Clinical Chemistry 61:12

1457–1465 (2015) Review

The Role of Exosomes in Breast Cancer

Michelle C. Lowry,1 William M. Gallagher,2 and Lorraine O’Driscoll1*

BACKGROUND: Although it has been long realized that common cancer (1 ). In the US it was estimated that
eukaryotic cells release complex vesicular structures into approximately 232 670 new cases of breast cancer would
their environment, only in recent years has it been estab- present in 2014. This staggering figure was predicted to
lished that these entities are not merely junk or debris, account for 29% of all new cancer cases (2 ). Of note,
but that they are tailor-made specialized minimaps of breast cancer is not restricted to the female population,
their cell of origin and of both physiological and patho- with approximately 1% of these cancers occurring in
logical relevance. These exosomes and microvesicles (ec- males. Primary breast tumors typically do not kill; this
tosomes), collectively termed extracellular vesicles (EVs), occurs as a result of cancer spread/metastasis to secondary
are often defined and subgrouped first and foremost ac- sites in the body. In fact, the 5-year survival rates are 99%
cording to size and proposed origin (exosomes approxi- for localized breast cancer, 84% for regional stage (nearby
mately 30 –120 nm, endosomal origin; microvesicles lymph nodes), and 23% for metastases (distant organs
120 –1000 nm, from the cell membrane). There is grow- and lymph nodes) (3 ).
ing interest in elucidating the relevance and roles of EVs Breast cancer is a highly heterogeneous disease and,
in cancer. although this review is not focused on describing these
subtypes but moreso on the relevance of exosomes, the
CONTENT: Much of the pioneering work on EVs in can- following information may help to add context for those
cer has focused on breast cancer, possibly because breast who have not previously studied breast cancer. In 2000,
cancer is a leading cause of cancer-related deaths world- Perou et al. (4 ) made a fundamental contribution to
wide. This review provides an in-depth summary of such defining breast cancer subclassifications, using DNA mi-
studies, supporting key roles for exosomes and other EVs croarrays. From this development, hierarchal clustering
in breast cancer cell invasion and metastasis, stem cell analysis revealed various subtypes based on gene expres-
stimulation, apoptosis, immune system modulation, and sion patterns, i.e., basal-like [mostly classified as triple-
anti– cancer drug resistance. Exosomes as diagnostic, negative breast cancer (TNBC),3 although basal-like and
prognostic, and/or predictive biomarkers and their po- TNBC are not synonymous (5 )], human epidermal
tential use in the development of therapeutics are growth factor receptor 2 (HER2)/neu-overexpressing,
discussed. normal-like, and luminal epithelial/ER⫹ (estrogen recep-
tor positive). Subsequent to this study, the luminal sub-
SUMMARY: Although not fully elucidated, the involve- class was further subdivided into luminal A and luminal
ment of exosomes in breast cancer development, progres- B. Without treatment being considered, the poorest sur-
sion, and resistance is becoming increasingly apparent vival rates have been associated with basal-like and
from preclinical and clinical studies, with mounting in- HER2-overexpressing tumors (6, 7 ). Because of the het-
terest in the potential exploitation of these vesicles for erogeneous nature of breast cancer and different stages of
breast cancer biomarkers, as drug delivery systems, and in diagnosis from individual to individual, treatment typi-
the development of future novel breast cancer therapies. cally involves multimodality approaches with surgery,
© 2015 American Association for Clinical Chemistry chemotherapy, radiation therapy, hormone therapy, and
other newer targeted treatments, including monoclonal
antibodies and small molecules.
According to the WHO, cancer is a leading cause of
death worldwide, with breast cancer being the fifth most

3
Nonstandard abbreviations: TNBC, triple-negative breast cancer; HER2, human epider-
mal growth factor receptor 2; EV, extracellular vesicle; miR, microRNA; EGFR, epidermal
1
School of Pharmacy and Pharmaceutical Sciences & Trinity Biomedical Sciences Institute, Trin- growth factor receptor; CAF, cancer-associated fibroblast; TIMP, tissue inhibitor of metal-
ity College Dublin, Dublin 2, Ireland; 2 School of Biomolecular and Biomedical Science, Uni- loproteinases; CSC, cancer stem cell; MSC, mesenchymal stem cell; VEGF, vascular en-
versityCollegeDublinConwayInstitute,UniversityCollegeDublin,Belfield,Dublin4,Ireland. dothelial growth factor; ADSC, adipose tissue–derived stem cells; IL-6, interleukin 6; SEB,
* Address correspondence to this author at: School of Pharmacy and Pharmaceutical Sci- staphylococcal enterotoxin B; EXO/SEB, exosomes from MDA-MB-231 cells were an-
ences & Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland. Fax chored with SEB; DC, dendritic cell; NK, natural killer; NKT cells, natural killer T cells;
+353-1-8962810; e-mail lodrisc@tcd.ie. NKG2D, NK group 2, member D; TAM, tumor-associated macrophage; NF-␬B, nuclear
Received March 11, 2015; accepted August 14, 2015. factor-␬B; EGCG, epicathecin gallate; TLR2, Toll-like receptor 2; hTERT, human telomer-
Previously published online at DOI: 10.1373/clinchem.2015.240028 ase reverse transcriptase; P-gp, P-glycoprotein; Adr, adriamycin; Doc, docetaxel; CTL,
© 2015 American Association for Clinical Chemistry cytotoxic T lymphocyte; PTEN-CT, PTEN C-terminus.

