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Seminars in Cell & Developmental Biology xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Review

Exosomes: Key mediators of metastasis and pre-metastatic niche

formation
Richard J. Lobb a,1 , Luize G. Lima a,1 , Andreas Möller a,b,∗
a
Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, QLD 4006, Australia
b
School of Medicine, University of Queensland, Brisbane, QLD 4072, Australia

a r t i c l e i n f o a b s t r a c t

Article history: While tumour cells are classically known to communicate via direct cell-to-cell contact and the secretion
Received 16 September 2016 of soluble protein-based factors such as cytokines and growth factors, alternative novel mechanisms that
Received in revised form promote tumour progression have recently emerged. Now, new critical components of the secretome
22 December 2016
thought to be involved in tumour progression are exosomes, small vesicles of endocytic origin that carry
Accepted 6 January 2017
a variety of bioactive molecules, including proteins, lipids, RNA, as well as DNA molecules. Cancer cell-
Available online xxx
derived exosomes have been shown to participate in crucial steps of metastatic spread of a primary
tumour, ranging from oncogenic reprogramming of malignant cells to formation of pre-metastatic niches.
Keywords:
Cancer
These effects are achieved through the mediation of intercellular cross-talk and subsequent modification
Extracellular vesicles of both local and distant microenvironments in an autocrine and paracrine fashion. Here, we summarise
Pre-metastatic niche the recent findings that implicate this non-canonical signalling within the tumour as a critical driver
Metastasis of metastatic disease progression, and discuss how understanding the molecular mechanisms involved
Exosomes in exosome-mediated metastasis is of great value for the development of new therapeutic strategies to
prevent cancer progression.
© 2017 Published by Elsevier Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. The role of exosomes in the primary tumour microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Exosomes and angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Oncogenic transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Exosomes and immune suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Exosome-related pro-metastatic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. The role of tumour-exosomes in pre-metastatic niche formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.2. Exosomes and vascular remodelling in pre-metastatic niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.3. Effects of cancer-derived exosomes on other non-immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.4. Impact of cancer-derived exosomes on immune cells in pre-metastatic niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.5. Modification of the metabolic environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.6. Determination of organotropic homing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00
3.7. Non-tumour-derived exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abbreviation: BM, bone marrow; BMDCs, bone marrow-derived cells; EVs, extracellular vesicles; FasL, fas ligand; FGF2, fibroblast growth factor 2; GM-CSF, granulocyte
macrophage colony-stimulating factor; HGF, hepatocyte growth factor; HIF-1, hypoxia-inducible factor 1; HUVECs, human umbilical vein endothelial cells; IBA-1, ionized
calcium-binding adapter molecule 1; IL-6, interleukin 6; MDSCs, myeloid-derived suppressor cells; MMP, matrix metalloproteinase; MVBs, multivesicular bodies; PGE2,
prostaglandin E2; PKM, pyruvate kinase; PlGF, placental growth factor; PTEN, Phosphatase and tensin homolog; TGF-␤, transforming growth factor beta; TLR, toll like
receptor; TNF-␣, tumour necrosis factor alpha; TRAIL, TNF-related apoptosis-inducing ligand; VEGF, vascular endothelial growth factor; VEGFR-1, vascular endothelial
growth factor receptor 1; VLA-4, very late antigen 4; ZO-1, zonula occludens.
∗ Corresponding author at: 300 Herston Road, Herston, QLD, 4006, Australia.
E-mail address: andreas.moller@qimrberghofer.edu.au (A. Möller).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.semcdb.2017.01.004
1084-9521/© 2017 Published by Elsevier Ltd.

Please cite this article in press as: R.J. Lobb, et al., Exosomes: Key mediators of metastasis and pre-metastatic niche formation, Semin
Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.01.004
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4. Clinical utility of exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction systemically modify distant organs to provide favourable microen-

vironments for the dissemination and outgrowth of metastases
Despite significant advancements in therapies, a large propor- [13–15]. These effects are achieved by the bioactive molecular
tion of cancer patients will ultimately pass away due to metastatic constituents of exosomes mentioned earlier, which are in turn
disease progression. It is therefore a key goal of cancer research determined by the cell of origin that the exosomes are secreted
efforts to further our understanding of metastatic disease progres- from [10,13–15]. For this very reason, exosomes have become a
sion and its dynamics. Based on recent research findings, it is clear valuable target in identifying novel cancer biomarkers that could
that metastatic disease is not a random process. That is, the seeding potentially diagnose cancer, predict patient outcome or response,
of cancer cells at secondary organs does occur in an organ-specific and understand, and perhaps prevent, cancer progression.
