When exosomes were first identified, about 35 years ago, they were thought to be simply vehicles for clearing cellular waste. But these nano-sized vesicles, made up of a lipid bilayer containing proteins, RNAs, and bioactive lipids, have turned out to be far more than that. Exosomes form a vast array of intracellular messengers that can communicate between cells of the same type and cells of other types, carrying signals of both health and disease. Much like in a cellular data network transmitting text messages, exosomes package up and send molecular “messages” from one cell to another.
Most of these messages are benign—indeed, important to normal function. But just as a simple text message can transmit information that’s used to commit a crime, exosomes can be hijacked by pathogens to transmit disease to other cells. For example, one type of exosome, known as tumor-derived exosomes, appears to facilitate tumor growth and metastasis, transporting nucleic acids and proteins from tumor cells to neighboring and distant cells.
Because they contain within their tiny (40–200 nanometers) spherical vesicles important molecular information, such as microRNAs and messenger RNAs, exosomes have also proved to be an exciting potential target for liquid biopsy. The field is younger than other advanced tools in the field, such as circulating tumor cells (CTCs) and circulating cell-free DNA (ccfDNA); at present, exploration of exosomes for liquid biopsy is almost entirely limited to the research space. No one is using exosomes to, for example, detect metastasis of liver cancer in a clinical setting. Yet.
But exosomes do appear to have several key advantages over tools like CTCs and ccfDNA that may move the field rapidly forward.
Advantages of exosomes
“To the extent that you want to interrogate what cells are using to communicate with each other—across all cell types, in disease and normal states—exosomes are an excellent way, perhaps the best way, to find out how cells are talking to each other,” says Jerry Williamson, CEO of NanoView Biosciences, which will release a novel technology platform, ExoView™, to identify and characterize exosomes by the end of the first quarter of 2019. “Every cell exhibits exosomes for normal cell communication, and diseased cells use exosomes to recruit other cells to grow and become diseased.”
Circulating tumor cells, on the other hand, are very hard to find in liquid biopsy. A tumor seeking to create a metastatic site will send out whole cells that adhere to a tissue in a distant part of the body. “But you need a lot of blood to identify just a single CTC,” Williamson observes. “There’s a similar problem with cell-free DNA, which derives from the process of cell death. As any cell dies—diseased or not—it dumps DNA into the bloodstream. Finding a needle circulating tumor DNA in a haystack of cell-free DNA from the normal cell death process involves a lot of unknowns, although a lot of good progress is being made in the field.”
And CTCs and ccfDNA both require fresh samples and must be processed within hours, says Constanze Kindler, Ph.D., associate director global product management at QIAGEN, who specializes in liquid biopsy technologies. QIAGEN offers solutions for all three types of liquid biopsy, including the exoEasy membrane affinity binding kit and exoRNeasy for serum or plasma samples. “With exosomes, you can use biobanked material, frozen or fresh samples, and you require only about 1 mL of blood to isolate them—although there may be cases that you also need up to 4 mL if the target is only in low amounts in your sample. By contrast, you’ll only find about 1–10 CTCs in 5 mL of blood.”
Not only are exosomes more abundant, they’re more directed, says Williamson. “In an exosome, you may be looking for nucleic acid, including microRNAs associated with exosomes for a particular cell type. They have biomarkers that you can use to characterize what’s going on with that cell. They’re much more specific than any other biomarker approach to looking at disease, provided, of course, that you know what cell you’re looking for.”
Enrichment of exosomes has often been done by ultracentrifugation, which can be time-consuming and difficult, says Milan Dieris, product manager for bioanalysis with MACHEREY-NAGEL, which offers its own exosome precipitation solutions. “Ultracentrifugation can take a whole day, and it requires a lot of equipment. Not everyone has an ultracentrifuge in their lab.”
And ultracentrifugation introduces its own bias in downstream analysis, Williamson adds. “You’re basically selecting for particles that are 40–50 nanometers, which means that if you didn’t do your preparation right, you’re not going to have specific results and you won’t get your analysis right. And to confirm that you’re finding your target of interest, you need to do additional analysis—with proteins, for example, you’ll have to run a western blot.”
The tools from QIAGEN, MACHEREY-NAGEL, and soon to come from NanoView all claim to conquer these challenges. “Our precipitation solution involves just one centrifugation step, which takes a total of 45 minutes to give you your exosomes in a pellet, which you then can use with a standard microRNA purification kit—ours or someone else’s,” says Dieris.
“Our affinity assay allows you to pull down out of solution only the exosomes you’re looking for,” says Williamson. “By the way they’re formed, exosomes have common biomarkers on the surface, and we use those to select for exosomes over other particulate matter like protein aggregation and cell debris that might be of that size. Because we can pull down and select only exosome molecules and nothing else, you can use the unpurified sample and don’t have to enrich it, so you’re not introducing bias.”
In addition to its off-the-shelf products, QIAGEN also offers a genomics core to conduct analysis for customers. “Especially when you’re dealing with precious samples or small quantities, customers sometimes prefer to send them to an expert,” says Langsdorff. “We can do sample prep, sequencing, and comprehensive data analysis.”
It’s still likely to take some time before exosomes become viable for liquid biopsy in the commercial diagnostics field. “I don’t think there’s yet been a lot of commercial activity on that front,” says Williamson. “But many in the clinical research community have begun to recognize their value. Hopefully, with expanding and improving new technology specific diagnostic opportunities will soon prove out.”
Hero image of genetic and molecular interactions courtesy of Keiichiro Ono, University of California, San Diego/ NIH image gallery.