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number of clones sequenced or, alternatively, by comparative CT methods involving
2
−D D CT
calculation. When profiling circulating miRNAs from minimal sample mate-
rial, qPCR-based detection platforms have shown superiority in sensitivity com-
pared to microarray platforms [
193
] .
13.4.6
Increased Secretion of Exosomes in Cancer
First discovered during reticulocyte maturation decades ago [
194
] , exosome vesi-
cles are now widely recognized as being secreted also by all other cell types [
195
]
and to be more actively secreted from malignant cells. For example, medullablas-
toma cell lines were found to secrete 13,400-25,300 vesicles per cell in 48 h, com-
pared to 3,800-6,200 vesicles secreted from normal fibroblasts [
185
] . Interestingly,
the level of RNA in the vesicles was 210- to 310-fold higher in the medullablastoma
cell-derived vesicles compared to those from normal fibroblasts. Increased endo-
somal release and uptake by cancer cells were exhibited at low pH indicative of the
tumor microenvironment in metastatic melanoma cells [
154
]. In a xenograft mouse
model of melanoma, the level of exosomes detected in plasma correlated with the
tumor burden [
196
]. Accordingly, an increase in exosome concentration has been
observed in the bloodstream of cancer patients with lung cancer or melanoma [
196,
197
]. Specific cancer-associated markers detected on a subset of the vesicles dem-
onstrated that these vesicles were indeed tumor derived, as opposed to the result of
a general host response. Several studies have now shown that exosome constituents
are altered in plasma- and urine-derived exosomes in a variety of cancers that pro-
motes the possibility of using these constituents in noninvasive tests [
155,
166,
196-
198
] .
Epithelial cell adhesion molecule (EpCAM)-positive vesicles were found in the
serum of ovarian cancer patients but absent in healthy individuals and increased
with the stage of the disease. Furthermore, although EpCAM-positive vesicles, iso-
lated using an immune-affinity capture technique, were present in both benign and
malignant disease, the miRNA profiles from these vesicles were distinct for patients
with benign ovarian disease and patients with ovarian cancer [
166
] . Of note, prote-
olytic cleavage by metalloproteinases of EpCAM on exosomes has been observed
in a subset of serum samples from breast cancer patients [
199
] . This emphasizes
that tumor-derived exosomes are heterogeneous in composition and that isolation of
cancer-derived exosomes using an EpCAM-bead subfractioning method should be
considered carefully.
13.4.7
Circulating miRNAs as Diagnostic Cancer Biomarkers
Circulating miRNAs have now been associated with several cancer types (Table
13.3
).
As indicated in the table, three of the listed studies have identified exosome-derived
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