manner, depending on the cancer type, verifying the so called “seed Cancer cell-derived exosomes have been shown to significantly
and soil” hypothesis, which was originally proposed over 100 years contribute to different aspects of metastatic dissemination of a
ago by Stephen Paget [1,2]. This concept of metastatic growth speci- primary tumour, including invasion of the surrounding tissues,
ficity has since been validated experimentally and clinically [3], angiogenesis, modulation of immune responses, and formation of
and decisively shown that even when cancer cells can be found the pre-metastatic niche [16]. The roles exosomes have in pre-
in the vasculature of multiple organs, only selective sites consis- metastatic niche formation are diverse, and range from metabolic
tently develop metastatic tumour deposits [4]. It is now widely reprogramming, to recruitment of numerous immune and non-
accepted that the spread of cancer cells to secondary organs is immune stromal cells in order to facilitate metastatic outgrowth.
indeed promoted by the prior formation of a specialised environ- The aim of this review is to discuss the particular multi-faceted roles
ment at distant sites, termed the pre-metastatic niche. of these extracellular particles that impact both cancer cell spread
Originally, pre-metastatic niche formation was shown to consist from primary to secondary organs, resulting in the outgrowth of
of bone marrow-derived hematopoietic progenitor cells (BMDCs), macroscopic metastatic lesions (Fig. 1), and lastly, provide new
which accumulate at pre-metastatic sites in organs different to the insights into how this can be translated to clinical applications.
site of the primary tumour and before the arrival of any cancer cells
[5]. These BMDCs expressed VLA-4, and were recruited by tumour- 2. The role of exosomes in the primary tumour
derived secreted factors, which resulted in the accumulation of the microenvironment
VLA-4 ligand fibronectin at pre-metastatic organs [5]. Since then,
numerous studies have established that variable tumour-secreted 2.1. Exosomes and angiogenesis
factors are responsible for establishing a permissive secondary
organ through the modulation of a number of stromal cell-types [6]. A common process known to be involved in tumour progres-
However, these studies have largely focused on canonical intercel- sion is hypoxia. Hypoxia is the relative reduction in oxygen tension
lular communication based on classical cytokines, chemokines and and occurs in all solid tumours larger than 1 cm3 . This results
growth factors; yet, it is becoming evident non-canonical signalling from inadequate blood supply due to the aberrant microcirculation
pathways are also involved in pre-metastatic niche formation [7]. found in most tumours [17,18]. The microcirculation is essential
More recently, a new mechanism of intercellular communi- in normal tissues homeostasis, as it is responsible for balancing
cation via the secretion of extracellular vesicles (EVs) has been the supply of nutrients and removal of cellular waste products
established as an integral part of complex tumour-host interac- [19,20]. In the context of a solid tumour, neo-angiogenesis, or
tions [8]. Clinical interest in EVs has heightened due to the fact that the physiological process of new blood vessel growth, is neces-
continuing evidence supports the notion that EVs represent a new sary for continued tumour growth in response to hypoxia [19,20].
paradigm of intercellular communication [9]. EVs are constitutively Typically, solid tumours have been described to induce vascu-
secreted by most likely all different cell lineages in the body, and lar remodelling through the secretion of soluble factors, such as
carry diverse molecular contents, including proteins, lipids, RNA VEGF and PIGF. However, exosomes are also capable of being
(mRNA, miRNA, lncRNA, and other RNA molecules), as well as DNA key mediators between cancer cells and the surrounding vas-
molecules (dsDNA, ssDNA, mtDNA) [10]. There are a variety of EVs culature by eliciting pro-angiogenic responses [21,22]. Hypoxic
subtypes, and their accurate annotation is currently an ongoing, glioblastoma-derived exosomes are capable of inducing microvas-
contentious problem in the field [11]. There are clearly two dis- culature sprouting and vascularisation of xenografts models [22].
