REVIEW

REVIEW

TOPIC

Neuro-oncologic Applications of Exosomes,

Microvesicles, and Other Nano-Sized
Extracellular Particles
David D. Gonda, MD
Johnny C. Akers, PhD
Ryan Kim, BS
Steven N. Kalkanis, MD
Fred H. Hochberg, MD
Clark C. Chen, MD, PhD*
Bob S. Carter, MD, PhD*
Department of Neurosurgery, University of California, San Diego, California;
Center for Theoretic and Applied
Neuro-oncology, University of California,
San Diego, California; Department of
Neurosurgery, Henry Ford Health System,
Detroit, Michigan; Pappas Center, Massachusetts General Hospital, Boston,
Massachusetts
*These authors have contributed equally
to this article as senior authors.
Correspondence:
Clark C. Chen, MD, PhD,
3855 Health Science Dr, No. 0987,
La Jolla, CA 92093-0987.
E-mail: clarkchen@ucsd.edu
Received, September 3, 2012.
Accepted, December 11, 2012.
Published Online, December 28, 2012.
Copyright 2012 by the
Congress of Neurological Surgeons

The discovery that tumor-derived proteins and nucleic acids can be detected in nanosized vesicles in the plasma and cerebrospinal fluid of patients afflicted with brain
tumors has expanded opportunities for biomarker and therapeutic discovery. Through
delivery of their contents to surrounding cells, exosomes, microvesicles, and other
nano-sized extracellular vesicles secreted by tumors modulate their environment to
promote tumor growth and survival. In this review, we discuss the biological processes
mediated by these extracellular vesicles and their applications in terms of brain tumor
diagnosis, monitoring, and therapy. We review the normal physiology of these extracellular vesicles, their pertinence to tumor biology, and directions for research in this
field.
KEY WORDS: Biomarkers, Extracellular vesicles, Exosomes, Microvesicles
Neurosurgery 72:501510, 2013

DOI: 10.1227/NEU.0b013e3182846e63

he discovery that tumor-derived proteins

and nucleic acids can be detected in nanosized extracellular vesicles (EVs) in the
plasma and cerebrospinal fluid of patients with
brain tumors has opened a new field of biomarker
discovery and therapeutics.1-4 In this review, we
discuss the biological processes mediated by
these EVs and their applications as biomarkers
for brain tumor diagnosis, monitoring, and
therapeutic development.

DEFINITIONS AND GENERAL

BIOLOGY OF EVs
Terminology Related to EVs
In this review, we use the term EVs as a general
descriptive term referring to membrane-encapsulated cellular components that are released outside
the cell (Figure 1). EVs isolated from the various
biofluids have been called exosomes, exosomelike

ABBREVIATIONS: AMPA, a-amino-3-hydroxy-5methyl-4-isoxazole-propionic acid; EGFR, epidermal growth factor receptor; EGFRvIII, epidermal
growth factor receptor variant III; EV, extracellular
vesicle; GPCR, G-protein coupled receptor; miRNA,
micro-RNA; MMP, matrix-degrading metalloproteinase; RVG, rabies viral glycoprotein

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vesicles, microvesicles, shedding microvesicles,

epididimosomes, argosomes, promininosomes,
prostasomes, dexosomes, texosomes, archaeosomes, and oncosomes. Much of this nomenclature is based on the specific mechanism of
vesicular biogenesis or the source biofluid from
which the vesicles were prepared.5 For instance,
exosomes are vesicles formed in the endosomal
compartment and are ultimately secreted into the
extracellular space. This mechanism is one of the
many cellular processes that generate EVs.6-8
Thus, the EVs isolated from any given biofluid
(serum, cerebrospinal fluid, etc) likely include
exosomes and other vesicles that are not generated by the exosomal biogenesis pathway, including apoptotic bodies6,7 or retroviral particles.8 To
the extent that there is no generally agreed-on
physical method for distinguishing 1 type of EV
from another, we use the general term EV when
describing vesicles with mechanisms of biogenesis
that remain unknown. We reserve terms such as
exosomes, microvesicles, apoptotic bodies, and
retroviral particles for vesicles with biogenic
mechanisms that are clearly defined.
Isolation of EVs
EVs have been isolated from all bodily fluids,
including blood, urine, cerebrospinal fluid, lymphatics, tears, saliva and nasal secretions, ascites,

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GONDA ET AL

FIGURE 1. Scanning electron microscopic images of a primary glioblastoma cell at lower (A) and higher (B) magnifications show
extracellular vesicles on the cell surface. Reprinted from Skog et al2 with permission from the publisher. Copyright  2008, Nature
Publishing Group.

and semen. There is no consensus on the best method for isolation.