1457
Review

Fig. 1. The role of exosomes in breast cancer.

Exosomes are released from breast cancer and stromal/cancer–associated fibroblast cells into the extracellular milieu and tumour microenvi-
ronment. Exosomes have been found to play roles in cancer cell invasion and metastasis, stem cell stimulation, apoptosis, interaction with cells
of the immune system, and drug resistance.

Exosomes and Breast Cancer gressive isogenic subclone Hs578Ts(i)8 (13 ) to investi-
gate the exosomal transfer of phenotypic traits (14 ). Here
Exosomes, cell-derived vesicles, were initially described as we found that exosomes from Hs578Ts(i)8 cells confer
vesicles released during the maturation process of reticu- their more aggressive phenotypic traits to all secondary
locytes (8 ). Exosomes and microvesicles (also termed ec- breast cancer cells assessed (including the Hs578T parent
tosomes) are typically differentiated on the basis of size, cells, SKBR3, MDA-MB-231, and HCC1954). These
origin (endosomal or cell membrane), markers, and con- characteristics included increased proliferation, migra-
tents (9 ). Exosomes are typically described as 30 –120 tion, and invasion, as well as increased angiogenesis,
nm, and microvesicles/ectosomes are often described as when these exosomes were applied to human endothelial
120 –1000 nm (10 ). Collectively these vesicles are cells (14 ). Additionally, regarding the TNBC cell line
termed extracellular vesicles (EVs). MDA-MB-231 and the noninvasive mammary epithelial
Exosomes/EVs have been described as a means of cell line HMLE, microRNA (miR)-10b was found to be
communication between tumor cells and other cell types, expressed at significantly higher concentrations in the
including those of the microenvironment and beyond, in TNBC cells (15 ) and, in turn, HMLE cell invasion was
breast, as well as other cancer types (11 ). Dysregulation promoted upon their exosomal uptake of MDA-MB-
in this cell-to-cell communication and thus undesirable 231– derived miR-10b. Similarly, MDA-MB-231–
cellular cross-talk is understood to contribute to cancer derived exosomal miR-105 was successfully transferred
development and progression. The multiroles of exo- to an endothelial cell line, HMVEC, in vitro, resulting in
somes/EVs in breast cancer are reviewed here and sum- impaired endothelial monolayer tight junctions, reduced
marized in Fig. 1. vessel sprouting, and increased migration (16 ). Advanc-
ing to in vivo studies, mice were pretreated with these
Exosomes in Invasion and Metastasis MDA-MB-231– derived, miR-105–loaded exosomes (or
PBS as control) by intravenous injection into the tail vein
The most common sites for breast cancer metastasis are and, following subsequent intracardiac injection of
to bone, brain, liver, and lung (12 ). A number of inter- MDA-MB-231 cells, lung and brain metastases were ob-
esting studies, using cell lines, preclinical in vivo studies, served in these mice (16 ). Additionally, MDA-MB-231–
and/or clinical samples, have started to unravel the role of derived exosomes have been shown to transfer miRNAs
exosomes/EVs in such breast cancer invasion and to MCF10A cells, causing miR-10b and miR-21 to be
metastasis. upregulated in the MCF10A cells. This resulted in cell
The first published study of exosomes in TNBC viability, cell proliferation, and colony-forming capacity
included the TNBC cell line Hs578T and its more ag- to be increased in a Dicer-dependent manner. Further-