tinct subtypes of EVs that can be separated effectively through In response to this angiogenic phenotype, tumours displayed accel-
differential centrifugation: microvesicles are the larger subclass erated growth kinetics, demonstrating a role for exosomes during
(200–1000 nm) and sediment between 10,000 and 20,000g, while early tumour progression events. Hypoxia content changes in
smaller EVs known as exosomes exhibit a more homogenous size multiple myeloma derived exosomes also induce angiogenesis in
distribution of 30–150 nm, and sediment at 100,000g [9,11,12]. recipient endothelial cells [23]. Under hypoxia conditions, multiple
Exosomes are constitutively released by a variety of cell lineages, myeloma cells increase the secretion of miR-135b contained within
including cancer cells [10]. The biogenesis of exosomes is classically exosomes in order to promote an angiogenic response. Endothelial
described as originating from the endosomal compartment and are cells that received miR-135b-containing exosomes had signifi-
formed as intraluminal vesicles by inward budding of the limit- cantly increased HIF-1 alpha levels, as miR-135b binds the 3 -UTR of
ing membrane of late endosomes, forming multivescular bodies factor-inhibiting hypoxia-inducible factor-1 (FIH-1). This induced
(MVBs). The subsequent fusion of MVBs with the plasma membrane a hypoxic response and greatly accelerated angiogenesis [23]. In
allows the release of exosomes into the extracellular environment. further support of the role that exosomes play during angiogene-
This fusion and release into the extracellular milieu, allows can- sis, CD105+ vesicles from renal carcinoma cells have been shown
cer cells to utilise exosomes to modify local microenvironments, or to induce proliferation of HUVECs in vitro and in vivo [21]. These

Please cite this article in press as: R.J. Lobb, et al., Exosomes: Key mediators of metastasis and pre-metastatic niche formation, Semin
Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.01.004
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Fig. 1. Exosomal mechanisms of metastasis promotion. (a) Exosomes secreted by cancer cells contribute to the intercellular cross-talk and subsequent reprogramming of
the primary tumour microenvironment in an autocrine and paracrine fashion. Bioactive content of exosomes have been implicated in different aspects of primary tumour
progression, including angiogenesis, modulation of immune responses, and oncogenic reprogramming. (b) Moreover, exosomal factors are capable of mobilising bone
marrow-derived cells (BMDCs) to the primary tumour, which in turn facilitate the establishment of a supportive microenvironment. (c) This non-canonical signalling within
the primary tumour has also emerged as a critical driver of pre-metastatic niche formation. Tumour-derived exosomes can home to specific secondary organs and directly
modify the local microenvironment by interacting with distinct resident cell populations. A variety of factors contained within exosomes were shown to participate in a range
of biological processes, from modulation of immune and non-immune stromal cells to metabolic reprogramming. (d) Chemokines and other pro-inflammatory molecules
are also upregulated in pre-metastatic sites in response to exosome-mediated signalling, leading to the recruitment and activation of BMDCs. Altogether, exosome-induced
pre-metastatic niche modifications generate a microenvironment conducive to metastatic outgrowth (e), thereby facilitating cancer cell dissemination and colonisation of
distant tissues in an organotropic specific manner.

data provide novel insights into mechanisms of how tumours can
circumvent the clinical inhibition of angiogenic proteins (for exam-
ple through VEGF-neutralizing antibodies), and induce vascular
remodelling, allowing tumour-induced neo-angiogenesis through
exosomal transfer.
2.2. Oncogenic transfer
Tumours typically consist of numerous subclones that are genet-
ically and phenotypically different from one another [24]. It is
therefore possible that different subclones can transfer onco-
genic traits via exosomes to other cancer cells in the primary
tumour microenvironment. Indeed, glioma cells have been shown
to enhance tumourigenesis of recipient cells through the transfer
of mutant epidermal growth factor receptor (EGFRvIII) [25]. Recip-
ient cells were then shown to have a higher proliferation index and
were more resistant to apoptosis. Mutant KRAS can also be trans-
ferred via exosomes and increases the three-dimensional growth
of wild-type recipient colon cells [26]. Furthermore, metastatic
melanoma-derived exosomes are capable of inducing a metastatic
phenotype of non-metastatic melanoma recipient cells [27]. It
remains unclear whether this protein inheritance changes tumour
growth and metastasis in a permanent or an acute manner, yet it
provides interesting insights into how tumour cells can communi-
cate and promote tumour progression through exosomal transfer
of oncogenic proteins.
2.3. Exosomes and immune suppression
Rudolf Virchow was the first to observe leukocyte infiltration
into tumours in the 19th century [28]. Since then, experi-
mental and clinical investigations have demonstrated the close
association between inflammation and tumourigenesis [29,30].
However, inflammation can have paradoxical roles during tumour
progression, the outcome depending on the balance between
tumour-promoting and tumour-suppressing activities in the local
microenvironment [30]. Classically, chemokines and cytokines
secreted through canonical pathways have been thought of as
the master regulators of inflammatory processes in the tumour
microenvironment, yet it has recently emerged that exosomes add
another layer of complexity to cancer-associated inflammatory
reactions. Cancer cell-derived exosomes contain molecular con-
stituents that can induce immune cell dysfunction and contribute
to silencing of anti-tumour immune responses by several different
ways [31], either by directly altering immune cell function, or indi-
rectly through modifying non-immune cell stromal phenotypes.