Isolation methods include differential centrifugation to remove
larger cellular debris followed by ultracentrifugation at 100 000g
to pellet the nano-sized vesicles or suspension in sucrose
gradients.9 Other isolation methods include the use of serial
filters (exoMIR), immune isolation with magnetic beads, or
microfluidic separations. The isolated particles are too small to be
seen by light microscopy and therefore are typically visualized by
electron microscopy (Figure 2). Proprietary commercial technologies (NanoSight; IZON) are also available to quantify and
provide measures of EV size and zeta potential surface charge.10
General Description of Biological Functions of EVs
The secretion of EVs subserves 3 general biological functions.
First, it affords a mechanism for remodeling or removal of cellular

components. Toxic metabolites or unneeded membrane components can be packaged into EVs and removed from the cell.11
Second, EVs serve as a platform for intercellular communication.
Information can be conveyed to the recipient cell either by direct
uptake of the EV or by ligand-receptor interactions.12-16 Finally,
EVs have been shown to modulate the microenvironment to
facilitate cell migration, angiogenesis, or immune regulation.
Examples of each of these biological functions are reviewed
below.
Remodeling and Removal of Cellular Components
An illustrative example of the importance of EVs in normal
physiological processes involves the maturation from spherical
reticulocyte to a biconcave erythrocyte. During this process,
approximately one-third of the plasma membrane is lost by

FIGURE 2. Scanning electron microscopic images of exosomes isolated by serial centrifugation of cerebrospinal fluid at lower (A)
and higher (B) magnifications. Higher magnification shows the typical cupped disk shape of the exosome.

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EXTRACELLULAR VESICLES IN NEURO-ONCOLOGY

secretion of plasma membrane, transferrin receptors, and acetylcholine receptors in the form of EVs.5,17,18 This process is
essentially the direct reversal of endocytosis. In fact, the term
exosome was coined to describe the EVs formed by reversed
endocytosis.19 Other examples of cell surface remodeling by EV
secretion have been reported since this seminal finding. For
example, expression of major histocompatibility complex class II
molecules on the surface of dendritic cells and a-amino3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor on the surface of cortical neurons is closely regulated by EV
secretion.20,21
Secretion of EVs also affords an opportunity to remove
metabolites that are otherwise toxic to the cell.22 For instance,
human ovarian cancer lines can attain resistance to cisplatin by
concentrating this chemotherapeutic agent in EVs and exporting
them.23 EVs thus constitute a selective export mechanism for
both soluble factors and membrane constituents.
Intercellular Communication
There is an increasing awareness that EVs serve as a platform by
which proteins and genetic materials can be transferred from one
cell to another. This transfer is achieved either by direct uptake of
the EV or by ligand-receptor interactions.12-16 In the former case,
genetic materials such as mRNA may be released into the
cytoplasm of the recipient cell and translated into protein.2 In the
latter case, signal transduction in the recipient cell may be
initiated by ligand/receptor interactions or transfer of protein
between the EV and the recipient cell surface1,24 (Figure 3).
Examples of such communication are reviewed below.
Within the central nervous system, EVs serve to mediate
reciprocal communication by the delivery of specific proteins,
mRNAs, and other contents. For instance, EVs released from
microglia are taken up by neurons. This uptake enhances
sphingolipid metabolism of the neuron, increasing the probability
that glutamate will be released at the neuronal terminal.25 As
another example, EVs derived from oligodendrocytes are taken
up by neurons. These EVs deliver proteins and mRNA that exert
trophic effects on recipient neurons and serve to maintain
specialized neuronal structures.26
Smalheiser27 proposed EV transfer of proteins and RNAs
between neurons as an important contributor to the development
of synaptic plasticity. Agnati et al28 later called this process of
interneuronal communication roamer type of volume transmission because EVs are like roaming extracellular vehicles
containing important materials. Membranous structures analogous to those that produce exosomes exist at the presynaptic and
postsynaptic terminals of neurons.29a EVs from neuronal cells are
secreted in response to calcium influx and from glutamatergic
activity.29b Their contents include AMPA21 and G-protein
coupled receptors (GPRCs), which are important for long-term
potentiation.30 The transfer of EV proteins and RNAs between
cells may serve to complement classic synaptic communication.
Within the coagulation system, EVs mediate intercellular
communication by direct transfer of membrane proteins and

