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J Cancer Res Clin Oncol (2016) 142:1395–1406
DOI 10.1007/s00432-015-2029-8
REVIEW – CANCER RESEARCH
Membrane microparticles: shedding new light into cancer cell
communication
Paloma Silva de Souza1 · Roberta Soares Faccion1 · Paula Sabbo Bernardo2 ·
Raquel Ciuvalschi Maia1
Received: 12 June 2015 / Accepted: 5 August 2015 / Published online: 19 August 2015
© Springer-Verlag Berlin Heidelberg 2015
Keywords Microparticles · Multidrug resistance ·
Cancer · Intercellular communication · MicroRNAs · Akt
and ERK signaling pathways
Introduction
A comprehensive classification of the different types of
shed vesicles is a main concern in the intercellular com-
munication research field. However, the complexity of this
task begins with confusing nomenclature, which includes
oncosomes, microvesicles (MVs), microparticles (MPs),
endosomes, exosomes and ectosomes. Usually, microvesi-
cle is considered to be the collective term for both MPs and
exosomes. MPs may be called ectosomes or, as recently
proposed, oncosomes, in the case of cancer-derived MPs.
The diameter of MPs may range between 0.2 and 2 µm
(Piccin et al. 2007; Simak et al. 2004), and exosomes are
usually approximately 50–100 nm in diameter. The meth-
odologies available to distinguish MPs from exosomes
by size may result in overlapping pools of the different
microvesicles. Biochemically, MPs are derived from the
plasma membrane and thus usually display membrane bio-
markers such as PS or lipid raft proteins, whereas exosomes
are derived from intracellular endosomes and present dis-
tinct biomarkers. In this review, we focus on studies that
specifically isolate MPs to describe their role in cancer
communication and their content.
Membrane microparticles: What are they?
MPs are small vesicles formed from the cytoplasmic mem-
brane that can be released from normal or cancer cells
(termed oncosomes) (El Andaloussi et al. 2013; Hugel
Abstract
Background Microparticles (MPs) or ectosomes are small
enclosed fragments (from 0.2 to 2 μm in diameter) released
from the cellular plasma membrane. Several oncogenic
molecules have been identified inside MPs, including solu-
ble proteins XIAP, survivin, metalloproteinases, CX3CL1,
PYK2 and other microRNA-related proteins; membrane
proteins EGFR, HER-2, integrins and efflux pumps; and
messenger RNAs and microRNAs miR-21, miR-27a,
let-7, miR-451, among others. Studies have shown that
MPs transfer their cargo to neoplastic or non-malignant
cells and thus contribute to activation of oncogenic path-
ways, resulting in cell survival, drug resistance and cancer
dissemination.
Discussion and Conclusion This review summarizes
recent findings on MP biogenesis and the role of the MPs
cargo in cancer and discusses some of the RNAs and pro-
teins involved. In addition, the discussion covers evidence
of (1) how and which signaling pathways can be activated
by MPs in recipient cells; (2) recipient cell-type selectivity
in incorporation of proteins and RNAs transported by MPs;
and (3) how upon stimulation, stromal cells release MPs,
promoting resistance to chemotherapeutics and invasive-
ness in cancer cells.
Paloma Silva de Souza, Roberta Soares Faccion and Paula Sabbo
Bernardo have contributed equally to this work.
* Raquel Ciuvalschi Maia
rcmaia@inca.gov.br
1 Laboratório de Hemato-Oncologia Celular e Molecular,
Programa de Hemato-Oncologia Molecular, Brazilian
National Cancer Institute (INCA), Rio de Janeiro, Brazil
2 Programa de Pós-Graduação em Oncologia, INCA, Rio de
Janeiro, Brazil
1396 J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
et al. 2005). Commonly, MPs are characterized by expo-
sure of phosphatidylserine (PS) residues on their outer sur-
faces. However, because MPs are preferentially released
from membrane lipid rafts, they may also contain raft-
related proteins, such as P-selectin glycoprotein ligand-1
(PSGL-1) and tissue factor (TF) in monocyte-derived MPs
(Del Conde et al. 2005) and other markers, depending on
the cell type, such as CD44 for breast cancer cell-derived
MPs (BCC-MPs) and ezrin for leukemic cell-derived MPs
and BCC-MPs (Jaiswal et al. 2013). Phospholipids are
distributed in a specific manner in the bilayer membrane,
but during MP shedding, an energy-dependent mechanism
rearranges the PS to outer membrane. In this process, the
flippase, floppase and scramblase enzymes respond to
increasing intracellular levels of Ca2+ and promote phos-
pholipid redistribution. In parallel, MP maturation is initi-
ated by membrane restructuring and cytoplasmic protru-
sions with cytoskeletal rearrangement and degradation.
These events are mediated by proteins such as gelsolin
and calpain allowing MP shedding (Del Conde et al. 2005;
Enjeti et al. 2008; Salzer et al. 2002).
In tumor cell-derived MPs, others proteins also contrib-
ute to MP shedding. The ADP-ribosylation factor (ARF),
which belongs to ARF family of small GTPases, has been
associated with membrane traffic, including internaliza-
tion of ligands and the organization of the actin cytoskel-
eton (D’Souza-Schorey and Chavrier 2006). Muralidharan-
Chari and colleagues demonstrated that ARF6 modulates
MP shedding via the phospholipase D, extracellular-sig-
nal-regulated kinases (ERK) and myosin light chain path-
way. They also observed that ARF6 inhibition blocks MP
shedding (Muralidharan-Chari et al. 2009). In addition,
Pasquier and colleagues showed the importance of ARF6
upregulation in MPs in mediating a vascular metastatic
microenvironment. Inhibition or deletion of ARF6 trig-
gered a reduction in MP shedding by endothelial and can-
cer cells (Pasquier et al. 2014). Furthermore, Schllienger
et al. (2014) demonstrated that ARF1 activity is required
for the shedding of MPs, invadopodia maturation and
MMP-9 activity, suggesting that ARF1 is important not
only to MP biogenesis but also to MP-mediated cancer cell
invasiveness. They showed that ARF1 modulates RhoA and
RhoC activity, which in turn activate myosin light chain
(MLC) phosphorylation.