Tumour-derived exosomes can suppress T cell immunity and
direct immune cells towards a tumour-promoting phenotype. The
effects on T cells are partly achieved through causing apopto-
sis of cytotoxic T-cells via the transfer of the death ligands FasL
and TRAIL [32,33]. Furthermore, exosomes are capable of editing
the immune composition of the primary tumour microenviron-
ment by recruiting and expanding myeloid-derived suppressor
cells (MDSCs). These cells have been identified in both humans and
mice as a heterogeneous population of immature myeloid cells,
capable of mediating immunosuppressive functions within the
tumour microenvironment [34]. In mice, MDSC subsets are char-
acterised by the expression of CD11b and Gr-1, while in humans
they are usually identified by a CD11b+ /CD33+ /HLA-DR- phenotype
[34]. Breast carcinoma-derived exosomes containing prostaglandin
E2 and TGF-␤ skew the development of myeloid cells toward
a MDSC phenotype, thus promoting tumour development [35].
Whether these and other cytokines/chemokines are indeed con-
tained in tumour-derived exosomes or are merely contaminants

Please cite this article in press as: R.J. Lobb, et al., Exosomes: Key mediators of metastasis and pre-metastatic niche formation, Semin
Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.01.004
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from either tumour or non-tumour cell sources, caused by the exo-
some isolation procedure, is however still a matter of debate [36].
Nonetheless, these data collectively suggest that exosomes play
a key role in deciding the tumour-promoting fate of infiltrating
immune cells.
Tumour-derived exosomes can further promote an immune-
suppressed tumour microenvironment through the expansion of
CD4+ /CD25+ regulatory T cells [37]. This is mechanistically linked
to the functional expression of CD39 and CD73 on exosomes, and
consequent ATP-dependent adenosine production in recipient cells
[38]. Melanoma exosomes also impair dendritic cell (DC) differ-
entiation from monocytes, and simultaneously convert these cells
toward an immune-suppressive phenotype [39]. In support of these
observations, an increased frequency of CD14+ /HLA-DR− myeloid
cells associated with a TGF-␤-mediated inhibitory activity on T
cells was found in the peripheral blood of melanoma patients [39].
Even though these studies have focused on the role of immune
cell uptake in the primary tumour microenvironment, it is inter-
esting to speculate that similar mechanisms could be happening at
pre-metastatic organs.
Finally, it seems that modulation of immune cells behaviour by
cancer-derived exosomes can also be triggered by other mecha-
nisms, such as transfer of functional mRNA between tumour and
host cells [40]. Transplanting glioma and lung carcinoma cells,
engineered to express Cre recombinase, into mice with a Cre-
lox background, the uptake of Cre mRNA-contaning exosomes by
CD11b+ Gr-1+ MDSCs present in the primary tumour microenvi-
ronment could be tracked [40]. It is noteworthy that recombined
MDSCs showed alterations in both their immunosuppressive phe-
notype and miRNA profile [40]. These observations suggest a
long-term, stable genetic reprogramming of myeloid cells by exo-
somes.
3. Exosome-related pro-metastatic mechanisms
In earlier studies, the majority of cancer research has focused
on the pathogenesis of the primary tumour. However, metastatic
disease is responsible for over 90% of cancer-related deaths, and
very few therapies have proven successful in the clinic for treat-
ing patients with metastatic deposits [41]. Therefore, we need
to understand the underlying mechanisms of metastasis in order
to generate novel, effective therapies for patients with advanced,
aggressive cancers. The metastatic process is composed of a number
of sequential events that tumour cells are required to accomplish
in order to successfully disseminate to secondary organs. Known
as the metastatic cascade, a series of steps involving local inva-
sion, survival and evasion of immune responses, intravasation into
the circulation, and extravasation at secondary organs are required
for the formation of macroscopic metastatic lesions. Importantly,
exosomes are capable of enhancing the systematic entry and
progression of cancer cells along the metastatic cascade [19,20],
highlighting the importance of understanding exosome biology
in metastatic disease progression. In the following paragraphs,
we will further examine the role of cancer-derived exosomes in
diverse mechanisms relating to metastasis, which can orchestrate
pre-metastatic niche establishment at secondary organs and con-
sequent metastatic outgrowth (Fig. 1).
3.1. The role of tumour-exosomes in pre-metastatic niche
formation
Changes at secondary organs prior to tumour cell metastasis
were first described to be caused by matrix metalloproteinase
9 (MMP-9), which is specifically induced in pre-metastatic lung
endothelial cells and macrophages by distant primary tumour cells
Please cite this article in press as: R.J. Lobb, et al., Exosomes: Key mediators of metastasis and pre-metastatic niche formation, Semin
Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.01.004
[42]. The term ‘pre-metastatic niche’ was coined later by demon-
strating that tumour-derived conditioned media alone is capable
of inducing migration of VEGFR-1-expressing BMDCs into organ-
specific pre-metastatic sites, as well as posterior formation of
BMDCs clusters that precede tumour cell arrival [5]. These clusters,
in turn, create a favourable microenvironment for incoming tumour
cells, in a mechanism dependent on the expression of fibronectin,
VLA-4, Id3, and MMP-9 [5]. Cell-free conditioned medium from
hypoxic breast cancers was also shown to drive the accumulation of
a specific subset of BMDCs, known as CD11b+ MDSCs, in the lungs of
immune-competent mice [43]. There, the formation of an immune
suppressed environment through the reduced cytotoxic capacity
of Natural Killer (NK) cells, in turn promote the metastatic out-
growth of both breast cancer and melanoma tumours [43]. Since
then, a variety of BMDC lineages, among other organ resident cell
lineages, have been shown to contribute to the generation of pre-
metastatic niches, and a large number of chemokines, cytokines
and growth factors have been connected to specific pre-metastatic
niche-related processes [44,45]. In recent times, exosomes have
been described to have unique and central functions during pre-
metastatic niche establishment and maintenance.