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FIGURE 3. Mechanisms of extracellular vesicle (EV) intercellular communication. EVs interact with target cells through a number of mechanisms, including
direct ligand-receptor binding; fusion with the target cell, thus delivering the
vesicular nucleic acid and protein contents into the cell; and/or the direct
transference of extracellular vesicle membrane proteins and receptors to the cell
surface.

initiation of protein-ligand interactions.31 For instance, platelets

secrete EVs containing the platelet-endothelium attachment
receptors (CD41, CD61, and CD62), cytokine receptors
(TNF-RI, TNR-RII, and CD05), and ligands such as CD40L
and PF-4. Many of these receptors can be transferred to recipient
cells to facilitate platelet adhesion and to initiate intracellular
signaling cascades.32,33 The clinical correlate of these interactions is
that elevations of platelet-derived EVs are found in patients with
cardiovascular disease, transient ischemic attacks, lacunar infarcts,
and multi-infarct dementia.34 For instance, decreased clearance of
platelet-derived EVs is associated with hypercoagulation.35 Similarly, a defect in platelet secretion of EVs is associated with
a clotting deficiency known as Castaman disorder.36,37
Microenvironment Modulation
Secretion of EVs by tumors has been shown to modulate the
tumor microenvironment and to influence processes of cellular
invasion, angiogenesis, and host immune response. Select examples of these modulations are reviewed below.

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GONDA ET AL

EVs from tumor cells have been implicated as mediators of

tumor migration and invasion.38 Analysis of EVs from a spectrum
of tumors reveals high levels of matrix-degrading metalloproteinases (MMP-2, MMP-9, membrane type 1 (MT1)-MMP) and
urokinase-type plasminogen activator.4,39-41 These EVs have been
shown to possess proteolytic activities required for remodeling of the
extracellular matrix.40 This remodeling, in turn, increases migration
and invasion of tumor cells in experimental models.42 Other studies
suggest that tumor EVs travel to the bone marrow where they
deliver cytokines promoting the differentiation of bone marrow
derived cells. These reprogrammed bone marrow-derived cells, in
turn, return to the tumor site facilitating vasculogenesis, tumor
invasion, and metastasis.43,44 In the case of melanomas, EVs have
been shown to condition lymph nodes in ways that facilitate
metastatic spread of distant melanoma cells.45
EVs from tumor cells are also highly enriched in angiogenic
proteins and mRNAs to facilitate angiogenesis. EVs derived from
glioblastoma cells have been shown to facilitate angiogenesis in at
least 2 ways. First, uptake of EVs by endothelial cells can lead to
translation of proangiogenic mRNA, resulting in increased
endothelial proliferation.2 Second, the epidermal growth factor
receptor (EGFR) variant III (EGFRvIII), a frequently encountered oncogenic variant in glioblastoma, constitutively signals to
initiate a proangiogenic cascade.1 EVs of EGFRvIII-expressing
glioblastoma cell lines harbor EGFRvIII. These EVs are taken up
by endothelial cells. The consequent EGFRvIII signaling within
the endothelial cells promotes proliferation and subsequent angiogenesis.24 Thus, tumor EVs transfer proangiogenic factors and
mRNAs in a horizontal manner, endowing nearby endothelial cells
with increased growth capacity.
Finally, substantial data suggest that EVs released from tumor
cells modulate the immune response within the microenvironment. The modulatory effect can be either inhibitory or stimulatory, depending on the local context.46 In terms of inhibitory
effects, EVs derived from a number of tumors induce the
expansion of human T-regulatory cells and stimulate T-regulatory
function. These T-regulatory cells generally suppress the host
immune response.47,48 For instance, ovarian cancer-derived EVs
induce increased production of Fas ligand, interleukin-10, transforming growth factor-b1, cytotoxic T-lymphocyte antigen-4,
granzyme B, and perforin in T-regulatory cells. The Fas ligandbearing EVs trigger Fas-dependent apoptosis of lymphocytes, thus
attenuating their antitumor activity. Additionally, EVs contain
cytokines such interleukin-10 and cytotoxic T-lymphocyte
antigen-4 that suppress other aspects of host immune responses.
Thus, tumor-derived EVs suppress host immune response
through a variety of mechanisms.49,50
In terms of immune-stimulatory properties, some EVs are
enriched with molecules involved in antigen presentation, including
major histocompatibility complex class I and II molecules.51
Macrophages infected by various pathogens, including mycobacterium and toxoplasmosis, and endothelial cells infected by cytomegalovirus release EVs expressing pathogen-derived antigens.52,53
Antigens presented by these EVs are able to directly initiate host

