The Application of MicroRNAs in Cancer Diagnostics - RNA Interference from Biology to Therapeutics - page 270

as an alternative to profiling of solid tumors. At present, detection of cancer relapse
is often carried out by invasive testing (e.g., frequent cystoscopies for bladder can-
cer). Ideally, such procedures could be replaced by noninvasive testing based on
monitoring of circulatory miRNAs in biological fluids. miRNAs have been detected
in all body fluids examined so far, including urine, plasma, serum, semen, saliva,
and cerebrospinal fluid. The miRNAs are present in nanovesicles such as exosomes,
as well as in association with free protein complexes. The possible biological role
of circulating miRNAs is presently the focus of numerous studies. According to
available data, circulating miRNAs appear to possess a regulatory function beyond
cells from which they originate and in recipient cells that take up the miRNA com-
plexes. Exosome-mediated transfer of miRNAs has been demonstrated in cell cul-
ture models. Furthermore, in vivo models suggest that cancer cell-derived exosomes
may modulate the microenvironment at distant sites into pre-metastatic niches and,
thereby, promote metastasis through yet unknown mechanisms [ 152 ] . Whether
present in exosomes or in free protein complexes, the detection of these miRNAs
serve as a potential diagnostic tool for cancer evaluation.
Circulating tumor-specific miRNAs were first discovered in 2008, where
increased levels of miR-210, miR-155, and miR-21 were detected in serum samples
from patients with diffuse B-cell lymphoma [ 153, 154 ] . The same year, Mitchell
and coworkers reported the detection of miRNAs in plasma samples. Plasma-derived
miRNAs showed remarkable stability and were stable upon repeated freeze/thaw-
ing, exposure to low/high pH, and extended storage. They were also resistant toward
endogenous RNase degradation, in contrast to synthetic spike-in miRNAs [ 155,
156 ]. Highly expressed miRNAs, such as miR-16, miR-15b, and miR-24, were
detected at similar levels in both plasma and serum. Furthermore, human tumor
cell-derived miRNAs, with no murine orthologs, could be detected in the blood-
stream of xenografted mice, and increased serum levels of miR-141 were seen in
patients with prostate cancer compared to healthy controls [ 155 ] . A study by Chen
et al . described 63 miRNAs in serum from lung cancer patients that were not
detected in healthy individuals [ 156 ]. Remarkably, a comparative analysis revealed
highly similar miRNA profiles in blood cells and serum in normal individuals (90%
identical miRNAs in the two fractions), but not in lung cancer patients (~50% of
detectable miRNAs were only present in serum). Accordingly, disease-specific
miRNAs present in serum seem to derive directly from cancer cells rather than
reflecting an altered miRNA profile in blood cells. This reiterates the potential of
circulating miRNA profiles for cancer diagnosis.

13.4.2 Circulating miRNAs Are Highly Stable and Present

in Protein Complexes and Vesicles

Early work suggested that circulating miRNAs were not cell-associated [ 155 ] . It is
now known that miRNAs are encapsulated in small vesicles, such as exosomes,
microvesicles, and microparticles, as well as in free association with proteins such
as Argonaute 2 (AGO2), Nucleophosmin 1 (NPM1), and high-density lipoprotein