3.2. Exosomes and vascular remodelling in pre-metastatic niches
Modulation of vascular permeability, and stimulation of
neo-angiogenesis, are key steps during pre-metastatic niche estab-
lishment that favour the initial extravasation and subsequent
metastatic growth of tumour cells into secondary organs [45]. The
ability to modulate endothelial cell activity could also represent a
crucial element in the repertoire of pro-metastatic roles of tumour-
derived exosomes. Recently, the importance of extracellular miRNA
contained within exosomes in vascular remodelling has been
highlighted. Metastatic MDA-MB-231 breast cancer cells secrete
exosomes enriched for the miRNA miR-105 [46]. The presence of
this miRNA in breast cancer-derived exosomes increases metastasis
by facilitating endothelial cell barrier destruction through down-
regulation of the tight junction zonula occludens 1 (ZO-1) protein.
The reduction of ZO-1 by miR-105 increases endothelial barrier per-
meability and facilitates cancer cell migration in vitro and in vivo.
Perturbation of vascular cell barrier function is a key step in allow-
ing cancer cells to not only intravasate into the circulation, but also
extravasate at secondary sites to form metastatic lesions. Indeed,
exosomal miR-105 increased vascular permeability at secondary
organs, thereby increasing metastasis by facilitating colonisation
of new tissues [46].
In support of these observations, exosomes purified from the
supernatant of CD105+ renal cancer stem cells that induced vascu-
lar remodelling by increasing angiogenesis at the primary tumour,
also induced an angiogenic response at pre-metastatic sites [21].
CD105+ vesicles enhance lung metastasis in vivo due to the
uptake and parallel up-regulation of VEGF and MMP-2 in lung
endothelial cells [21], thereby demonstrating the importance of
vascular remodelling at pre-metastatic organs. Melanoma-derived
exosomes also accumulated at common sites of melanoma metas-
tasis, and subsequently caused vascular remodelling by enhancing
endothelial cell permeability and up-regulation of the expression of
many genes previously associated with a microenvironment con-
ducive to metastatic outgrowth [15].
Taken together, these results demonstrate that tumour exo-
somes exhibit a strong impact on endothelial cell function, which
could contribute to remodelling of the microenvironment in order
to promote tumour progression. It is noteworthy, however, that
induction of vascular permeability by tumour exosomes in dis-
tant organs, such as the lung, is not always accompanied by lung
parenchyma uptake or facilitation of metastasis [14]. These obser-
vations indicate that vascular remodelling alone is not sufficient
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to promote tumour outgrowth in secondary organs, although it is Exosomes derived from breast cancer cells were shown to be
regarded as a necessary step during the metastatic cascade. preferentially taken up by CD11b+ Gr-1+ BMDCs, which in turn
accumulated in the spleen and lung of exosome-treated mice [57].
Further in vitro assays demonstrated that exosomes impair the dif-
3.3. Effects of cancer-derived exosomes on other non-immune
ferentiation of GM-CSF-stimulated BM precursor cells into DCs, as
cells
well as their maturation, which is partially dependent on exosome-
induced IL-6 production [57]. A crucial role for the MyD88-TLR
Apart from endothelium modifications, tumour-derived exo-
signalling pathway in MDSCs activation has also been evaluated
somes may be capable of altering the metastatic niche by directly
[58]. After treatment with tumour exosomes, MyD88 knockout
targeting other non-immune stromal cells, including mesenchymal
mice displayed a less prominent accumulation of MDSCs in pre-
stem cells, fibroblasts and epithelial cells and, in this way, promote
metastatic lung and liver tissues when compared to wild-type
tumour-supportive processes through extracellular matrix (ECM)
animals [58]. This was associated with reduced tumour exosome-
remodelling and induction of angiogenesis. For example, exosomes
promoted lung metastasis, as well as with decreased TNF-a and
produced by human chronic lymphocytic leukemia cells were
IL-6 levels in the serum, and low CCL2 production in the lung
shown to be taken up by mesenchymal stem cells in vitro [47]. As a
[58]. Mechanistically, it seems that the heat shock protein HSP70
result, the cell proliferation of the recipient cells is increased, as is
is involved in tumour exosome-mediated MDSCs induction in
their migration and adhesion to tumour cells [47]. In addition, these
secondary organs, possibly by triggering p-Stat3 in MDSCs in a
recipient mesenchymal stem cells are associated with enhanced
TLR2-MyD88-dependent manner [61].
