The furor over personalized medicine and the push for minimally invasive patient sampling procedures have made liquid biopsies the darlings of translational research. Hidden in blood, sputum, urine and other easily accessible matrices is a wealth of information about whether someone has had a heart attack or will develop Alzheimer’s disease, or if a particular therapy regimen will be effective against his or her particular cancer.
To get at such evidence, researchers turn to a variety of sources, such as cell-free DNA (cfDNA) and its subset, circulating tumor DNA (ctDNA); metabolites; microbes; extracellular vesicles; and cells themselves. Various techniques, using assorted instrumentation, are being harnessed to do so. Here we look at some of what can be gleaned by applying flow cytometry to the liquid biopsy.
Why flow?
Flow cytometry is a powerful, high-throughput technique to analyze (and sometimes sort) particles based on their fluorescent and light-scattering properties. By using a panel of fluorescently tagged antibodies, different surface and interior markers (typically proteins) can be labeled; the particle’s size is read out as forward scatter, and side scatter roughly indicates granularity.
For example, cells from the buffy coat of whole blood can be stained with antibodies against the epithelial cell adhesion molecule (EpCAM), cytokeratins (CK) and the CD45 molecule. Upon running these through a flow cytometer (and selecting those with the appropriate size and granularity), those rare cells that express CK and high levels of EpCAM, but not CD45 (which is a marker for leukocytes), can be quantified. This is the basis for CELLSEARCH, an FDA-cleared system for identifying circulating tumor cells (CTCs) from a simple blood test.
Flow cytometry, though, is “only one of several ways to pull the needle out of the haystack,” notes Julie Lang, director of the Breast Cancer Program at the Keck School of Medicine of the University of Southern California (USC). During the last few years, quite a number of microfluidic filtration products that separate CTCs on the basis of cell-surface markers (such as EpCAM) have launched; other products base the separation on size, deformability and other physical properties. For logistical reasons—such as scheduling issues and the preparation time necessary for sorting on a flow cytometer—Lang’s team now uses the Angle Parsortix system to retrieve CTCs for downstream analysis. “RNA degrades rapidly, so if the specimen is waiting for more than a couple of hours, it’s no longer useful for cancer research; there is no blood preservation tube that can simultaneously preserve the cell-surface markers as well as the RNA,” Lang explains.
Yet the multidimensionality of flow cytometry enables unsurpassed specificity, notes Jonni Moore, professor of pathology and laboratory medicine at the Hospital of the University of Pennsylvania. There are tumor cells that may have downregulated EpCAM and thus do not meet the CELLSEARCH criteria, and many of these would also fall through the cracks of physical separation methods. And “sometimes the markers (EpCAM being one) are broadly expressed” in nontumor cells, Moore says; the physical sorting criteria are not 100% accurate either, and so would lead to false-positive results. “One of the reasons people are going back to flow cytometry is the ability to look at more markers,” Moore says, for example, to assure that a putative breast cancer cell is from the breast.
The multidimensionality of flow cytometry enables unsurpassed specificity.
CTCs are shed from primary and metastatic tumors and are thought to be the direct precursors of metastatic disease. Characterizing these cells by multidimensional flow cytometry enables researchers to discern the evolution of the cancer, perhaps yielding treatment insights that would differ if only the primary tumor biopsy were to be examined. Similarly, insights into efficacy of therapy can be gained by looking at CTCs longitudinally.
Of course, such studies needn’t be either/or analyses. The ideal combo is both the specificity of flow cytometry with the sensitivity of genomics, at least for now, remarks Moore.
Instrumentation
Many researchers have taken to enriching their samples for EpCAM+ cells, or depleting them of CD45+ cells, prior to running them through a flow cytometer. This can be done using a column or with magnetic beads. BD Biosciences is developing the FACSFocus instrument with inline immunomagnetic separation and acoustic washing. “So there’s no centrifugation … and that means we don’t lose cells,” Moore says.
Moore and her colleagues have used the instrument to detect and characterize clusters of circulating pancreatic cancer cells, which are indicative of metastatic potential. “You can’t do that with current technology other than flow. It’s not too amenable to chip-based tech at the moment, but I wouldn’t say that it won’t be,” she says.
Flow Cytometer Search Tool
Find, compare and review flow
cytometers from different suppliers Search
There is rapid development in flow cytometry instrumentation. “More and more, in the field they’re getting small-volume cytometers, chip-based cytometers, that allow us to query small samples, whether they come from the blood or the tissue itself, in conjunction with biopsy.”
Some new flow cytometers, such as Apogee Flow Systems’ Micro Flow Cytometer, focus on small particles, says Karen Gylys, a neuroscientist and professor at the University of California, Los Angeles (UCLA), School of Nursing. “That, at least theoretically, gets down into the exosome range, which is about 100 nm.” Other vendors are building instruments which, although not dedicated, are able to discriminate small particles, as well.
Extracellular vesicles
Circulating extracellular vesicles (ECVs) are small, membrane-bound particles believed to have separated from cells. They may contain microRNA, messenger RNA, DNA and other nucleic acids and proteins (including cytokines), and their membranes are enriched in certain surface markers, such as CD9, CD63 and CD81. ECVs are alternatively thought to represent cellular debris/waste products or a means of intracellular communication and other “functions.” Distinct terminology may refer to particular subsets, but for most practical purposes the terms “ECVs,” “EVs,” “microparticles,” “macrovesicles” and “exosomes” are used interchangeably.
ECVs have been investigated as biomarkers for cancer, brain injury, cardiovascular disease and a variety of other disorders. Flow cytometry has been used in only a handful of these studies to date—often as proof of principle or as confirmatory assays.
One of the difficulties in using conventional flow cytometry to look at ECVs is that sample preparation has traditionally been very tedious, typically involving ultracentrifugation. In addition, because of the particles’ diminutive size, the flow rate needs to be carefully controlled to allow for individuation.
But with newer instrumentation, reagents, protocols and informatics tools coming online, that should change. Several vendors now offer intact-exosome isolation kits. And Miltenyi Biotec recently introduced its MACSPlex Exosome Kit, which captures exosomes on beads using pan-exosome markers, allowing the characterization of 37 other exosome surface markers in a single experiment by running the beads in a flow cytometer, explains Johannes Fleischer, global product manager for flow cytometry reagents.
Varghese John, associate professor of neurology at the David Geffen School of Medicine at UCLA, hopes to use a modified pulse-laser flow cytometer to isolate pure neuronal exosomes and in turn “either diagnose Alzheimer’s disease, or come up with new [drug] targets, or predict therapeutic efficacy of treatment.”
And notwithstanding the challenges in analyzing ECV signatures—Moore says that “right now, you need biocomputational expertise as much as you do with genomics”—ECVs “may really be the gold mine here. That’s your source. That’s your liquid biopsy.” Within two years, Moore predicts, “people will be looking at ECVs more than cells for biomarker research and the like.” Whether or not this holds true, there is an exciting time ahead for flow cytometry-based liquid biopsy research.
Image: Shutterstock Images