1458 Clinical Chemistry 61:12 (2015)

Exosomes and Breast Cancer
Review

more, tumor formation occurred upon implantation of regulated in RNA from highly metastatic breast cancer
MCF10A cells into the mammary fat pads of the female tumor samples (n ⫽ 76) compared to normal breast tis-
athymic nu/nu mouse when coinjected with MDA-MB- sue (n ⫽ 34) (22 ).
231– derived exosomes (17 ). Conversely, coinjection of Fibroblast-derived exosomes have been shown to
MCF10A cells with these exosomes, but with Dicer an- play a role in increasing breast cancer metastasis and mo-
tibodies, resulted in reduced tumor growth. Because exo- tility via the Wnt pathway (23 ). As reviewed by Zardawi
somal Dicer protein concentrations have been found to et al. (24 ), the Wnt signaling pathway plays a role in cell
be increased in exosomes from the serum of breast cancer migration, cell adhesion, stem cell maintenance, tissue
patients (n ⫽ 11) compared to healthy controls (n ⫽ 8), patterning, and carcinogenesis. CD81⫹ exosomes were
this result suggests that exosomes may promote cancer found to be present in conditioned medium from mouse
development and progression through miRNA biogene- fibroblast L cells. Inhibition of breast cancer cell (MDA-
sis and through the oncogenic transformation of normal MB-231) motility was observed following siRNA (small
adjacent cells. Further research into exosomal-cell com- interfering RNA) knockdown of CD81 in L cells, there-
munication and miRNA transport is necessary to greater fore suggesting that the fibroblastic induction of breast
understand their roles in invasion and metastasis and to cancer cell motility is regulated by CD81. Following
their possible exploitation for TNBC diagnostics and/or coinjection of an orthotopic breast cancer mouse model
therapies. with MDA-MB-231 cells and CD81 knockdown L cells,
Breast cancer cell– derived exosomes, released under MDA-MB-231 cell metastasis was significantly sup-
hypoxic conditions, have been associated with increasing pressed. Breast cancer cell motility was found to be reg-
the invasive and metastatic potential of breast cancer cells ulated by L-cell–secreted CD81-positive exosomes. The
(18 ). In studies of human breast cancer cell lines mechanism of action was found to be dependent on au-
(MCF-7, MDA-MB-231, and MDA-MB-435) under tocrine Wnt–planar cell polarity signaling (23 ). Because
hypoxic conditions, expression of RAB22A [an Rab GT- the Wnt signaling pathway has been shown to be acti-
Pase, which is a membrane-bound protein and functions vated in TNBC patient tumors (n ⫽ 130) and Wnt/␤-
as a molecular switch, oscillating between its active and catenin signaling has been associated with a greater risk of
inactive states to integrate intracellular signaling and lung metastasis (25 ), it would be important to investigate
membrane trafficking events (19 )] was found to be reg- Wnt in cancer-associated fibroblast (CAF)-derived exo-
ulated through hypoxia-inducible factors that, in turn, somes that may be causally involved in these events. Ev-
increased the release of microvesicles. Conversely, idence to suggest CAF-derived exosomes may promote
shRNA (short hairpin RNA) knockdown of RAB22A in cancer cell motility is supported in a study in which
both MDA-MB-231 and MDA-MB-435 cells resulted in ADAM10-rich exosomes were found to promote the ac-
a decrease in microvesicle formation and thus a decrease tivation of oncogenic signaling (26 ). Here, the CAF-like
in their in vivo invasion and lung colonization (18 ). cell state was investigated to determine the role of the
RAB22A may thus have potential as a novel therapeutic tissue inhibitor of metalloproteinases (TIMP) family in
target for suppressing tumor cell invasion and metastasis. the maintenance of the extracellular matrix. TIMP
In keeping with these observations, King et al. (20 ) re- knockout fibroblasts were found to produce ADAM10-
ported hypoxic conditions to significantly increase the rich exosomes which, in turn, increased expression of
quantities of exosomes released from MDA-MB-231 and cancer stem cell (CSC) markers, including aldehyde de-
T47D breast cancer cell lines. Here, exosomal miRNA hydrogenase as well as integrin ␣6, and also increased
concentrations (miR-16, let7a, miR-21, and miR-210) cellular motility. Conversely, loss of the metalloprotei-
were investigated under normoxic and hypoxic condi- nase ADAM10 suppressed lung metastasis in the MDA-
tions and exosomal miR-210 was found to be signifi- MB-231 xenograft model. The clinical relevance of this
cantly upregulated under hypoxic conditions, suggesting study is supported by the fact that ADAM10 is signifi-
a role for this miRNA in promoting tumor progression in cantly upregulated in breast tumor stroma (n ⫽ 51) com-
response to hypoxic conditions (20 ). Advancing on this, pared to stroma from normal healthy breast reduction
after subcutaneous injection of MCC70-let-7a cells into tissue (n ⫽ 6) (26 ).
RAG2⫺/⫺ mice, let-7a miRNA was delivered to the cells
via epidermal growth factor receptor (EGFR)-targeting Stem Cells
exosomes. The successful delivery of let-7a resulted in
inhibition of tumor development in vivo (21 ). This Stem cells from mesenchyme, from fibroblasts, and
study suggests that nucleic acid therapeutics may be from cancer cells themselves—and their associated
transported via exosomes to target EGFR-expressing tu- exosomes—have been implicated in breast cancer.
mors and may also have antimetastatic potential. The Mesenchymal stem cells (MSCs) have homing capa-
clinical potential relevance of this observation is further bilities and can regenerate and differentiate into tissues
supported by the fact that let-7a is reported to be down- such as bone, cartilage, muscle, ligament, tendon, fibro-