secretion of inflammatory cytokines that seemed to contribute
More recently, we have shown the tissue-specific immune-
to tumour cell survival [47]. In fact, TGF-␤-containing exosomes
suppressive effects of exosomes derived from breast cancer cells
obtained from various cancer cell lines (including mesothelioma,
with differing metastatic potential [62]. Fluorescently-labelled
prostate and gastric cancer cells) can drive distinct changes in
tumour exosomes injected into syngeneic mice accumulated pre-
primary fibroblasts and mesenchymal stem cells consistent with
dominantly in the lung and were preferentially taken up by CD11b+
their differentiation into a myofibroblast-like phenotype [48,49].
BMDCs, DCs and macrophages, regardless of the metastatic pheno-
This includes high production of FGF2, VEGF, HGF and MMPs, and
type of the cell of origin. Nevertheless, conditioning of tumour naïve
acquisition of both pro-angiogenic and tumour-promoting charac-
animals with exosomes derived from highly metastatic breast can-
teristics [50,51]. The cellular release of soluble factors, such as IL-6
cer cells induced a significant recruitment of MDSCs to lung and
by cholangiocarcinoma-derived exosome-treated BM mesenchy-
liver compared to non-metastatic isogenic exosomes, which was
mal stem cells, also altered tumour cell proliferation, which was
accompanied by a reduced frequency of NK cells [62]. The formation
partially dependent on IL-6R activation [52]. Finally, EMMPRIN,
of this immune-suppressed microenvironment was then associ-
an integral membrane protein shed on lung carcinoma-derived
ated with a higher metastatic burden in both secondary organs,
exosomes, was found to stimulate the production of MMPs by
highlighting that metastatic cancers are highly efficient at prepar-
fibroblasts, outlining another mechanism of tumour-stromal cell
ing a permissive microenvironment that enhances metastatic
interaction that could influence ECM remodelling in the tumour
disease progression [62].
microenvironment [53].
Strikingly, exosome uptake by BMDCs appears to permanently
Recently tumour exosomal small RNA has been demonstrated
alter cellular phenotype. This alteration was shown with a recon-
to be required for neutrophil recruitment to the pre-metastatic
stitution experiment of lethally irradiated control mice with stem
niche after activating resident epithelial cells [54]. This neutrophil
cells from exosome-treated mice that resulted in a significant
recruitment is mediated by the expression of toll like receptor
increase in the number and size of melanoma metastases, as well
(TLR) 3 in alveolar type II lung epithelial cells. Small RNA species
as in the number of metastasis-associated BMDCs [15]. A more
contained within exosomes stimulate activation of TLR3 in lung
detailed assessment of the composition of the bone marrow after
epithelial cells, thereby increasing chemokine production (CXCL1,
reconstitution showed an elevated frequency of pro-angiogenic
CXCL2, CXCL5, and CXCL12) and neutrophil recruitment to pre-
c-kit+ Tie2+ cells after exosome conditioning [15]. Compared to
metastatic sites. Neutrophil recruitment has been shown to be
exosomes derived from poorly metastatic melanoma cells, exo-
involved in cancer metastasis [55,56]. Subsequently, neutrophil
somes from highly metastatic cell lines expressed higher levels
recruitment initiates pro-metastatic inflammation and is related
of metastasis-related proteins, including the MET tyrosine kinase
to the expression of the key metastatic molecules S100A8 and
receptor. The transfer of MET was implicated in the pro-metastatic
S100A9 [54]. This reprogramming of the pre-metastatic niche by
changes of bone marrow progenitors, given the key role of MET
small RNAs in exosomes is conducive to metastatic invasion and
signalling in migration, and angiogenesis [63].
colonisation of secondary organs. These interactions of tumour-
Selective in vivo uptake of pancreatic cancer exosomes by Kupf-
derived exosomes with pre-metastatic niche cells, which in turn
fer cells was also shown to play a key role in liver pre-metastatic
recruit BMDCs indicates that pre-metastatic niche formation is not
niche formation [13]. Kupffer are specialised macrophages localised
simply a binary process, but rather a complicated orchestration of
to the liver, and the transfer of macrophage inhibitory factor (MIF)
signalling involving numerous cells.