Another important protein is P2X7, a non-selective cat-
ion channel belonging to the family of P2X receptors. This
family is associated with several cellular processes such as
cell-to-cell communication, secretory activity and mem-
brane excitability. Its activation induces a sustained Ca2+
influx and consequently changes in phospholipid asym-
metry followed by the release of MPs (Roger et al. 2014).
Several groups have previously demonstrated that P2X7
stimulation induces MP release in normal hematopoietic
cells (Qu and Dubyak 2009). Qu et al. (2007) even sug-
gested that P2X7 itself might be carried by MPs. In the
context of cancer, Constantinescu et al. (2010) further dem-
onstrated that P2X7 activity stimulates MP release from
murine erythroleukemia cells, while inhibition of P2X7
activity significantly decreases MP release. Although they
did not describe the contents of the MPs, they provided the
first evidence that P2X7 is important for cancer-derived MP
release.
Other proteins described as being involved in MP bio-
genesis are transglutaminase 2 (TG2) and neurokinin 1
receptor (NK1R). Transglutaminases (TGs) are enzymes
involved in protein transamidation. TG2, also called tis-
sue TG (tTG), is a member of the TG family that plays a
role in cellular signaling and in cytoskeletal organization.
TG2 mediates cell adhesion in association with integrins
and fibronectin (Lorand and Graham 2003). In addition,
van der Akker et al. (2012) suggested that cross-linking of
TG2 with fibronectin and the association of this complex
with cytoskeletal elements are required to form a structural
foundation for the MP in smooth muscle cells. Conversely,
Antonyak and colleagues (2011) demonstrated that TG2
was not necessary for cancer MP biogenesis and release,
on the basis that its inhibition did not alter MP shedding
(Antonyak et al. 2011). Regarding NK1R, activation of this
protein induces rapid cell shape changes (membrane bleb-
bing) (Meshki et al. 2009). Chen et al. (2012) observed that
activation of NK1R in human embryonic kidney cells, but
not in human glioblastoma cells, induces membrane bleb-
bing followed by MP release via a ROCK and dynamic
activity-dependent mechanism. Together, these studies sug-
gest that tumor cell-derived MP biogenesis deserves further
analysis. As few reports have investigated the role of TG2
and NK1R, at this point it is not clear whether tumor cells
may present different mechanisms of MP biogenesis than
normal cells.
Although the mechanism of MP release has not been
clearly characterized, the endosomal sorting complex
required for transport (ESCRT) machinery has been shown
to be required. Like other membrane-related processes, MP
membrane fission and consequently MP release involve the
core ESCRT machinery, assembled in three types of com-
plexes on the membrane. These complexes are recruited by
site-specific adaptors as well as other associated proteins
such as the ATPase and vacuolar protein sorting 4 (VPS4).
Mechanistic models have been proposed in which ESCRT-
III filaments and VPS4 interact to catalyze membrane fis-
sion (Jouvenet 2012; McCullough et al. 2013).
The interaction between MPs and recipient cells is a
fundamental process in cancer cell communication. Some
studies have hypothesized that the exposed PS on the MP
surface may bind to cellular PS receptors, leading to MP
recognition and consequently, cargo transfer. Binding of
1397J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
PS to PS receptors has been described as an important pro-
cess for the identification and removal of apoptotic cells
and apoptotic bodies (large apoptotic vesicles) (Fadok et al.
1998; Hoffmann et al. 2001). Park and colleagues demon-
strated that BAI1 receptor binds to PS on apoptotic cells
and then promotes the phagocytic removal of their apop-
totic bodies (Park et al. 2007). Tim4 and Tim1 were also
identified as PS receptors associated with engulfment of
cells undergoing apoptosis (Miyanishi et al. 2007).
Usually, membrane fusion events are dependent on
ligand–receptor interactions. However, studies have sug-
gested that MP may also be engulfed by the endosome
pathway or the cargo may be transferred by a physical
interaction between MP and the recipient cell, as reviewed
by (Mause and Weber 2010).
Microparticles and receptor proteins
EGFR
The epidermal growth factor receptor (EGFR) is a trans-
membrane glycoprotein member of the ErbB family, which
consists of four members: EGFR (also known as ErbB1
or HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4
(HER4). Similar to the other family members, EGFR acti-
vation culminates in changes in gene expression, rearrange-
ment of the cytoskeleton, increased cell proliferation and
inhibition of apoptosis. In several epithelial tumors, EGFR
is constitutively activated. The EGFRvIII mutation pro-
duces a constitutively active receptor, which is associated
with an unfavorable cancer prognosis. The EGFRvIII muta-
tion is the most frequent genetic alteration in glioblastoma
(GBM), accounting for 34–63 % of the cases. However,
it has been shown to arise in a variety of other epithelial
tumor types such as breast (20–78 % of cases) and lung
carcinomas (16–39 % of cases). The EGFRvIII mutated
protein can activate a variety of signaling pathways, such
as Src/Stat3, PI3K/Akt, Raf/MEK1/2/ERK1/2, Beclin and
NFκB, among others (Gan et al. 2013; Jutten and Rouschop
2014).