Clinical Chemistry 61:12 (2015) 1459

Review

blasts, and adipose [as reviewed by Chamberlain et al. indicates that tumor-derived exosomes may contribute to
(27 )]. The roles of stem cell– derived exosomes in breast the conversion of ADSCs into tumor-associated myofi-
cancer progression and as potential therapeutics are be- broblasts, thus contributing to the progression of tumor
coming evident. Following treatment with human MSC- cells in the microenvironment (31 ).
derived exosomes, MCF-7 cells underwent migration as- CSCs are proposed to be involved in breast cancer
sessed by transwell assay, via mechanisms that involve the relapse (32 ) and, in preclinical studies, have been shown
Wnt signaling pathway and are dose dependent on the to play a role in spontaneous metastases in an orthotopic
exosomes added (28 ). EVs (termed exosomes by the au- breast cancer mouse model (xenotransplantation of pa-
thors) from MSCs have also been shown to suppress an- tient tumor samples into nonobese diabetic/SCID mice)
giogenesis in the highly metastatic, invasive, and tumor- (33 ). Following treatment of tumor-associated mam-
igenic murine 4T1 mammary carcinoma cell line. mary gland fibroblasts with breast cancer cell (MCF-7)–
Specifically, 4T1 cells were coincubated with murine derived exosomes, mRNA concentrations of CSC
bone marrow– derived MSC-derived exosomes, and activation markers, CD44, interleukin 6 (IL-6), and apo-
␣-amanitin was co-added to suppress transcriptional ac- lipoprotein E were all found to be significantly upregu-
tivation caused by the addition of exosomes. miR-16, lated (34 ). When treated with nuclear receptor agonists
assessed by quantitative reverse transcription–PCR, was (i.e., peroxisome proliferator activated receptor-␥ and
shown to be transferred from MSC-derived exosomes to retinoid X receptor), exosomes derived from hypoxic
4T1 cells and subsequently reduced vascular endothelial MCF-7 cells reduced mammosphere formation and
growth factor (VEGF) expression. BALB/c mice were Notch3 protein expression compared to normoxic con-
injected with 4T1 cells alone or 4T1 cells with MSC- ditions. Thus, it was proposed that peroxisome prolifera-
derived exosomes, and tumor growth was significantly tor activated receptor-␥ and retinoid X receptor agonists
inhibited upon coinjection of 4T1 cells and MSC- may have potential in targeting the CSC niche (34 ).
derived exosomes (29 ). Although substantial work has
yet to be done to further investigate this finding, these Apoptosis
studies suggest that MSC-derived exosomes may be use-
ful in a therapeutic setting as antiangiogenics or in the
Exosomes have also been implicated in evading apopto-
transfer of antiangiogenic miRNAs. It is noteworthy,
sis. For example, exosomes from 4T1 breast cancer cell
however, that these studies showed somewhat contrast-
mouse models were found to increase proliferation and
ing responses to exosomes from MSCs, i.e., stimulation
suppress apoptosis of CD133⫹ 4T1 cells, supporting the
of breast cancer cell migration (28 ) but reduced angio-
potential role of exosomes in tumor progression through
genesis and suppressed tumor progression (29 ), respec-
evading apoptosis (35 ). Other studies, including human
tively. Although there may be other contributing factors
for these results, we propose that these differing findings cells/clinical samples supporting a role for exosomes in
may, at least in part, be attributable to the former study evading apoptosis, are outlined below.
being of human breast cancer cells and exosomes from The superantigen staphylococcal enterotoxin B
human MSC cells for which the exosomes were isolated (SEB) induces T-cell activation and proliferation and is
using filtration and ultracentrifugation techniques and involved in Fas-mediated apoptosis (36, 37 ). When exo-
the latter study having included murine breast cancer somes from MDA-MB-231 cells were anchored with
cells and extracellular vesicles (isolated using ExoQuick- SEB (EXO/SEB) and cocultured with MDA-MB-231
TC) from murine MSCs. This highlights the relevance of cells or with white blood cells and embryonic kidney cells
activities by groups such as ME-HaD (European Net- as normal controls, coculture of EXO/SEB with MDA-
work on Microvesicles and Exosomes in Health and Dis- MB-231 cells resulted in decreased proliferation of the
ease) that are aimed at standardization of techniques for tumor cells. Conversely, EXO/SEB coculture with both
isolation, characterization, and analysis of EVs, including normal cell types resulted in no cytotoxic or antiprolif-
exosomes, so that true biological differences can be sepa- erative effects. This is suggested to be due to EXO di-
rated from technical differences (30 ). rectly influencing tumor cells, but not normal cells. In
Adipose tissue–derived stem cells (ADSCs), ob- the MDA-MB-231 cells, after 24 h coculture, caspase-3
tained from lipoaspirates from individuals undergoing and caspase-9 were significantly increased, indicating the
liposuction, were cocultured with exosomes derived from mechanism involved to be via activation of the mito-
breast cancer cell lines (MCF-7 or MDA-MB-231). Fol- chondrial apoptotic pathway (38 ).
lowing treatment with exosomes, ADSCs displayed a The role of S100 family genes, responsible for en-
myofibroblastic phenotypical change with functional coding calcium binding proteins (39 ), has been investi-
characteristics through induced or greatly increased ex- gated in exosomal studies and in the study of breast can-
pression of stromal cell–derived factor-1, CCL5 protein, cer progression. A number of S100 genes were found to
transforming growth factor-␤, and VEGF. This study be involved in breast cancer progression, with S100A11