from tumour-derived exosomes to these hepatic cells promoted
TFG-␤ production. This generated a fibrotic microenvironment
3.4. Impact of cancer-derived exosomes on immune cells in through increased fibronectin expression from hepatic stellate
pre-metastatic niches cells, as well as further arrest of BM-derived macrophages and neu-
trophils in the liver. These myriad changes ultimately enhanced
Among all the BMDCs subsets, CD11b+ MDSCs are the most metastatic burden, providing key insights into pancreatic cancer
commonly associated with tumour progression and pre-metastatic liver metastasis [13].
niche generation. Exosomes isolated from different types of tumour Additional studies are required to further our understanding
cells (including breast cancer [35,57–59], melanoma [58], thymoma of the effects of tumour exosomes on immune cells functions, as
[59], colon carcinoma [59] and multiple myeloma [60]) have been well as their exact contribution to pre-metastatic niche formation.
shown to modulate BMDCs behaviour, by either directly skewing However, these findings collectively indicate that tumour-derived
their differentiation into MDSCs (as aforementioned) or affecting exosomes promote metastasis by conditioning the BMDCs pheno-
BMDCs homing to pre-metastatic sites. type and/or directly interact with immune cell lineages present in

Please cite this article in press as: R.J. Lobb, et al., Exosomes: Key mediators of metastasis and pre-metastatic niche formation, Semin
Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.01.004
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the pre-metastatic niche. However, some questions still remain to
be addressed such as: (1) if similar observations can be extended to
all types of cancer; (2) which immune cell populations are precisely
affected by which subsets of circulating exosomes after their target
to different organs; (3) the alterations in tissue-specific immune
composition induced by cancer exosome accumulation; (4) which
exosomal content is responsible for driving the opposing effects
on anti-cancer immune regulation; and (5) the consequences of
exosomal content on immune surveillance during metastatic dis-
semination. In this way, expanding our knowledge about how
exosomes impact immune cells to alter their function and induce a
tumour-supportive environment may improve the efficacy of cur-
rent cancer immunotherapeutics, a highly promising novel class of
treatment.
3.5. Modification of the metabolic environment
In a somewhat unique aspect of pre-metastatic niche biol-
ogy, exosomal miRNA has also been demonstrated to reprogram
the metabolic environment of the pre-metastatic niche. Circulat-
ing miR-122 has been associated with metastasis in breast cancer
patients [64]. Interestingly overexpression of miR-122 in an exper-
imental breast cancer model reduced primary tumour growth,
yet enhanced metastasis [65]. miR-122 is contained within breast
cancer-derived exosomes and taken up by recipient cells in the
pre-metastatic niche of the brain and the lung in vivo [65]. Uptake
of miR-122 by recipient cells specifically reduces pyruvate kinase
(PKM) expression, and results in decreased GLUT1 and glucose
uptake. When miR-122 containing exosomes were injected into
mice, the expression of PKM and GLUT1 in the brain, and the
lung was reduced. Metabolic reprogramming of the pre-metastatic
niche enhanced metastasis, presumably due to the increased nutri-
ent availability for arriving tumour cells. Cancer cells typically
have a high energy demand, and this unique line of investigation
demonstrates that cancer cells can systemically suppress nutrient
utilisation of residing cells in secondary organs in order to favour
incoming cancer cells.
3.6. Determination of organotropic homing
Organotropic homing of cancer cells to specific organs is of sig-
nificant importance in metastatic research. This phenomenon has
largely been demonstrated with tumour-derived soluble factors.
That is, mice treated with melanoma-derived conditioned medium
redirected metastatic outgrowth of lung carcinoma cells to sites
typical of melanoma but not lung cancer metastasis [5]. This sug-
gested that tumour-derived factors could be capable of inducing
organotropic specific homing. In this context, exosomes have been
recently identified as drivers of organotropic metastatic spread,
through preferential accumulation at future metastatic sites. This
was first established with melanoma-derived exosomes which
accumulated in sentinel lymph nodes, whereas control liposomes
(man-made lipid-based nanoparticles) evenly spread to regional
and distant nodes [66]. Exosome localisation to these sites was
accompanied by several modifications in the microenvironment,
such as matrix deposition and vascular proliferation, and thereby
facilitated lymphatic melanoma metastasis, implicating exosomes
in not only pre-metastatic niche formation, but specific organ-
otropic modulation [66]. Organotropism of exosomes is supported
by the assessment of the biodistribution and molecular profiles of
exosomes obtained from several cancer cells with distinct organ-
otropism [14]. Exosomes accumulate in different tissues, however,
exosome localisation recapitulated the organ-specificity of secret-
ing cells [14]. More strikingly, exosomes obtained from lung-tropic
breast cancer cells could promote the dissemination of a bone-
Please cite this article in press as: R.J. Lobb, et al., Exosomes: Key mediators of metastasis and pre-metastatic niche formation, Semin
Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.01.004
tropic cancer-cell population into the lung, demonstrating their
ability to redirecting metastatic spread of tumour cells [14].