Al-Nedawi et al. (2008) were the first to detect EGR-
FvIII in GBM-derived MP. They observed that: (1)
EGFRvIII expression in glioma cells induces the release
of microvesicles containing flotillin-1, which is a com-
mon lipid raft protein. This observation indicates that
these microvesicles are indeed MPs derived from lipid
rafts-rich membrane regions, (2) these MP merge with
the plasma membrane of cells that express the wild-type
EGFR, and (3) this fusion further confers the oncogenic
activity of EGFRvIII to the recipient cells, which begin to
display activation of MAPK and Akt pathways, enhanced
expression of EGFRvIII-upregulated genes, morphological
transformation and increased anchorage-independent
growth ability. The authors concluded that these glioma-
derived MPs may be loaded with EGFRvIII and that
blockage of MP exchange (for instance, using annexin
derivatives) may have therapeutic potential (Al-Nedawi
et al. 2008).
In a subsequent study, Al-Nedawi et al. (2009) fur-
ther demonstrated that these EGFR-containing MPs may
also be taken up by other cell types, such as non-malig-
nant endothelial cells, and showed that (1) the recipient
cells begin to exhibit EGFR-dependent signaling includ-
ing MAPK and Akt phosphorylation and (2) MP transfer
can indeed be inhibited by PS blockers such as annexin V.
Furthermore, they observed that EGFR transfer through
glioma-derived MPs is associated with the onset of VEGF
(vascular endothelial growth factor) expression and auto-
crine activation of its receptor (VEGFR), consistent with
previous findings that microvascular density was reduced
by PS blockers. Diannexin (an annexin analogous) treat-
ment even resulted in reduced tumor growth. Thus, they
concluded that the uptake of glioma-derived MPs by nor-
mal endothelial cells renders these cells cooperative with
the tumor, which in turn provides the endothelial cells with
an angiogenic stimulus that favors tumor development (Al-
Nedawi et al. 2009).
Together, these studies provided great insight into
tumor cell communication within the tumor microenviron-
ment because they demonstrated the lack of a requirement
for cell–cell contact in order to transfer the EGFRvIII
mutation phenotype horizontally. They argued that glioma
cells can share EGFRvIII with wild-type tumor cells, via
MPs converting them to a more aggressive phenotype,
and with stromal cells, switching them to a tumor-prone
phenotype.
HER2
Human epidermal growth factor receptor 2 (HER2/neu/
Erb2) is also a member of the EGFR family. It shares many
of its properties with EGFR. HER2 overexpression is a
common feature of breast (15–30 % of cases) and ovarian
(20–30 % of cases) cancer, but it also occurs in a number of
others malignancies (head and neck, esophagus, stomach,
colon, bladder, lung, etc.), and it may be associated with
gene amplification (Iqbal and Iqbal 2014).
Recently, Liebhardt et al. (2010) identified HER2
expression in MPs from breast cancer patients. Although
it did not reach statistical significance, the level of HER2-
positive MPs were elevated in patients with lymph node
metastasis, which suggests that the presence of this Erb
family member may be involved in the metastatic ability of
breast cancer cells (Liebhardt et al. 2010). This observation
deserves further investigation.
1398 J Cancer Res Clin Oncol (2016) 142:1395–1406
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Mac‑1 integrins
Macrophage1 antigen, or Mac-1, is an αMβ2-integrin, also
known as complement receptor 3 (CR3) or CD11/CD18b.
It is a leukocyte adhesion receptor of the β2-integrin fam-
ily, which consists of major players in the leukocyte adhe-
sion cascade (Mitroulis et al. 2015). Although normally
expressed only in neutrophils and monocytes, Mac-1 has
been found to play an important role in MP-driven metastasis
of murine hepatocarcinoma cells (Ma et al. 2013). Ma et al.
(2013) observed that upon activation with PMA (phorbol
myristate acetate), myeloid splenic cells produce and release
MPs loaded with a variety of immune cell markers, including
CD3, CD19, F4/80 and Gr-1. When co-incubated with hep-
atocarcinoma cells, these MPs are taken up by tumor cells,
but the MPs do not co-localize with lysosomes, ER or Golgi,
suggesting an endosome-independent pathway of uptake
that preserves the MPs in the cytoplasm. Moreover, tumor
cells that received MPs derived from PMA-treated tumor
cells (PMA-MP) grow in several tissues and yield a worse
prognosis. Furthermore, PMA-MP-treatment of both mela-
noma and hepatocarcinoma cells causes a tenfold increase in
migration and invasion, suggesting that the MPs derived from
activated immune cells could enhance the invasiveness of
diverse tumor cell types. Blocking either the CD11b or CD18
subunits of Mac-1 inhibits transwell migration of most cells,
diminishes adhesion of tumor cells to the endothelium and
improves the overall survival of mice, suggesting that Mac-1
is a fundamental player in the metastasis promoted by acti-
vated immune cell-derived MPs. Thus, the authors suggested
that activated immune cell-derived MPs may be taken up by
tumor cells, providing the tumor with immune cells typical
migration features, and facilitating metastasis. Interestingly,
Mitroulis et al. (2015) recently reviewed endogenous inhibi-
tors that modify integrin functions and could thus be impli-
cated as promising therapeutic targets (Mitroulis et al. 2015).
CXCR4
CXCR4 is a membrane receptor for CXCL12 (stromal
cell-derived factor-1), which is a potent chemoattractant
for human progenitor cells. CXCL12 mediates homing to
the bone marrow, survival, proliferation and even egress
to the circulation (Lapidot et al. 2005). Overexpression of
CXCR4 on human CD34+ progenitors increases their pro-
liferation, migration and repopulation in mice (Kahn et al.
2004), whereas disruption of CXCL12/CXCR4-mediated
cell anchorage induces the release of hematopoietic progen-
itors as well as mature cells from the bone marrow into the
circulation (Lapidot et al. 2005). In cancer, the CXCL12/
CXCR4 interaction has been implicated in acute myeloid
leukemia (AML) progression and in solid tumor growth
and metastasis (Juarez and Bendall 2004; Zlotnik 2004).
