Exosomes—a subset of extracellular vesicles (EVs)—carry a collection of information that can be analyzed with flow cytometry (FC). In most cases, the analysis explores the outside of exosomes, but some methods look inside as well. Exosomes come in many types, such as nuclear-derived ones, which can contain DNA, and the source of an exosome determines how it can be used by researchers.
“Flow cytometry is a powerful technology enabling the analysis of several markers in parallel and ideally on a single-vesicle level,” says Stefan Wild—group leader, research and development at Miltenyi Biotec. “Especially, the information on different proteins being present on the same vesicle can be extremely useful to identify extracellular vesicle subpopulations and potential functions.”
EVs comes from many sources. “In complex biological fluids like plasma, EVs originating from a broad variety of cell types are present,” Wild says. “As EVs comprise parts of the cellular membrane, known cell-surface markers can be used to assign EV subpopulations to the parent cell population.”
The information gleaned from analyzing these structures can be used in many ways, including basic research and clinical applications. “Changes in EV composition are expected to enable insight into physiological changes that might not be easily addressed on a cell or tissue level,” Wild explains. “Such liquid biopsies could facilitate indications on pathological changes, like autoimmune diseases or cancer, even in routine screenings.”
FC-based obstacles
The method behind FC—using scattered light to characterize cells by size or density—creates some challenges with exosomes. “Small EVs hardly scatter light, and therefore only very limited information on the size of the vesicles can be gained by flow cytometry,” Wild explains. “As a consequence, potential artefacts like doublets, aggregates, or swarm effects need to be carefully considered.”
Scientists who use FC to analyze exosomes point out other challenges. As Irfan Rahman—a biochemist at the University of Rochester Medical Center in New York—and colleagues—research assistant professor Isaac Sundar and postdoctoral fellow Krishna Maremanda—noted: “We can use flow-based isolation and characterization of exosomes, subject to the availabilities of very specific antibodies with specific cell-type of origin.” Still, these scientists point out three key challenges:
- lack of image-stream methods for flow-based exosomes
- lack of specific antibodies
- lack of cell-based specific exosome assays
- lack of gating based on sizes.
Consequently, scientists often develop custom techniques. This is particularly required with applications that necessitate extremely sensitive detection and analysis.
Figuring out the flow
Making FC solve a specific exosome-based challenge requires various steps. For example, Anil Sood, a gynecologic oncologist at The University of Texas MD Anderson Cancer Center, wanted to analyze nuclear-derived exosomes. “The challenges were how to look at single exosomes accurately and how to detect specific exosomes, such as DNA-positive or specific protein-positive exosomes.” That raised some tricky obstacles.
First, it can be challenging to even find these exosomes. “It was known that nuclear-derived exosomes should be a small population, but we wanted to focus on this subpopulation,” says Sood. But that’s not the only challenge. “Exosomes are tiny vesicles and can be difficult to visualize,” Sood adds. “For this reason, the single-exosome analysis has been considered to be very challenging.” Still, Sood and his colleagues developed an effective FC-based approach. He says, “We tested rigorously in many ways and many times and concluded that our flow method enables us to see single exosomes.”
Sood and his colleagues believe that their set-up can be very useful. “We can say the flow-based method is a powerful tool to analyze the diversity of exosomes, because the results by flow can estimate the percentage of a subpopulation,” Sood explains. “This data can provide us novel, fundamental insights into exosomes.”
Developing new products
Beyond scientists developing in-house methods, vendors offer many options.
From Miltenyi Biotec, for example, Wild says, “Our contribution has been the development of the MACSPlex Exosome Assay using bead-bound antibodies to assist surface-marker screening of EVs by flow cytometry.” In this assay, he explains, “EVs are bound to differently colored beads according to the respective surface markers present on the vesicles.” Then, the bound vesicles can be stained by dye-conjugated antibodies and analyzed with FC. “Without single-vesicle resolution, the assay facilitates a broad overview of 37 surface markers on EVs focusing on epitopes related to immune cells and cancer,” Wild notes. “Due to the antibody pairs used for capture and detection, additional information on two different markers being present on the same vesicles can be gained.”
Scientists can consider other surface-related products, as well. For example, Abcam’s Exosome Isolation and Analysis Kits can be used to analyze external proteins on exosomes. “This can be used to better understand the purity of exosomes or the sources of exosomes,” says Michael Tackett, Abcam’s director of assay development.
Image: Diagram illustrates FirePlex immunoassay workflow, from biofluid input through to multiplex data analysis. Image courtesy of Abcam.
The inside angle
Besides looking at the surface of exosomes, scientists can learn a lot from what’s inside these structures. In particular, scientists might want to analyze the microRNA—miRNA—or other small molecules inside of exosomes. Doing that requires an extremely sensitive method. “Often, you get a very small number of exosomes, and the amount of micro-RNA is really tiny—dozens or hundreds of micro-RNAs,” Tackett says.
By looking inside exosomes, scientists can learn even more. “We want to understand the contents of exosomes, because an exosome is carrying that content from one cell type to another,” Tackett explains. “If you isolate exosomes from a specific population and analyze the micro-RNA, you can get a really high signal-to-noise ratio.” He adds, “It’s a powerful way to look at biomarkers.” Tackett also says that Abcam can help scientists develop methods to explore the contents of specific exosome-based samples.
In fact, FC is a powerful way to explore exosomes inside and out. The findings can be used to expand the basic understanding of cellular biology, such as how cells communicate. Also, the markers inside and outside of exosomes can be used to diagnose disease or assess the efficacy of a treatment. Given the small size of exosomes, sophisticated technology is essential to reveal even more about these structures, what they carry, and what they do.