The organotropic nature of exosomes is largely attributed to a
specific repertoire of exosomal surface integrins that was found to
dictate exosome homing to distant organs, as well as uptake by dis-
tinct target cells, thereby promoting a favourable pre-metastatic
niche [14]. For instance, ␣6 ␤4 - and ␣6 ␤1 -expressing exosomes
preferentially interacted with fibroblasts and epithelial cells in the
lung, whereas ␣v␤5 integrin-enriched exosomes were preferen-
tially taken up by Kupffer cells within the liver microenvironment
[14]. Perhaps most interesting was the finding that integrins ␤4
and ␣v expression in exosomes isolated from the plasma of can-
cer patients could predict metastasis to lung and liver, respectively.
Interestingly, this finding suggests that circulating tumour-derived
exosomes can be utilised as biomarkers to determine organotropic
metastatic outgrowth [14]. While this work greatly extended our
understanding of the molecular mechanisms involved in organ-
specific metastasis, and corroborated the role of exosomes on the
formation of pre-metastatic niche as well as on tumour progression,
it also raised several questions. Bone-tropic exosomes, for exam-
ple, were unable to redirect lung-tropic cell metastasis, and also
displayed a limited integrin repertoire [14]. Thus, integrin profiles
alone are unlikely to account for the promotion of organotropic
metastasis in all types of cancer, suggesting that additional organ-
specific traits of exosomes will still need to be defined. Given these
data, it is apparent further investigation is required to determine
how these achievements can be translated to the clinical man-
agement of cancer patients, especially on the use of exosomal
molecular profiles in both cancer diagnostics and therapeutics.
3.7. Non-tumour-derived exosomes
Tumour-derived exosomes are certainly capable of promot-
ing metastasis, however it is also apparent that autocrine and
paracrine signalling at secondary microenvironments – indepen-
dent of cancer cells – are integral in the metastatic seeding and
outgrowth of cancer cells [67]. Recently, exosomes derived from
cells residing at secondary microenvironments have been shown
to be essential for metastatic outgrowth after extravasation [67]. In
particular, astrocyte-derived exosomes were shown to be respon-
sible for inducing PTEN loss in brain metastases. PTEN loss was
induced by astrocyte-derived exosomal miR-19a, and was essential
for metastatic outgrowth in the brain due to the reciprocal secre-
tion of CCL2 and recruitment of IBA1-expressing myeloid cells. This
specific response to the microenvironment of the brain enhanced
cancer cell proliferation and reduced apoptosis, highlighting the
importance of understanding not only tumour-derived exosomes,
but also exosomes derived from other cell types at secondary
organs.
4. Clinical utility of exosomes
The evident and rapid emergence of numerous publications
on extracellular vesicles has highlighted the importance of these
extracellular components as biomarkers of disease, particularly
cancer. There is currently a large unmet clinical need to develop
minimally-invasive prognostic or diagnostic markers for most can-
cer types. Given that exosomes contain a myriad of novel proteomic
and RNA biomarkers [68,69], which have important functions,
particularly in metastatic dissemination and pre-metastatic niche
formation, tumour-derived exosomes are being touted as the next
significant advancement in the biomarker quest [69]. However,
there are still several hurdles to overcome for retrospective and
prospective clinical investigations. Technical standardisation is of
the upmost importance for utilising exosome biomarkers [70]. We
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R.J. Lobb et al. / Seminars in Cell & Developmental Biology xxx (2017) xxx–xxx
have previously demonstrated that different isolation protocols
will deliver significantly different purities, and therefore, con-
tain various abundances of non-exosomal contaminants that may
confound subsequent analysis [12]. Given the disparate isolation
techniques currently being employed worldwide, a complete stan-
dardisation of isolation techniques is critical, particularly in order
to reproduce retrospective trials, and of course, prospective clin-
ical trials, to confirm the validity of the putative exosome-based
biomarker.
Even given these limitations, we are at an exciting stage of
biomarker discovery utilising exosomes. We have discussed the
integral role exosomes serve in tumour progression at the primary
tumour, but also how exosomes are capable of enhancing metas-
tasis and numerous steps of the metastatic cascade. Exosomes are
capable of these feats through the specific RNA and protein con-
tent they carry, and we are now in a unique position to hijack the
information carried by exosomes in order to understand, and per-
haps interfere with their “intended” purposes, to improve patient
outcome through diagnostic measures and superior clinical man-
agement.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the members of the Tumour
Microenvironment Laboratory for critically reading the manuscript.
These authors are supported by the National Health and Medical
Research Council Australia (NHMRC, APP1068510) and the Rio-
Tinto-Ride-To-Conquer-Cancer (6156).
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