Kalinkovich et al. (2006) described CXCR4 as well as
its ligand, CXCL12, in acute myeloid leukemia (AML)-
derived MP. CXCR4 molecules could be transferred from
MPs to AML cells, enhancing migration toward CXCL12
in vitro and homing to the bone marrow of mice. CXCR4
inhibition diminished these effects, suggesting that
CXCR4-loaded MPs are involved in the progression of
AML. The levels of CXCR4 and CXCL12 in MPs were
also enhanced in the serum of AML patients compared
with normal subjects, indicating that these proteins could
be used as biomarkers for the disease (Kalinkovich et al.
2006).
Osteopontin
Osteopontin (OPN) is a small integrin-binding ligand
N-linked glycoprotein (SIBLING). SIBLING proteins bind
to cell surface integrins and CD44 in normal tissues and
function as signal transducers to promote cell adhesion,
motility and survival through the activation of kinase cas-
cades and transcription factors. They can also regulate pro-
cesses such as inflammation, immune response and cancer
(Bellahcene et al. 2008).
In both non-malignant and cancer cells, OPN has been
shown to activate the PI-3K/AKT/NFκB pathway, which
promotes cellular proliferation and differentiation, and
inhibits apoptosis (Gimba and Tilli 2013). Interaction with
CD44 and integrins has been shown to activate this OPN-
dependent signaling. In cancer, OPN is also involved in
invasion, extracellular matrix degradation, metastasis,
inflammation/complement evasion and angiogenesis (Bel-
lahcene et al. 2008; Ramchandani and Weber 2015).
Recently, Fremder et al. (2014) detected a high OPN
content in tumor cell-derived MP. Although OPN deple-
tion from MP is not sufficient to reduce tumor growth, they
observed that when bone marrow-derived pro-angiogenic
cells (BMDC) from paclitaxel-treated mice were injected
into OPN-depleted tumor-bearing mice, tumor growth and
BMDC infiltration were greatly inhibited (Fremder et al.
2014). These data suggest that OPN-loaded MPs may play
important roles in tumor homing and BMDC mobilization,
which facilitates angiogenesis.
Microparticles and cancer resistance
Multidrug resistance (MDR) is one of the major obstacles
for chemotherapy in several types of neoplasia. The main
mechanism of MDR is the overexpression of P-glycopro-
tein (Pgp/ABCB1), a transporter protein involved in the
efflux of anticancer drugs. Pgp belongs to the ABC super-
family of transporters, and others members of this family
including multidrug resistance-associated protein 1 (MRP1
1399J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
or ABCC1) and breast cancer resistance protein (BCRP or
ABCG2) are also associated with the efflux of chemical
compounds in cancer (Gottesman et al. 2002; Vasconcelos
et al. 2011).
In 2009, Bebawy and colleagues demonstrated that Pgp
is carried by MPs derived from drug-resistant cancer cells
and can be transferred to drug-sensitive cells. Their work
also showed transfer of functional Pgp and consequently
MDR phenotype acquisition in recipient cells after MPs
interaction (Bebawy et al. 2009). In another study, the same
group identified the Pgp mRNA and also ABCC1 mRNA in
MPs derived from resistant cancer cells and their transfer
to recipient cells (Lu et al. 2013). They also showed MDR-
related miRNAs in the contents of drug-resistant cell-
derived MPs (Jaiswal et al. 2012a).
Jaiswal and co-workers (2013) hypothesized that Pgp
transfer by MPs can occur selectively. They showed that
MPs derived from resistant breast cancer cells could only
transfer their MDR protein content to malignant breast
cells and not to non-malignant cells, while resistant leu-
kemic cell-derived MPs could transfer MDR proteins to
both malignant and non-malignant cells. These results sug-
gest MPs derived from breast cancer cells display cell-type
selectivity on the transfer of MDR features. In this regard,
the authors suggested that the CD44 carried by MPs derived
from breast cancer cells could contribute to the horizontal
transfer of MDR proteins (Jaiswal et al. 2013). Addition-
ally, our group showed that Pgp protein and mRNA as well
as miRNAs related to the MDR phenotype are carried in
MPs derived from myeloid leukemia cells and that these
molecules can be transferred to sensitive breast and lung
cancer cells, displaying no cell-type selectivity (de Souza
et al. 2015). Moreover, Pasquier et al. (2014) showed that
endothelial cells are recipient cells for the MPs derived
from both epithelial and mesenchymal cancer cells, sug-
gesting that endothelial cells might be targets for MPs of
both origins.
A proteomic study demonstrated that nineteen cytoskel-
etal proteins and ten proteins involved in regulation of
the actin cytoskeletal pathway were detected in the MPs
derived from MDR breast cancer cells. These results sug-
gest that these proteins are important for stabilizing Pgp in
MPs (Pokharel et al. 2014). Among the identified proteins,
CD44, ezrin, radixin and moesin appeared to be involved
in the resistance phenotype and metastasis (Donatello et al.
2012). Moreover, CD44 has been demonstrated to interact
with Pgp and also to be associated with cell migration and
invasiveness (Miletti-Gonzalez et al. 2005), suggesting that
the presence of CD44 in MPs may be required at least to
facilitate the MDR phenotype transfer from solid tumor
cells to drug-sensitive recipient cells (Pokharel et al. 2014).
Ezrin, radixin and moesin proteins were shown to
be important for the interaction between the plasma
membrane and cytoskeletal proteins (Arpin et al. 2011).