1460 Clinical Chemistry 61:12 (2015)

Exosomes and Breast Cancer
Review

(S100 calcium binding protein A11)4 and S100A14 cer stages and corresponding exosomes. Overall, these
(S100 calcium binding protein A14) associated with pa- studies support a role for exosomal (as well as tissue-
tient outcome (40 ). The S100 protein hornerin, which based) survivin and its splice variants as diagnostic and
has been detected in exosomes (41 ), was found to be prognostic biomarkers with potential for their exploita-
expressed in breast cancer tissue, including mammary tion in a therapeutic setting (43 ).
epithelial cells and stromal cells, and found to undergo An in vitro study of miR-373–transfected MCF-7
proteolytic cleavage and differential subcellular compart- cells showed estrogen receptor expression to be down-
mentalization. Premalignant, malignant, and metastatic regulated and an inhibitory effect on apoptosis was ob-
MCF10A cells were developed by transfecting the pri- served. To add a clinical perspective, significantly in-
mary cell line with active H-Ras; cells were then selected creased concentrations of exosomal miR-373 have been
according to increased tumor growth from xenograft tu- reported in serum of TNBC patients (n ⫽ 168) com-
mors. Hornerin was found to be decreased in the more pared to serum from age-matched healthy controls (n ⫽
aggressive tumor tissue (invasive ductal carcinoma) com- 28). Although, again, further studies are necessary—
pared to invasive lobular carcinoma. Furthermore, in- ideally including in vitro, preclinical, and clinical studies
creased expression and fragmentation of hornerin was of each breast cancer subtype to truly determine the po-
observed in breast cancer cells following the induction of tential of exosomal miR-373—this link to increased
apoptosis/necrosis with H2O2. Hornerin, possibly trans- breast cancer aggressiveness suggests its potential as a di-
ported via exosomes, may play a role in promoting apo- agnostic biomarker with possible benefit for exploitation
ptosis and suppressing tumor progression (41 ). in a therapeutic approach (45 ).
The antiapoptotic protein survivin has been de-
tected within exosomes and found to be released in the Immune System
extracellular space via exosomes. Initially, exosomes de-
rived from the ovarian cancer cell line HeLa were found Many tumors develop immune evasion mechanisms to
to secrete survivin under basal conditions and this was survive. Such mechanisms include secretion of tumor
increased following proton irradiation stress, suggesting proteins, T-cell evasion, promotion of T regulatory cells,
the involvement of an exosomal pathway in the release of
and reduction of the expression of antigen-presenting
survivin in cancer cells (42 ). More recently, the use of
proteins (46, 47 ). Studies have shown that breast cancer
serum-based exosomal survivin splice variants as early
cell– derived exosomes can influence the immune system
diagnostic biomarkers has shown promise in breast can-
through interactions with T cells, dendritic cells (DCs),
cer. With the use of acetylcholinesterase activity assays,
macrophages, and T regulatory cells.
the amount of cancer exosomes was found to be signifi-
Natural killer (NK) cells, natural killer T (NKT)
cantly higher in breast cancer patient serum (n ⫽ 40)
than in control serum from female patients who had un- cells, CD8⫹ ␣␤ T cells, ␥␦ T cells, and macrophages all
dergone neoadjuvant treatment followed by surgery with express the NK group 2, member D (NKG2D) receptor
no recurrence (n ⫽ 10) (43 ). Considering the roles of that, upon ligand binding, activates NK-cell cytotoxic
survivin and its splice variants in cancer (44 ), for which killing and provides a costimulatory signal in T cells.
survivin is an inhibitor of apoptosis and survivin-⌬Ex3 Lymphocyte NKG2D receptor expression and CD8⫹ T
and survivin-2B have apparently retained and lost anti- cell cytotoxic ability have been reported to be signifi-
apoptotic potential, respectively, Western blot analysis cantly inhibited following coculture of peripheral blood
revealed an increase in antiapoptotic survivin and leukocytes with breast cancer cell (T47D)-derived exo-
survivin-⌬Ex3 protein in breast cancer patient serum– somes (48 ). Further studies based on these interesting
derived exosomes. Survivin and survivin-⌬Ex3 proteins initial results, including clinical samples, could be very
were also found to be increased in breast cancer tissue useful.
(n ⫽ 23) compared to control samples (female patients Advancing from these findings, investigators have
who had undergone neoadjuvant treatment followed by shown that bone marrow–derived precursor myeloid
surgery with no recurrence) (n ⫽ 10). Additionally, dif- cells take up tumor exosomes. This was shown by inject-
ferential expression of proapoptotic survivin-2B was evi- ing PKH67-labeled TS/A-cell (metastatic murine cell
dent in patient exosomes and tumor tissue compared to line)– derived exosomes into BALB/c mice for which
that of healthy controls. Specifically, low expression of subsequent FACS (fluorescence-activated cell sorter)
survivin-2B was found in the most aggressive breast can- analysis revealed the majority (94%) of PKH67-labeled
exosomes to be present in myeloid cells. In keeping with
this observation, MDA-MB-231 cell line– derived exo-
somes, cocultured with human CD14⫹ monocytes, were
4
Human genes: S100A11, S100 calcium binding protein A11; S100A14, S100 calcium found to inhibit DC differentiation through the induc-
binding protein A14; PTEN, phosphatase and tensin homolog. tion of IL-6 (49 ). These studies suggest that exosomes