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immune responses through activation of T cells.54 Pertinent to

oncology, EVs isolated from malignant effusions carry tumor
antigens and are able to induce production of tumor-specific T cells
when pulsed with dendritic cells.55 The factors that determine
whether EVs are immune-stimulatory or immune-inhibitory remain
poorly understood. Elucidation of these factors will be critical for
EV-based immunotherapy (see below).

CONTENTS OF EVs
Holmgren et al56 initially suggested the presence of oncogenic
material within EVs in a seminal article describing the presence of
DNA within apoptotic bodies. In 2006, Baj-Krzyworzeka et al4
confirmed their suspicion, definitively demonstrating the presence of tumor-derived mRNA and proteins within EVs. There is
evidence that nucleic acids/proteins are selectively imported or
incorporated into the EVs and therefore could be captured for
biomarker analysis in relevant body fluids such as serum, plasma,
or cerebrospinal fluid.
Ribosomal RNA, which makes up roughly 80% of total RNA
within cells, is essentially undetectable inside the EV. Although
mRNA, micro-RNA (miRNA), DNA, and select proteins are
found within the EV, the level at which they are found may differ
from those levels found in the cells of origin.57 As an example,
microarray analysis of the EV mRNA from primary glioblastoma
multiforme cell lines identified a . 5-fold differential expression
level of nearly 3500 transcripts compared with the cells of origin,
indicating a selective enrichment process during the packaging
of EVs.2 Similarly, select analysis of the expression levels of
7 angiogenic proteins showed that 6 of these proteins were
overexpressed in the EVs relative to the glioblastoma cell lines
from which they are derived.2 In some cases, however, the levels
of certain proteins in the EV seem to parallel levels in the cell of
origin. Nano-sized EVs from patients harboring glioblastoma
overexpressing the EGFRvIII mRNA also harbored expression
of the mutated protein.1,2 Similarly, proteomic analysis of EVs
isolated from the media of the brain tumor cell line SMA560vIII
identified 36 tumor-derived proteins with expression patterns that
parallel the cell of origin.58 Because the mechanism underlying
the selective EV import remains unclear, it is currently not
possible to predict which proteins or mRNAs are likely to be
selectively concentrated in the EVs.
Much of the genetic content within EVs consists of small RNA
species. Bioanalyzer profiles of the RNA from glioblastoma
multiforme-derived exosomes demonstrated a high level of RNA
in the , 300-nucleotide range.59 Sequencing efforts of these
RNA revealed a diverse range of RNA species, including retroviral
RNA repeat regions, mRNA fragments, transfer RNA fragments,
noncoding RNA, small nuclear RNA, small nucleolar RNA,
small cytoplasmic RNA, silencing RNA, and miRNA.60 Interestingly, most of the sequenced RNAs from EVs consisted of
noncoding RNAs, suggesting these noncoding RNAs may be
secreted to mediate intercellular communication.