The presence of all of these proteins in MPs may facilitate
the transfer and intracellular trafficking of Pgp. Probably,
other membrane proteins are also involved in Pgp trans-
fer to drug-sensitive cancer cells via MPs (Pokharel et al.
2014). In addition, ezrin plays an important role in Pgp
membrane insertion. This protein is carried in MPs, and
its expression is unchanged in recipient cells. Therefore, it
has been suggested that ezrin is related to Pgp stabilization
in MPs and incorporation by the drug-sensitive recipient
cells (Brambilla et al. 2012; Jaiswal et al. 2013; Luciani
et al. 2002).
Pokharel et al. (2014) also observed CD73 loaded in
the MPs derived from MDR breast cancer cells. Their data
suggest that Pgp interacts with CD73 along with CD44 in
MPs, which further suggests that CD73 may also partici-
pate in the MDR phenotype transfer from solid tumor cells
to recipient cells (Pokharel et al. 2014). Increased levels of
CD73 protein expression were found in several MDR cell
lines, and inhibition of its enzymatic activity reverses the
MDR phenotype and inhibits tumor cell growth, suggesting
that this enzyme is also involved in drug resistance (Ujhazy
et al. 1996). Moreover, in human breast cancer cells, CD73
has been shown to promote tumor angiogenesis (Wang
et al. 2013), invasion, migration, adhesion and growth
(Wang et al. 2008; Zhi et al. 2007).
The aforementioned studies demonstrate that MPs
derived from cancer cells are potent disseminators of the
drug-resistant phenotype, because MPs can interact with
sensitive cancer cells and non-malignant cells as well as
with endothelial cells. Studies have also reported the pres-
ence of MPs in the plasma of cancer patients (Fleitas et al.
2012; Savasan et al. 2004; Toth et al. 2008). Liebhardt et al.
(2010) analyzed the MPs in the plasma of breast cancer
patients and identified four major subpopulations of MPs
including a group positive for BCRP (BCRP+). The authors
observed that patients with lymph node metastases showed
elevated levels of BCRP+ MPs compared to patients with
initial disease and attributed this increase to enhanced can-
cer growth and dissemination in these patients (Liebhardt
et al. 2010).
The clinical relevance of the MPs role on pathological
processes has been comprehensively discussed by several
researchers. Gong and co-workers (2015) emphasized the
clinical importance of MPs on regarding drug resistance
and metastasis. On their review, the MPs potential on can-
cer diagnosis and prognosis was also discussed (Gong et al.
2015). In addition, El Andaloussi and co-workers (2013)
largely reviewed the role of MPs in tumor biology. They
argued about the importance of MPs in promoting cancer
progression, because MPs transport and transfer molecules
associated with tumor growth, immune system escape by
tumors and angiogenesis (El Andaloussi et al. 2013).
1400 J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
Microparticles and microRNAs
MicroRNAs (miRNAs) are small non-coding RNAs (18–
24 nucleotides) that regulate gene expression by binding
to mRNAs, leading to mRNA degradation or impairment
of translation. Briefly, miRNAs are processed by a com-
plex machinery composed by the endonucleases Drosha
and Dicer, and by a protein complex called RISC (RNA
silencing complex). The central components of RISC are
Argonaute proteins (Ago), primarily Ago-2. Ago proteins
mediate the binding of mature miRNAs to mRNA (Winter
et al. 2009). Each miRNA has hundreds of predicted tar-
gets, and it is believed that 60 % of mRNAs are regulated
by miRNAs (Jansson and Lund 2012). In cancer, they are
deregulated and involved in tumorigenesis, tumor pro-
gression, metastasis and resistance to treatment. Further-
more, miRNAs have recently been proposed as promising
biomarkers for cancer diagnosis and follow-up studies on
disease progression, because they can be detected in body
fluids, carried by MPs or associated with proteins that pro-
tect them from degradation (Fujiwara et al. 2014; Inns and
James 2015; Jackson et al. 2014; Lindner et al. 2015; Liu
and Xiao 2014; Pimentel et al. 2014). Several groups are
attempting to uncover the underlying mechanisms involved
in miRNA selection, release and transport to blood flu-
ids and their role in cancer (Arroyo et al. 2011; Li et al.
2012; Turchinovich et al. 2011). However, the majority
of the studies that have attempted to elucidate the mecha-
nism of miRNAs transfer among cells did not differenti-
ate between MPs and exosomes, analyzing them together
as MVs. Arroyo et al. (2011) demonstrated that only 15 %
of circulating miRNAs, including let-7a and miR-142-3p,
are found in MVs (exosomes and MPs). The majority of
the circulating miRNAs is found in non-vesicular frac-
tions associated with ribonucleoprotein complexes contain-
ing Ago-2 protein (Arroyo et al. 2011). The association of
miRNAs and Ago-2 in MPs was also identified in cancer
cells undergoing apoptosis and in those in which differ-
entiation had been induced, suggesting that Ago-2 confers
higher stability to miRNAs molecules (Li et al. 2012).
However, distinguishing between these types of vesi-
cles is very important because they transport different car-
gos, as demonstrated by Ji and colleagues (2014). These
authors separated exosomes from MPs derived from a
colon carcinoma cell line and demonstrated that some miR-
NAs including Let-7 family members and miR-451a are
common to all vesicles, but other miRNAs are specific for
either MPs or exosomes. They found four miRNAs selec-
tively carried in MPs: miR-675-5p, miR-7704, miR-98-5p
and miR-664a-3p (Ji et al. 2014).