Clinical Chemistry 61:12 (2015) 1461

Review

may therefore be important biomarkers and immuno- to ␥ irradiation) and, conversely, tumor-derived exo-
therapeutic targets in breast cancer. somes inhibited the release of exosomes from the normal
Exosomal transport of miRNAs has been shown to mammary epithelial cells, HMEC B42. A novel exo-
influence macrophages in the tumor microenvironment. somal release regulatory feedback pathway has thus been
The role of tumor-associated macrophages (TAMs) in proposed (58 ). Exosomes from HER2⫹ (human epider-
metastasis and cancer progression is still uncertain, al- mal growth factor receptor 2–positive) breast cancer cell
though they have been shown to be involved in tumor lines (SKBR3 and BT474) and the normal human mam-
angiogenesis in breast cancer patients (50 ). TAMs are mary epithelium 184A1 cell line were isolated to deter-
polarized to an immunosuppressive phenotype, through mine secretome profiling using 2-dimensional gel elec-
inhibitor of nuclear factor-␬B (NF-␬B) kinase subunit ␤ trophoresis and MALDI-TOF mass spectrometry (59 ).
(IKK␤)-mediated NF-␬B activation (51 ). The green tea In silico functional annotation using the Database for
component epicathecin gallate (EGCG) is reported to Annotation, Visualization and Integrated Discovery pre-
have antitumorigenic properties (52, 53 ). TAM infiltra- dicted exosomes derived from breast cancer cells to be
tion and M2 polarization were found to be suppressed in involved in energy metabolism, antigen processing, and
an in vivo and ex vivo EGCG-treated breast cancer study antigen presentation (59 ). Wet laboratory research into
(54 ). In studies aimed at elucidating the mechanism of the role of exosomes in tumor immunosurveillance and
events involved, miR-16 was found to be upregulated energy metabolism is warranted.
following EGCG treatment of 4T1 cells in vitro; miR-16 In a preclinical in vivo study of a 4T1 breast cancer
was transported to TAMs (derived from established mu- metastasis model, primary breast tumor– derived exo-
rine breast cancer) via tumor-derived exosomes, and so somes successfully communicated/interacted with im-
inhibited macrophage infiltration and polarization in the mune cells. Specifically, tumor-infiltrating leukocytes
tumor microenvironment (54 ). were cocultured with murine breast cancer 4T1 cell–
By microarray analysis, overexpression of miR-223, derived exosomes, and fibronectin was found to be suc-
miR-565, and miR-660 was evident in human monocyte– cessfully absorbed by/recruited into the exosomes (60 ).
derived macrophages compared to breast cancer cell When placed in vivo, tumor cell invasion was found to be
lines (SKBR3 and MDA-MB-231). IL-4 –activated enhanced through the regulation of CD25⫹ T regulatory
monocyte-derived macrophages were found to overex- cells and GR-1⫹ myeloid-derived suppressor cells, by the
press miR-223, but this was not seen in the breast cancer fibronectin that had been recruited into the exosomes
cells. miR-223 was thus advanced to further investigate (60 ). The cellular cross-talk evidenced in this study
its expression levels in breast cancer cells upon coculture should be further expanded to increase our understand-
with IL-4 –activated monocyte-derived macrophages. ing of the mechanisms of exosomal communication and
For this purpose, breast cancer cells were cocultured with protein sorting into exosomes.
TAM-derived exosomes and miR-223 was found to be
shuttled by the TAM exosomes, resulting in increased Drug Resistance
miR-223 in the cancer cells. miR-223 was subsequently
reported to promote the invasive potential of breast can- Drug resistance is a major obstacle in breast cancer treat-
cer cells (55 ). In agreement with that observation, a lower ment and exosomes/EVs are of major interest in drug
rate of overall survival was found in TNBC patients resistance studies. Stromal cells were found to initiate
whose tumors had high concentrations of infiltrating cross-talk with breast cancer cells (MDA-MB-231) via
TAMs (56 ). Although yet to be investigated, it is possible exosomes. Exosomes were found to be transferred from
that exosomal miR-223 from the TAMs may be playing stromal cells to breast cancer cells, thereby activating an-
an adverse role here. In a recent study, exosomes from tiviral RIG-I (retinoic acid-inducible gene 1 enzyme) sig-
breast cancer cell lines (MDA-MB-231 and MCF-7) naling and in parallel activating NOTCH3 pathways to
were reported to stimulate macrophages through the ac- regulate the expansion of therapy-resistant tumor-
tivation of NF-␬B via TLR2 (Toll-like receptor 2) (57 ). initiating cells. It has been proposed that this mechanism
This suggests bidirectional communication (exosomes of exosomal transfer is regulated through the stromal
from macrophages influencing cancer cells; exosomes cell–induced increase in RAB27B and the activation of
from cancer cells influencing macrophages), with the RIG-I signaling via the transfer of exosomal 5⬘tripohos-
possible exosomal alteration of partner cells adding fur- phate RNA. In further investigation of these findings,
ther to the complexity of breast cancer. MDA-MB-231 xenograft female nude mice were coin-
Exosomes from the hTERT (human telomerase re- jected with nontransformed MRC5 human diploid fi-
verse transcriptase)-immortalized human normal mam- broblasts (as stromal cells), and in these mice STAT1
mary epithelial cells, HMEC B42, inhibited the release of (signal transducer and activator of transcription 1) ex-
exosomes from its clonal breast cancer subline B42 clone pression was increased, with reduced cell death and in-
16 (which was developed by exposing HMEC B42 cells creased tumor growth evident in this model (61 ).