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EXTRACELLULAR VESICLES IN NEURO-ONCOLOGY

Of the noncoding RNAs, miRNAs are of particular interest

given that their biological functions are better understood relative
to other RNA types. miRNAs are 19- to 22-nucleotide noncoding
RNAs that bind to the 39 untranslated region of mRNA species
and mediate mRNA degradation or translational repression.61
The available data suggest that a single miRNA may be able to
target . 100 different genes. This regulatory function plays
a critical role during development and oncogenesis.62 Thus, it is
not surprising that the expression of miRNAs is often dysregulated in glioblastoma cells.57,63,64 In particular, miR-21 is a wellstudied miRNA that is highly overexpressed in glioblastomas65
and downregulates the expression of proteins required for
apoptosis and cell cycle regulation.65 EVs isolated from the
serum of glioblastoma patients harbor a . 40-fold-higher level
of miR-21 relative to EVs isolated from nononcologic patients.2
Another type of RNA sequence that is highly represented in the
EVs derived from tumor patients is the RNA derived from
retrotransposons.66 Retrotransposons are DNA sequences that
can amplify themselves and make up nearly half of the human
genome. Retrotransposons copy themselves to RNA through
a reverse transcriptase and then back to DNA that is then inserted
into the genome.67 This process is generally suppressed in normal
cells but becomes activated during periods of cellular stress,
including oncogenesis.68 The finding that RNA intermediates of
retrotransposons are highly represented in EVs66 suggests that
intercellular communication may mediate gene transfer of
retrotransposon sequences and provides another opportunity
for biomarker development.

CLINICAL APPLICATIONS
The biology mediated by EVs and their content expand
opportunities for the development of biomarkers and therapeutic
strategies in neuro-oncology. These opportunities are reviewed
below.
Biomarker Development
The genomic and physiological profiles of tumors change with
time, alterations in the microenvironment, and therapy.69
Therapeutic interventions effective at one time may serve as no
more than placebo at another.70 In this context, real-time or
nearly real-time monitoring of the physiological state and genetic
constitution of the cancer is absolutely essential. However,
longitudinal molecular studies of brain tumors throughout
treatment obligate repeat tissue specimens and serial biopsies of
the brain, which is not practical. The observation that many brain
tumor types secrete EVs harboring genetic material mirroring
aspects of the secreting cell offers a unique opportunity in terms
of biomarker development that may afford such monitoring.
The ability of EVs to shelter proteins and genomic material
from the harsh destructive environment of the extracellular space
makes them a promising source of potential biomarkers. Their
varied contents make them amenable to several fields of biomarker
testing, including protein typing assays, microarray assays, and

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DNA sequencing studies. Of these potential assays, several have

reached the threshold of potential clinical utility.
Proteins
Shao et al71 developed a microfluidic chip that uses size and
immunoaffinity to label EVs with target-specific magnetic nanoparticles and then quantifies their presence by a micro-nuclear
magnetic resonance system. Using this technology allows detection
of EV proteins with a sensitivity exceeding other protein detection
methods, including Western blotting and enzyme-linked immunosorbent assays, by several orders of magnitude. Comparative
profiling of EV proteins derived from glioblastoma cells and their
host controls identified a distinct molecular signature of elevated
EGFR, EGFRvIII, podoplanin, and IDH1 R132H proteins in the
glioblastoma specimens.71 In an independent collection of blood
collected from 24 glioblastoma patients and 8 healthy volunteers,
elevated levels of these 4 proteins were found in the EVs derived
from the glioblastoma patients. Furthermore, glioblastoma patients
whose EVs harbored higher levels of these proteins were more likely
to fail standard temozolomide/radiation treatment (P , .005).71
These results suggest EV protein detection as a promising
diagnostic and prognostic/predictive platform.
Gene Expression Analysis
Microarrays can be used to compare expression levels of DNA,
mRNA, miRNA, methylation status, and proteins.72 Using microarray technology, Noerholm et al59 was able to show that the mRNA
profile of EVs from the serum of glioblastoma patients differed from
those of healthy volunteers. This difference was driven largely by
increased levels of ribosomal mRNAs in the healthy patient EVs.
Ribosomal mRNAs are highly expressed in lymphocytes, and the
differences observed correlated with the decreased white blood cell
counts seen in the glioblastoma patients. This highlights a challenge
in microarray genomic profiling of EVs obtained from serum or
other bodily fluids in which there may be an outsized degree of
background noise that must be sorted through. Even in oncologic
cases when EV production is upregulated, the representative number
of tumor-derived EVs in the serum may represent only a smallminority of the total EV population.
Point Mutations and Genetic Alterations in Brain Tumors as
Detected in EV Nucleic Acids
Sequencing of EV nucleic acids may be particularly valuable for
detecting tumor-specific mutations. A variety of studies have
demonstrated the potential for EVs to be used as markers of
systemic cancer and brain tumors. The field is in its infancy, and
whole-genome sequencing of tumor EVs in sera will likely increase
the list of evaluable tumor-associated markers and mutations. Brain
tumor-specific EV point mutations have been well identified. The
most clinically relevant of these are discussed below (Table).
EGFRvIII involves a deletion of exons 2 to 7 of the EGFR
mRNA.79 This deletion results in a constitutively active signaling
receptor inducing an aberrant signaling cascade not normally
found during EGFR activation.73,80 The mutation is found

