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from every living cell in the body. They have natural functions,
but also act as a preserving container for RNA,” says Kazinski.
Besides messenger RNA, microvesicles also contain microRNA
and proteins, providing information about both the transcriptional
and regulatory environments of their parent cells.
Regardless of the approach researchers are taking, Kazinski
reiterates the importance of careful sample handling: “What is
massively underestimated is the impact of the trivial question:
How do you take a blood draw, and how do you store it and how
do you ship it?” To address that, Qiagen has introduced a line of
specially treated blood collection tubes that chemically stabilize
samples for sensitive liquid biopsies.
Going with the fow
For investigators studying circulating tumor cells, the next step
after sampling is to isolate the desired cells as quickly and gently
as possible. Many tumors release cells with distinctive antigens on
their surfaces, and affinity-based techniques can pull these cells
out of the blood sample relatively easily. Indeed, that’s the basis
of the first FDA-approved liquid biopsy test, CellSearch, developed
by former Johnson & Johnson subsidiary Veridex (the test is now
owned by Menarini Silicon Biosystems in San Diego, California).
Unfortunately, CellSearch also became a cautionary tale for liquid biopsy developers. The FDA approved the test for determining
prognoses in breast, prostate, and colon cancers in 2008. When
subsequent studies found that CellSearch results had no influence
on clinical outcomes, though, insurers lost interest in paying for it.
While many researchers are still developing affinity-based
techniques for isolating circulating tumor cells, others are exploiting
the physical differences between normal and tumor cells. “[Picture]
when you walk near a river and there are some ... whirlpools in
the river that will trap leaves and branches; [we use] exactly the
same flow phenomenon, but at a much smaller scale,” says Elodie
Sollier-Christen, vice president of R&D at Vortex Biosciences in
Menlo Park, California.
Vortex’s system uses a microfluidic chip to generate tiny vortices
Spinning into the future
that trap larger, more deformable cancer cells from a blood plasma
sample. The chip itself is disposable, and the system is completely
automated. Users simply collect a blood sample from a patient,
Besides keeping the cells intact, the flow-based system also al-
lows investigators to isolate them for single-cell sequencing or other
analyses. That type of fine-grained control is also revealing that at
least some tumors may begin releasing cells sooner than scientists
had realized. “There was this initial thought that [circulating tumor
cells] are only present at late stage,” says Sollier-Christen, but she
notes that in the past year, several studies using more sensitive
techniques have found such cells much earlier in tumor develop-
ment, even before the tumor becomes visible by conventional imag-
The newest trend in liquid biopsy research, looking for circulating extracellular vesicles, grew partly from researchers’ frustrations with protein biomarkers. “We believe there are at least
10,000 proteins in our blood,” says W. Andy Tao, professor of
biochemistry at Purdue University in West Lafayette, Indiana. Tumors undoubtedly contribute distinctive proteins to this vast pool.
Unfortunately, the high background of normal proteins, along with
the presence of circulating phosphatases and other degradation
enzymes, consistently stump researchers searching for these rare
To address that, Tao and his colleagues focused on the subcellular vesicles released by the tumors. Because a majority of
tumors exhibit phosphorylation changes at some point in their
development, his lab looked specifically at protein phosphorylation
Many investigators use immunological markers to isolate extracellular vesicles from blood; Tao prefers the tedious but unbiased approach of differential centrifugation. His protocol uses progressively
higher centrifuge speeds to remove blood cells and debris, and then
isolate two different sizes of vesicles.
The next challenge is to identify the proteins in the isolated vesicles, and determine their phosphorylation states. Antibody-based
tests can also work for this purpose, but only after researchers
know what proteins and modifications they’re looking for. Scientists
doing more open-ended searches often use mass spectrometry,
generating lists of the extracellular vesicle–associated proteins in
samples from diseased and healthy patients or animals.
Although these surveys have recently revealed several promising
avenues for new liquid biopsy development, Tao warns that translating the results to clinical use will require some major changes. “In
the discovery stage, it’s perfectly fine using differential centrifugation followed by mass spectrometry, but when you go on to validation and verification [in] clinical samples, I think this is certainly not
practical.” Instead, he plans to narrow the list of useful biomarkers,
then develop antibody-based tests for clinical use.
Regardless of whether they’re looking at free DNA, intact cells,
or extracellular vesicles, researchers and physicians are optimistic
about the field’s prospects. Oxnard anticipates that “over the next
five years it will become routine, and just be something every [hos-pital] needs to offer to every cancer patient.”
Dana-Farber Cancer Institute
Harvard Medical School
Menarini Silicon Biosystems
Alan Dove is a science writer and editor based in Massachusetts.
Thermo Fisher Scientifc