Some studies have investigated the role of MPs in
the resistance to cancer treatment. Using parental acute
lymphocytic leukemia (ALL) and breast cancer cells along
with their MDR counterparts, Jaiswal and colleagues
(2012a) demonstrated the effect of MDR cell-derived MPs
on their respective parental cell lines. The authors identi-
fied transcripts of Drosha, Dicer and Ago-2 in MPs derived
from both parental and MDR cell lines. This study supports
the general MVs findings that suggest that miRNA machin-
ery proteins may be important to guarantee the activity of
the miRNA molecules (Jaiswal et al. 2012a). Additionally,
the authors also identified miR-1228*, miR-1246, miR-
1308, miR-149*, miR-455-3p, miR-638 and miR-923 in
MPs derived from both MDR cell lines. These miRNAs are
transferred to drug-sensitive cells and can activate impor-
tant pathways associated with the vesiculation of MPs,
including the calcium signaling pathway and regulation of
the actin cytoskeletal pathway, as well as pathways associ-
ated with MDR phenotype and oncogenesis (Jaiswal et al.
2012a; b).
miR‑503 downregulation and the resistance phenotype
Gong et al. (2014) identified miR-22-3p, miR-185-5p,
miR-503-5p, miR-652-3p, miR-1280 in MPs associated
with the acquisition of the MDR phenotype. Among these,
miR-503 was overexpressed in drug-sensitive cells and
downregulated in drug-resistant cells. The authors observed
that MPs derived from drug-resistant cells can inhibit miR-
503 expression in recipient cells and promote migration
and invasion (Gong et al. 2014). It was shown that miR-
503 inhibits PI3Kp85 and IKKβ and consequently, Akt
phosphorylation and NFκB activation (Yang et al. 2014).
In agreement with this, our group and others have dem-
onstrated that activation of these signaling pathways by
MPs confers a resistance phenotype (de Souza et al. 2015;
Pasquier et al. 2014; Wysoczynski and Ratajczak 2009).
miRNA-503 downregulation was also observed in lung and
hepatocellular cancers and in highly invasive breast can-
cer cells, and its overexpression induces G1 arrest, reduces
cell migration and invasiveness in vitro and in vivo (Gong
et al. 2014; Yang et al. 2014; Zhou and Wang 2011). One
study demonstrated that in response to a microbial chal-
lenge, epithelial cells overexpress CX3CL1 in a NFκB- and
Dicer-dependent manner. This effect was associated with
the downregulation of miR-503, which directly inhib-
its CX3CL1 expression (Zhou et al. 2013). CX3CL1 is a
membrane-bound chemokine that can be shed in a soluble
form or through release of MPs (Bazan et al. 1997; Cas-
tellana et al. 2009). In addition, Castellana et al. (2009)
showed that fibroblasts activated by tumor MPs in turn
release MPs containing CX3CL1 and induce a migratory
phenotype in recipient prostate cancer cells. Together, these
findings suggest that miR-503 downregulation in cancer
1401J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
cells may be mediated by Dicer, CX3CL1 and probably by
other MP cargo.
PYK2 regulation by CD44, miR‑494 and miR‑303‑3p
As miR-503 was transported by MPs but was not detected
in recipient cells, the opposite situation was observed for
proline-rich tyrosine kinase 2 (PYK2) (Gong et al. 2014).
PYK2 is a cytoplasmic tyrosine kinase of the focal adhe-
sion kinase subfamily (Fan and Guan 2011). PYK2 pro-
motes cell migration and drug resistance in many tumor
types including hepatocellular and breast cancer and is acti-
vated by the PI3K/Akt signaling pathway (Datta et al. 2015;
Geng et al. 2011). PYK2 mRNA and protein were detected
in recipient cells after co-culture with the MDR counterpart
cell line, although PYK2 was not detected in MPs. These
observations suggest that MPs carry and transfer interme-
diates that result in the inhibition of miR-503 and PYK2
expression in the recipient cells (Gong et al. 2014). Jaiswal
and colleagues demonstrated that CD44 is carried by the
MPs but is not detected in the recipient cells and that CD44
promotes PYK2 phosphorylation. This observation sug-
gests that somehow the interaction with CD44 stimulates
activation of endogenous PYK2 in the recipient cells. miR-
494 and miR-330-3p are regulators of PYK2 expression
(Gong et al. 2014) and were modulated in recipient cells.
miR-494, an inhibitor of a repressor of PYK2, was trans-
ported by MPs and incorporated into the recipient cells.
Conversely, miR-330-3p is a repressor of PYK2, and its
expression was inhibited after co-culture, suggesting that
PYK2 expression might be regulated by these miRNAs in
association with CD44 through cellular signaling pathways
(Jaiswal et al. 2013). In conclusion, the regulatory role of
MPs in cancer cells is a remarkable, complex and modestly
understood intercellular communication mechanism.
microRNAs involved with Efflux pumps
Likewise, the miRNAs involved with Pgp and MRP1 regu-
lation are transferred by MPs to drug-sensitive cancer cells.
miR-27a and miR-451 indirectly promote Pgp overexpres-
sion, while miR-326 inhibits MRP1 expression (Liang
et al. 2010). Zhu et al. (2008) demonstrated that miR-27a
and miR-451 positively regulate Pgp/ABCB1 expression.
However, other reports have demonstrated that miR-27a is
downregulated in Pgp-positive leukemia cells (Feng et al.