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TABLE. Potential Extracellular Vesicle-Contained Biomarkers of Gliomasa

Gene (mRNA)
EGFRvIII
IDH1/IDH2

BRAF V600E

BRAF-KIAA1549
fusion
C-myc
amplification

a
b

Predominant Tumor Type

(Incidence, %)

Detected in
EVs

Fluids
Examined

Primary glioblastoma (20-25)

Yes

Secondary glioblastoma (12)

Yes

CSF and
serum
CSF and
serum

Function
Receptor tyrosine kinase activation affecting cell
survival, proliferation, and motility
Impaired catalytic conversion of isocitrate to
a-ketoglutarate, leading to production of
2-hydroxyglutarate
Constitutive kinase activation of Ras/Raf/
MEK/ERK pathway affecting cell
proliferation, differentiation, and survival
Activates the ERK/MAPK pathway affecting cell
proliferation, differentiation, and apoptosis
Transcriptional regulator causes dysregulation
of cell cycle, proliferation, apoptosis, and
angiogenesis

Grade II xanthroastrocytoma (65)

References
2, 73
74-76

Not tested

77

Juvenile pilocytic astrocytomas (80) Not tested

78

Medulloblastoma (5-8)

Yes

Serumb

66

CSF, cerebrospinal fluid; EV, extracellular vescile.

Mouse xenograft model.

in 20% to 25% of glioblastoma specimens79,81 and is not found

in normal tissue, rendering EGFRvIII ideal as a biomarker. In
a landmark study, the EGFRvIII status of EVs derived from the
sera of 30 glioblastoma patients was examined by quantitative
polymerase chain reaction.2 Fourteen of these tumors harbored
the EGFRvIII mutation.2 EGFRvIII was found in EVs isolated
from the serum of 5 of these patients. Interestingly, 2 additional
patients with biopsy specimen scoring negative for EGFRvIII
harbored EGFRvIII in their sera EVs, suggesting that the initial
biopsy may have targeted a nonrepresentative area in an otherwise
heterogeneous tumor.2 Longitudinal sera collection was performed for the patients who scored positive for EGFRvIII in sera
EVs. In all cases, EGFRvIII was undetectable after resection.
These results open the possibility that sera-derived EGFRvIII
may be a valid biomarker platform.
Arguably, one of the most exciting discoveries in recent years in
glioblastoma research involves the finding of recurrent IDH1
mutations in a subset of glioblastoma samples.77 IDH1 encodes
a protein responsible for the production of a-ketoglutarate from
isocitrate. Interestingly, mutations found in glioblastoma specimens occur repeatedly at codon 132 of the IDH1 gene.78 The
pathophysiology of this mutation remains unknown.79 However,
the recurrent nature of the mutation at codon 132 and the fact
that these mutations are not found in normal tissue suggest IDH1
as another excellent candidate for biomarker development.
Preliminary data suggest that IDH1 mutations can be detected
in EVs isolated from cerebrospinal fluid of patients whose
glioblastomas harbor the mutation.80 In contrast, such mutations
are not found in healthy individuals.10 Validation of this finding
is currently underway through a consortium study involving 13
high-volume neurosurgical sites in the United States.
C-myc amplification frequently occurs during the pathogenesis
of medulloblastoma cells. C-myc encodes a transcription factor