1462 Clinical Chemistry 61:12 (2015)

Exosomes and Breast Cancer
Review

Exosomal transport of P-glycoprotein (P-gp) has Targeted Delivery Systems

been described as a possible mechanism in exosome-
mediated drug resistance. Our research group initially Because trastuzumab resistance is a major obstacle in the
showed this in relation to prostate cancer (62 ) and, treatment of HER2-overexpresssing breast cancers, there
more recently, exosomes from docetaxel-resistant is substantial focus on the development of clinically ef-
MCF-7 cells have been shown to transfer drug resis- fective antitumor vaccines, with Hao et al. (68 ) provid-
tance to docetaxel-sensitive MCF-7 cells. The sug- ing a promising approach. Here the use of ovalbumin-
gested mechanism of resistance is via exosomal deliv- pulsed DC-released exosomes stimulated cytotoxic T
ery of P-gp, because P-gp concentrations are higher in lymphocyte (CTL) responses. In this way, a CD4⫹ T-
exosomes derived from drug-resistant cells than in cell– based vaccine (OVA-TEXO) was found to stimulate
drug-sensitive cells (63 ). long-term CTL memory (69 ). Further to this study,
Adriamycin (Adr) and docetaxel (Doc) have been transgenic HLA-A2/HER2 mice were used to study
shown to have therapeutic efficacy in breast cancer pa- HER2-specific CD8⫹ CTL responses and antitumor ac-
tients, but drug resistance limits their clinical benefits. tivity following administration of the HER2-TEXO vac-
Drug-sensitive (MCF-7/S) and drug-resistant human cine (70 ). The HER2-TEXO vaccine was found to be
breast cancer cell lines (MCF-7/Adr and MCF-7/Doc) effective at killing trastuzumab-resistant BT474A2 tumor
were used to investigate possible exosomal transfer of cells in vitro and to eradicate the BT474A2 tumor in vivo
resistance. On coculture of MCF-7/Adr exosomes with (70 ). This study, therefore, provides early evidence for a
MCF-7/S cells, low levels of proliferation and high levels novel therapeutic approach involving the use of exosomes
of drug resistance were observed. Similarly, this effect was for trastuzumab-resistant HER2-overexpressing breast
seen for MCF-7/Doc– derived exosomes. Microarray cancer cells.
analysis identified miRNA profiles for both MCF-7/ Reducing immunogenicity and toxicity is essential
Adr– and MCF-7/Doc–resistant cells in which possible for the development of new cancer drug delivery systems.
To study their potential efficacy as therapeutic delivery
common pathways of resistance were observed, suggest-
systems, exosomes were isolated from mouse immature
ing that the transfer of miRNAs plays a role in this exo-
DCs that were engineered to express the fused protein
somal transfer of resistance (64, 65 ). In relation to ta-
Lamp2b-␣v integrin-specific iRGD peptide. The iRGD
moxifen, exosomes from tamoxifen-resistant MCF-7
exosomes were purified and loaded with doxorubicin.
cells were found to promote proliferation of MCF-7
These exosomes were shown to have high affinity for ␣v
wild-type cells. Functional assays (cell viability, apopto-
integrin⫹ MDA-MB-231 breast cancer cells and signif-
sis, and colony formation) assessed the involvement of
icantly inhibited MDA-MB-231 and MCF-7 cell pro-
miR-221 and miR-222 in the transfer of this tamoxifen liferation. Using an MDA-MB-231 tumor-bearing
resistance, which was found to be significantly blocked nude mouse model, tumor growth was significantly
using anti–miR-221/anti–miR-222 (66 ). inhibited using iRGD– exosome– doxorubicin treat-
HER2 concentrations are significantly increased in ment compared to controls (PBS, iRGD exosomes,
exosomes from breast cancer cell lines, and SKBR3 and blank– exosomes– doxorubicin), with no doxorubicin-
BT474 and HER2⫹ exosomes have been shown to bind associated cytotoxic effects reported. This study pro-
to and interfere with activity of the monoclonal antibody vides insight into the natural, nanoscale delivery of
trastuzumab in in vitro studies (67 ). In a study of samples chemotherapeutic drugs via exosomes. Although sub-
from HER2-overexpressing breast cancer patients (n ⫽ stantial work has yet to be done to further investigate
22), higher exosome–trastuzumab-binding capacity was this approach, this drug delivery system may prove to
evident in advanced disease–stage serum (n ⫽ 11) com- be effective in a clinical setting (71 ).
pared to serum from the early-stage cohort (n ⫽ 11). In Mutations in the tumor suppressor gene phospha-
contrast, exosomes did not interfere with the in vitro tase and tensin homolog (PTEN) deleted on chromo-
antiproliferative activity of the small molecule lapatinib some 10 are increased in breast cancer patients (72 ). The
that targets EGFR as well as HER2 (67 ). It is possible PTEN C-terminus (PTEN-CT) stabilizes PTEN.
that, in vivo, HER2⫹ exosomes may modulate trastu- DU145 prostate cancer cell– derived exosomes have been
zumab availability and so adversely affect patient out- found to secrete PTEN and transfer PTEN to DU145Kd
come. Although in vivo studies and more extensive anal- cells i.e., DU145 cells with PTEN knock-down (73 ).
ysis of relevant patient samples are necessary to further Exosomal delivery of PTEN-CT from HEK293 cells into
investigate this activity, this study supports a broad range the murine mammary carcinoma cell line 4T1 caused a
of important roles held by exosomes in breast cancer and reduction in cell viability and impaired colony-forming
highlights their potential in a diagnostic and therapeutic abilities (74 ). Although this finding has yet to be fully
setting. investigated with breast cancer cells, this exosomal