2011). Jaiswal et al. (2012b) demonstrated that a MDR
leukemia cell line transfers miR-326 to the sensitive coun-
terpart which is associated with MRP1 downregulation in
the cells. Moreover, the breast cancer MDR-derived MPs
transfer miR-326 and miR-27a, but the miR-326 expres-
sion did not correlate with MRP1 downregulation. MPs
carry miRNAs from the donor cells, but not all of them are
incorporated by the recipient cells. These results suggest
that the MP regulatory mechanisms that control MP for-
mation and incorporation differ among cell types (Jaiswal
et al. 2012b). Corroborating these findings, our group dem-
onstrated that the chronic myeloid leukemia (CML) cell
lines K562 and Lucena (a MDR-positive line derived from
K562 by continuous exposure to vincristine) release MPs
and that the Lucena-derived MPs carried Pgp protein and
mRNA (ABCB1). Furthermore, the co-cultures of Lucena
cell line or the Lucena-derived MP with recipient cells
(breast and lung cancer models) transferred miR-27a and
miR-451. However, an important oncogenic miRNA, miR-
21, was transferred only to the breast cancer cells, rein-
forcing the concept that transfer of cargo by MPs differs
among the recipient cell lines because it occurs in a cell-
type-dependent manner (de Souza et al. 2015; Jaiswal et al.
2013).
Signaling pathways and resistance mediated
by MPs
Several reports have demonstrated that the molecules car-
ried in cancer cell vesicles can activate diverse signaling
pathways including STAT, PI3K-Akt and MAPK, and pro-
mote angiogenesis, metastasis and treatment resistance in
the recipient cells (Lam et al. 2013). However, the major-
ity of these studies do not differentiate between MPs and
other vesicles. Here, we focus specifically on MP-related
mechanisms.
Uptream pathways: STAT, PI3K‑Akt and MAPK
STAT activation has been strongly associated with the
induction of angiogenesis, and this activation may be medi-
ated by MPs containing miRNAs. MPs released by pancre-
atic adenocarcinoma, colorectal adenocarcinoma and lung
carcinoma cells induce STAT3 activation in monocytes, an
important pathway for cytokine production and activation
of these cells (Baj-Krzyworzeka et al. 2007).
A non-pure fraction of MPs derived from human and
murine lung cancer cell lines activates the Akt and MAPK
pathways in human umbilical vein endothelial cells
(HUVEC) and in tumor-associated fibroblasts (TAF) induc-
ing the expression of pro-angiogenic factors, cytokines,
adhesion molecules and metalloproteinases (MMPs), with-
out inducing HUVEC proliferation. Once stimulated by
MPs, endothelial cells and fibroblasts, in turn, influence
tumor cells by inducing STAT3, Akt and MAPK activation
and by enhancing the metastatic potential of lung tumor
cells in vivo (Wysoczynski and Ratajczak 2009). Corrob-
orating these findings, MPs derived from prostate cancer
cells transfer MMPs to fibroblasts, activate ERK1/2 and
1402 J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
consequently induce an increase in active MMP-9. MMPs
are involved in extracellular matrix degradation allow-
ing cancer cell migration and dissemination. Additionally,
MP-induced chemoresistance in fibroblasts was shown to
be reversed by ERK1/2 inhibitors, which demonstrates the
role of this pathway in the acquired resistance. In turn, the
activated fibroblasts induce cancer cell migration and inva-
sion in a fibroblast MP-dependent manner (Castellana et al.
2009).
In a similar way, Antonyak and colleagues (2011) co-
cultured glioblastoma- and breast cancer cell-derived MPs
with non-tumoral cells. Cancer cell-derived MPs induced
Akt and ERK activation, promoting enhanced cellular
survival, growth in medium with low serum concentra-
tion and anchorage-independent growth, all of which are
characteristics of transformed cells. TG2 was found in the
outer leaflet of the MP membranes. The authors associ-
ated the oncogenic transformation of the non-tumoral
cells with the transfer of TG2 by the MPs. Nevertheless,
TG2 alone was not sufficient for cell transformation;
TG2 cross-linking with fibronectin was required. In addi-
tion, the breast cancer cell-derived MPs promoted aber-
rant growth of non-malignant mammary epithelial cells.
Finally, breast cancer cells from murine tumors secrete
MPs and induce transformation of fibroblasts in vivo
(Antonyak et al. 2011).
Pasquier et al. (2014) observed that MPs derived from
cells with a mesenchymal phenotype (M–MPs) induce pro-
liferation, motility and activation of endothelial HUVEC
cells, while MPs derived from cells with an epithelial phe-
notype (E-MPs) could not induce the same effect. M–MPs
induced Akt phosphorylation and activation of this path-
way by upregulation of Arf-6 expression. In addition, using
an endothelial cell model in which Akt was constitutively
activated, the authors demonstrated that MPs induced cel-
lular resistance to doxorubicin and taxol. The M–MPs also
induced the formation of spheres and epithelial-mesenchy-
mal transition (EMT) and as a consequence, an increase in
the pro-metastatic phenotype of tumor cells (Pasquier et al.
2014). In agreement with this study, we have previously
observed that after co-culture with MPs derived from an
MDR cell line, breast or lung cancer recipient cells become
resistant to paclitaxel and cisplatin. To further clarify the
pathways modulated in recipient cells by the MDR-posi-
tive MPs, activation of Akt pathway in breast cancer cells
and NFκB transcription factor nuclear localization in both
recipient cell lines were demonstrated (de Souza et al.
2015). Collectively, these studies strengthen the evidence
for a role of Akt pathway in the acquired resistance, dem-
onstrating once more that tumor-derived MPs can induce
a drug-resistant phenotype (de Souza et al. 2015; Pasquier
et al. 2014).
Inhibitor of apoptosis proteins
Furthermore, MPs derived from fibrosarcoma cells activate
the NFκB signaling pathway in recipient mesenchymal
stem cells. This activation is associated with the upregula-
tion of several targets of this pathway that are involved in
cell survival including MMP-1, MMP-3, BIRC3, NFκB1
and TNF. It is noteworthy that the authors of this work
studied MPs in a plasmatic membrane vesicle-enriched
fraction, containing approximately 25 % exosomes. Among
the activated genes, BIRC3 encodes a member of the inhib-
itor of apoptosis protein (IAP) family that is important for
cancer cell survival (Lozito and Tuan 2014; Verhagen et al.