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located on chromosome 8 and is frequently mutated or amplified

in a spectrum of tumors. The physiological roles of c-myc involve
regulation of cell proliferation, apoptosis, and angiogenesis. C-myc
amplifications have been identified in EVs isolated from the sera of
mice bearing human medulloblastoma xenografts.66
B-RAF encodes a serine/threonine kinase with a role in
mitogenic signaling. Activating mutations in B-RAF are frequently encountered in pediatric tumors. For instance, fusion of
the B-RAF protein to the KIAA1549 gene has been found in up
to 80% of pilocytic astrocytomas.78,83-86 Similarly, the activating
B-RAF-V600E mutation is frequently found in gangliogliomas
and about 65% of grade II xanthroastrocytomas.77 Like
EGFRvIII, IDH1 mutations, and c-myc amplifications, these
activating B-RAF mutations are not found in normal tissue.
Thus, their detection in serum- or cerebrospinal fluid-derived
EVs likely reflects the presence of active oncologic disease.
The massively parallel nature of next-generation sequencing
technology will allow hundreds of mutations to be interrogated
simultaneously and will provide an increased ability to detect
low-frequency mutations with high sensitivity. Quantitative
polymerase chain reaction methods are sensitive only to detect
low-frequency mutations down to a 1% presence against wild-type
background, whereas traditional Sanger sequencing is useful only
to a 30% presence of mutations. Ultradeep sequencing methods
are capable of detecting mutations present down to only 0.02% of
the transcripts. Deep sequencing methods will allow improved
detection of rare gene mutations upregulated into EVs by cancer
cells against a background of normal or wild-type genes.
Challenges
Although EVs hold tremendous promise as a platform for
biomarker development, there are a number of challenges inherent
within this approach. First, serum and plasma samples contain EVs

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EXTRACELLULAR VESICLES IN NEURO-ONCOLOGY

of platelet, neutrophil, and macrophage origin, the release of which

is likely influenced by age, infection, and inflammation. Relative to
this population, the EVs derived from tumors remain a smallminority of total EVs. Thus, the sensitivity of detection remains
a major challenge in tumor-specific biomarker development.59
Second, the procedures for optimal handling of specimens have
not yet been codified. EV secretion may be influenced by diurnal
rhythms, concomitant medications, the nature of venipuncture,
centrifugation, filtration, and handling. Finally, as the methods of
collection, handling, and analysis are standardized, the sensitivity
and specificity of pertinent bioassays will require rigorous
validation.
EV-Based Therapeutic Developments
The understanding of EV function opens at least 3 therapeutic
directions. The first focuses on blocking EV production, release,
and uptake. The second involves the use of EVs as a platform for
immunotherapy. The third direction uses EVs as delivery vehicles
for targeted drug or gene delivery.
Inhibition of EV Secretion and Uptake
As discussed, EV production is increased in many cancers. The
production of these EVs facilitates angiogenesis, suppresses
immune response, and facilitates tumor invasion. These effects
can, in principle, be reversed by suppression of EV production or
inhibition of their subsequent uptake by other cells. Al-Nedawi
et al1 interrupted uptake of EVs into surrounding cells, effectively
reducing the horizontal propagation of the EGFRvIII oncogene
using annexin V, a scaffolding protein that blocks phosphatidylserine exposure, thus inhibiting EV fusion to plasma membranes.
Interestingly, a number of drugs approved by the Food and Drug
Administration for the treatment of nononcologic diseases inhibit
the secretion of EVs, including the antihypertensive amiloride, the
antidepressant imipramine, and proton pump inhibitors.87-90
Amiloride reduces calcium influx into cells, thereby inhibiting
exosome release.91 Proton pump inhibitors are thought to disrupt
EV release by interfering with the tumoral acidic extracellular
environment.87,89 Imipramine impairs EV production through its
inhibition of sphingomyelinase, thus disrupting plasma membrane
fluidity.90 Consistent with the above proposed hypothesis, cancer
patients treated with amiloride exhibit decreased levels of EV
production and show evidence of decreased tumor-mediated
immunosuppression.88 Similarly, pretreatment of mice harboring
melanoma xenografts with proton pump inhibitors resulted in
increased sensitivity to cisplatin through reduced drug efflux
through EVs.89
As mechanisms of biogenesis become better understood, new
methods for disrupting EV production are being explored. Reduction of exosome secretion by 40% to 50% in a mouse melanoma
model was sufficient to prevent the education and mobilization of
bone marrow-derived cells, resulting in reduced primary tumor
growth and metastasis.44 Silencing of genes encoding proteins
required for EV production, including HRS, Rab27a, STAM, or
TSG101, by shRNA effectively decreases EV production through

