfitz
August 20, 2019 | Carbon nanotubes—super-strong cylindrical molecules with curious electronic properties—have found practical application in everything from aircraft parts to tennis rackets and baseball bats since their discovery in 1991. Now, researchers at Worcester Polytechnic Institute (WPI) have used the wonder material to create a liquid biopsy chip that can snare circulating tumor cells (CTCs) of all sizes and types in a stationary drop of blood, and reliably detect more of them than existing microfluidic technologies.
Members of the research team hail from the department of neurological surgery at the University of Massachusetts Medical School and the James Graham Brown Cancer Center at the University of Louisville School of Medicine, in addition to WPI.
As recently reported in the journal Lab on a Chip, the carbon nanotube-based device successfully captured between 89% and 100% of CTCs in a test run on blood spiked with cancer cells. It picked up 100% of CTCs in samples from actual breast cancer patients at every disease stage.
The early-stage catches are particularly noteworthy, since they have been associated with dramatic increases in progression-free survival and overall survival in cancer patients, says project lead Balaji Panchapakesan, professor of mechanical engineering and director of the Small Systems Laboratory at WPI.
The chip’s design takes advantage of the natural tendency of CTCs to attach—and to do so preferentially—to carbon nanotubes while white blood cells typically will not, which makes this a “game-changing technology,” says Panchapakesan. Red blood cells get removed via a process called lysing, which has no effect on CTCs, before a blood sample even gets added to the chip.
The new method combines biology and mechanical engineering to overcome many of the shortcomings of microfluidic devices for liquid biopsy, says Panchapakesan, including low sensitivity. “For some existing devices, the capture rate depends on the velocity of the cells flowing by; the faster the flow, the lower the capture rate. And existing microfluidic technology often requires diluting blood samples to prevent clogging inside the fluidic channels.” Also, due to the three-dimensional nature of the devices, imaging produces large files that need to be sorted manually, he adds.
The WPI technology, in contrast, has a “surface architecture” that simplifies the process of capturing, imaging and retrieving CTCs, Panchapakesan says.
The new liquid biopsy chip isolates circulating cells rather than trying to slow them down, he says. It’s a significant challenge, given that a single cancer cell may be circulating in a sea of a billion healthy blood cells and CTCs are only intermittently shed by cancerous tumors. Currently-marketed devices with any capability in this arena tend to get clogged with harmless red blood cells.
Individual cancer cells as well as clusters of CTCs get seized by the carbon nanotube chip, notes Panchapakesan. CTC clusters are comparatively rare but appear to play the greater role in seeding new tumors.
Contamination of samples by white blood cells is another issue for many liquid biopsy devices, making single-cell genomic sequencing problematic, Panchapakesan says. Captured cells often must be removed for laboratory analysis, sometimes by breaking the chip. For devices using narrow microfluidic channels cell removal can be especially tricky, and some of the smallest CTCs never make it through the transfer process. Manufacturing costs can also be steep.
Multiple Applications
The original idea a few years back was to look at the electrical signatures of cancer cells and classify them using advanced data mining, says Panchapakesan. During those early experiments, CTCs were found to have a greater affinity for carbon nanotubes than glass, enabling the capture of single cells.
Researchers pivoted to the creation of the new device featuring a layer of carbon nanotubes lining the bottom of multiple small test wells on a glass-and-silicon wafer, he continues. Only a small volume of lysed blood is placed in each well, making it possible to more accurately count the attached CTCs.
Like specks of sand in a fast-moving river, most CTCs in the bloodstream will be washed away without a rock-like matrix to quickly and easily latch onto and begin their mechanical work, Panchapakesan says. Softer materials, including collagen and other polymeric scaffolds, require them to expend more energy on attachment that they could instead be using for other processes such as survival.
The device uses a “passive leukocyte depletion strategy,” based on the density of cancer cells, to selectively send them to the bottom of the well where they encounter antibodies, he adds. The antibodies bind to the cancer cells, triggering a change in electrical conductance on the chip.
While the technique was used experimentally on triple-negative breast cancer cell line MDA-MB-231, it is not limited to specific cancer types, Panchapakesan explains. The “preferential adherence” methodology could theoretically also be used to capture colon, lung and prostate cancer cells. The device utilizes “antigen independent capture” to make the carbon nanotube surface capable of snagging cells of all different phenotypes, which is crucial for knowing the heterogeneity of captured cells.
Cells captured by carbon nanotube chips remain viable for culturing, Panchapakesan says, or researchers can stain and study the apprehended cells without removing them because the chips are transparent. “You can do microscopy from the bottom and from the top to capture all the cells, and because everything’s on a 3-millimeter chip it goes much faster.”
It takes about 20 minutes to put a blood sample on the liquid biopsy chip, plus a couple of hours to culture the captured CTCs, says Panchapakesan. Results are currently possible in 24-48 hours, due to the fragility of the CTCs, but researchers are still working on condensing the diagnostic process.
In the future, he predicts, diagnosticians will be able to use liquid biopsies to identify cancer earlier at both primary and metastatic sites. Tumor cells may “shed some DNA.” In early-stage breast cancer, malignant cells may only be present in the blood. Lesions aren’t detectable by CT or MRI imaging until they’re about 1 to 2 mm in size.
The device’s unique design might also make it useful in tracking the progression of cancers and patients’ response to radiation or chemotherapy, Panchapakesan says. Likewise, physicians might one day use the chip to make predictions about the likely course of a cancer to aid them in the selection of the most effective therapy.
A diagnostics startup company has licensed the novel chip technology and is now trying to secure funding to develop a point-of-care test, says Panchapakesan. A grant from the National Institutes of Health may be a prerequisite to bringing the test into routine clinical practice. “We want to start clinical trials in the U.S. and then with our research partners in other parts of the world, including India and China.”
Patients in a “diagnostic conundrum” are already inquiring about the carbon nanotube-based device, he says. “We can only hope the early enthusiasm is a signal that clinical trial recruitment wouldn’t be a holdup.”