2001).
Survivin and XIAP are the most studied and best char-
acterized IAP family members. These proteins are overex-
pressed in several types of cancer and are associated with
treatment resistance and poor prognosis (Faccion et al.
2012; Fulda and Vucic 2012). Our group was the first to
demonstrate that MPs transfer IAP proteins and mRNAs
including survivin, XIAP and cIAP1 to recipient cells (de
Souza et al. 2015). Honegger and colleagues demonstrated
that cervical cancer-derived extracellular vesicles carry
IAPs including survivin, cIAP1, XIAP and livin. However,
these vesicles were not a pure MP fraction. (Honegger et al.
2013). Our group also observed that after co-culture, lung
cancer cells expressed higher amounts of survivin, XIAP
and cIAP protein and mRNA (BIRC5, BIRC4 and BIRC2,
respectively), while breast cancer cells expressed higher
levels of XIAP with no increase in cIAP1 mRNA (BIRC2),
suggesting a selectivity with respect to MP cargo transfer
(de Souza et al. 2015). In addition, because XIAP mRNA
(BIRC4) was not detected in the MDR cell line-derived
MP, but was detected in recipient cells, others intermediates
carried by MPs may regulate the gene expression in recipi-
ent cells.
Activation of cellular signaling pathways by MPs is
also associated with miRNAs. The oncomiR (oncogenic
miRNA) miR-21 inhibits proteins associated with cell
survival and apoptosis pathways, including PDCD4 (pro-
grammed cell death 4) and PTEN. This inhibition induces
Akt and NFκB activation and results in cellular transfor-
mation (Iliopoulos et al. 2010). Our group identified miR-
21 as a content of leukemic MDR cell-derived MPs. We
analyzed miR-21 in recipient cells and observed an asso-
ciation of this miRNA with NFκB activation mediated by
Akt phosphorylation in these cells (de Souza et al. 2015).
In summary, MPs can mediate activation of signaling path-
ways associated with cancer survival in recipient cells by
carrying and transferring miRNAs or other regulatory mol-
ecules that modify the cellular gene/protein expression
patterns.
1403J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
Conclusion and future perspectives
In this review, we have described recent findings regarding
the contents of MPs and how these vesicles influence the
acquisition of an MDR or metastatic phenotype in recipi-
ent cells (Fig. 1). Gaining better understanding of MPs role
in cancer cell communication and its distinct role regarding
each cell type will provide future tools for cancer therapy.
Although MPs study and understanding are developing
fast, many issues concerning its role and properties have
not being clarified yet.
First, a notable and major challenge in the study of MPs
is the similarity between MPs and other cellular vesicles,
such as exosomes; because these are all small microvesi-
cles, their profiles may overlap, depending on the meth-
odology employed to study them. Thus, properly differ-
entiating the types of MVs is primordial to improve our
understanding of their specific roles. However, up until
now few studies analyze microvesicles properly, and in
most, a mixture of very different types of microvesicles
are included in the same analysis. In addition, terms such
as oncosomes, microvesicles, microparticles, endosomes,
Fig. 1 Schematic representa-
tion of a membrane microparti-
cles (MPs) budding from donor
cell and cargo content transfer-
ence to recipient cell. MPs are
released from donor cells after
increase in intracellular Ca2+
which promotes phospholip-
ids translocases response and
consequently cytoskeleton
disruption. MPs cargo can
include membrane and solubles
proteins, and microRNAs. MPs
content can be transferred to
recipient cells and promote
oncogenic pathways activation
1404 J Cancer Res Clin Oncol (2016) 142:1395–1406
1 3
exosomes and ectosomes present a challenge, as they often
appear in the nomenclature as synonyms when indeed they
are not. Thus, many controversial data are obtained because
the literature lacks standardized nomenclature. Mark-
ers to identify and differentiate these vesicles and refined
protocols for extraction and purification are also urgently
needed. To move forward and to fill the empty gaps with
reliable results, it is required to properly differentiate MVs
structures. Cellular MVs are in fact the collective of MPs
and exosomes, and they may have some common cargo
molecules. In this review, we made an effort to select stud-
ies that exclusively focus on MPs on the basis that there
are major biological differences between these and other
MVs and that studies on single forms of microvesicles are
required to properly understand the contribution of each
microvesicle type to cellular communication.
Second, modulation of MPs release, uptake and its surface
molecules have emerged as a promising therapeutic option
and several groups are trying to reduce MPs cancer cells
release and their uptake by recipient cells in order to impair
cancer progression as recently reviewed by El Andaloussi
(El Andaloussi et al. 2013). Furthermore, cellular engineer-
ing using MVs as a drug delivery system can be a less toxic
treatment option because MVs are biocompatible, can cross
biological barriers such as the blood–brain barrier (BBB), do
not activate immune responses against them and can deliver
nucleic acids and drugs, becoming an promissing tool for
gene therapy (Alvarez-Erviti et al. 2011; Zhuang et al. 2011).
Finally, further understanding the role of these vesicles in
the communication between cancer cells and the microenvi-
ronment (such as extracellular matrix elements, stromal and
immune cells) will contribute to elucidate an important gap in
our current understanding of the cancer cells network. How-
ever, there is still much more to be learnt in each one of these
fields. In conclusion, MPs play a remarkable, complex and
incompletely understood role in cancer cell communication.
Acknowledgments This work was supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação
para Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). PSS
and RSF were supported by postdoctoral fellowships from Ministé-
rio da Saúde/Instituto Nacional de Câncer. PSB was supported by a
“Nota 10” Ph.D. scholarship from (FAPERJ).
Compliance with ethical standards
Conflict of interest The authors declare to have no conflict of inter-
est.
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