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interference of the ESCRT (endosomal sorting complex required for

transport) machinery.92,93 These and other strategies for inhibiting
EV production may provide adjuncts to current treatment regimens.
EV-Based Immunotherapy
It is important to recall that EVs may serve to suppress or enhance
the immune response, depending on the setting. Understanding the
factors that tilt the balance of immunostimulation vs immunosuppression will be critical to the success of such approaches.
The presence of tumor-infiltrating lymphocytes is thought to
reflect the host immune response to tumor antigens. Accordingly,
investigators have explored strategies to expand and enhance
the immune response against neoplasms. The ability of tumor
peptide-pulsed dendritic cellderived EVs to stimulate cytotoxic T
lymphocytes independently of dendritic cells has made them an
attractive method of tumor immunotherapy. Animal studies have
shown successful eradication of established murine tumors in a T
cell-dependent manner through priming with EVs derived from
disease-laden mice.94 Two phase I trials have demonstrated the
feasibility and safety of injecting dendritic cell-derived EVs into
patients with melanoma and non-small-cell lung carcinomas.95,96
In both of these trials, a transient stabilization of disease was
demonstrated in a subset of patients. Another phase I clinical trial
showed the safety of injecting EVs isolated from ascites of patients
with colorectal cancer.97 The encouraging results from these early
trials set the foundation for the next generation of EV-based
immunotherapies.51
EV as Delivery Vehicles for Therapeutics
The engineering of EVs derived from dendritic cells to express
select membrane proteins has been successfully used to deliver siRNA
across the blood-brain barrier in murine models.98 This was achieved
by creating a fusion of rabies viral glycoprotein (RVG) and Lamp2b
protein, an endogenous membrane constituent of exosomes (the
term exosome is used here instead of EV given the established role
of Lamp2b as a constituent of exosomes). The RVG peptide,
designed to specifically bind neurons, is fused to the N-terminal
region of the extracellular portion of Lamp2b. The RVG-Lamp2b
fusion construct was transfected into murine dendritic cells.
Exosomes purified from these cells displayed RVG in their outer
surface. The exosomes cross the blood-brain barrier and then
display RVG peptides targeting the exosome for fusion with
neurons. In this way, siRNA loaded into the RVG-Lamp2b
particles were successfully delivered to the target neurons. This
proof-of-principle study sets the stage for other applications of
exosomes or other EVs as vehicles for drug delivery.99
Another therapeutic application being explored involves intercellular transfer of signaling molecules such as GPRCs through
EVs. The importance of GPRCs in cellular physiology is evidenced
by the fact that at least 50% of available drugs on the marker target
GPRCs.100 Guescini et al30 demonstrated the ability of EVs to
transfer functionally competent GPRCs safely from one cell to
another, thus affecting their response to pharmacologic interventions. Thus, it suggests the possibility of altering the decoding

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GONDA ET AL

capabilities of target cells by acting on the composition or release

of EVs in cancer patients.100

CONCLUSION
Cells secrete nano-sized EVs under both physiological and
pathological conditions. They likely provide intercellular transport
of encapsulated proteins and genetic material. EVs can be isolated
from all body fluids. Enhanced secretion of EVs by tumors offers
an opportunity to detect tumor-specific genetic material as
biomarkers for diagnostic or therapeutic monitoring. Brain tumor
therapeutic strategies may be developed by modulation of EV
biology. Opportunity exists to use EVs as delivery vehicles or
immune-modulatory tools. Awareness of these developments
should augment the armamentarium available to oncologydedicated neurosurgeons.
Disclosure
Dr Carter reports receiving consulting fees and grant support from Exosome
Diagnostic and ABC2 foundation. The other authors have no personal financial or
institutional interest in any of the drugs, materials, or devices described in this article.

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