Clinical Chemistry 61:12 (2015) 1463

Review

PTEN-CT delivery system may prove to be beneficial as of exosomes in breast cancer and their possible exploita-
a targeted therapeutic for PTEN-mutated breast cancers. tion in the development of future therapeutics in the
Although clinical trial studies of exosomes in breast interest of breast cancer patients.
cancer have not yet been reported, there is a precedent for
this approach in melanoma, colon, and lung (https://
clinicaltrials.gov NCT02310451, NCT01294072, and
Author Contributions: All authors confirmed they have contributed to
NCT01159288) cancers, giving hope of timely transla-
the intellectual content of this paper and have met the following 3 require-
tion of exosomes studies toward clinical utility in breast ments: (a) significant contributions to the conception and design, acquisi-
cancer. tion of data, or analysis and interpretation of data; (b) drafting or revising
the article for intellectual content; and (c) final approval of the published
Conclusion article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
This review brings together information on the roles of uscript submission, all authors completed the author disclosure form. Dis-
exosomes in breast cancer development, progression, closures and/or potential conflicts of interest:
drug resistance, and targeted drug delivery, including Employment or Leadership: None declared.
specific studies on their role in invasion, metastasis, stim- Consultant or Advisory Role: None declared.
ulation of stem cell populations, apoptosis, and modulat- Stock Ownership: None declared.
Honoraria: None declared.
ing cells of the immune system. These studies highlight Research Funding: W.M. Gallagher, the Irish Cancer Society
the growing interest, and growing understanding, of EVs Collaborative Cancer Research Centre BREAST-PREDICT Grant
in breast cancer. CCRC13GAL; L. O’Driscoll, the Irish Cancer Society Collaborative
Building on these interesting and exciting data, fur- Cancer Research Centre BREAST-PREDICT Grant CCRC13GAL,
ther research is now warranted, both to validate prelimi- H2020 Cooperation in Science and Technology, Microvesicles and
Exosomes in Health & Disease [ME-HaD; BM1202], and HEA
nary findings and to expand our knowledge in this area. PRTLI Cycle 5 funding of TBSI.
Although not yet exploited in a clinical setting, the exo- Expert Testimony: None declared.
some studies reviewed here demonstrate the many roles Patents: None declared.

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