International Journal of

Molecular Sciences

Review
Role of Extracellular Matrix in Development and
Cancer Progression
Cameron Walker 1,† , Elijah Mojares 1,† and Armando del Río Hernández 1, *
Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London,
London SW7 2AZ, UK; [email protected] (C.W.); [email protected] (E.M.)
* Correspondence: [email protected]; Tel.: +44 (0) 20-7594-5187
† These authors contributed equally to this work.




Received: 14 August 2018; Accepted: 28 September 2018; Published: 4 October 2018


Abstract: The immense diversity of extracellular matrix (ECM) proteins confers distinct biochemical
and biophysical properties that influence cell phenotype. The ECM is highly dynamic as it is
constantly deposited, remodelled, and degraded during development until maturity to maintain
tissue homeostasis. The ECM’s composition and organization are spatiotemporally regulated to control
cell behaviour and differentiation, but dysregulation of ECM dynamics leads to the development
of diseases such as cancer. The chemical cues presented by the ECM have been appreciated as
key drivers for both development and cancer progression. However, the mechanical forces present
due to the ECM have been largely ignored but recently recognized to play critical roles in disease
progression and malignant cell behaviour. Here, we review the ways in which biophysical forces of the
microenvironment influence biochemical regulation and cell phenotype during key stages of human
development and cancer progression.

Keywords: tumour microenvironment; cancer progression; extracellular matrix; matrix remodelling;

fibrosis

1. Introduction
The extracellular matrix (ECM) is most commonly defined as the non-cellular component of
tissue that provides both biochemical and essential structural support for its cellular constituents.
Rather than serving simply as an intercellular filling, the ECM is a physiologically active component
of living tissue, responsible for cell–cell communication, cell adhesion, and cell proliferation [1].
Fundamentally, the ECM is composed of and interlocking mesh of water, minerals, proteoglycans,
and fibrous proteins secreted by resident cells. However, every organ has a unique composition of
these elements to serve a particular tissue-specific purpose [1,2]. Indeed, this unique composition
arises through dynamic biophysical and biochemical feedback between cellular components and their
evolving microenvironment during tissue development [3,4]. For any specific tissue, components
of the ECM are created and arranged by resident cells in accordance with the needs of the tissue.
The production of essential fibrous proteins, such as collagen, elastin, and laminin are controlled by the
ECM and adapt during various stages of embryonic development and disease progression. As a highly
dynamic structure, the ECM is constantly undergoing a remodelling process, by which components
are degraded and modified, facilitated primarily by ECM proteinases [5,6]. The balance between
degradation and secretion of ECM, orchestrated by ECM-modifying cells, is responsible for tensional
homeostasis and the properties of each organ, such as elasticity and compressive/tensile strength.
In vitro, most animal cells are known to only maintain viability when adhered to a substrate [7].
In this regard, cells rely heavily on their sense of touch to survive by protruding, adhering, and
spatially interacting with the surrounding ECM. Various cellular growth factor receptors and adhesion

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Int. J. Mol. Sci. 2018, 19, 3028 2 of 31

molecules along the cell membrane, such as integrins, are responsible for the cell’s ability to adhere
and communicate with its environment [8,9]. Indeed, cells have been shown to transduce cues
from the ECM, such as spatial context and mechanical rigidity, to coordinate crucial morphological
organization and signalling events through regulation of gene transcription. This process in which a
cell converts external mechanical stimuli into a downstream intracellular chemical signal is known as
mechanotransduction [10]. The sensitivity by which cells respond to biophysical and biochemical cues
of the ECM demonstrates the importance of tissue homeostasis in the maintenance of healthy resident
cells. Accordingly, dysregulation of ECM remodelling has been shown to contribute significantly to
cell fate through various fibrotic conditions, characterized by excess ECM deposition and increased
rigidity [11]. Due to increased interstitial pressure, unresolved loss of tissue homeostasis has been
linked to an elevated risk of various conditions, such as osteoarthritis, cardiovascular disease, and
cancer [11]. In this review, we will discuss the role of the ECM in critical physiological processes, such
as tissue development and cancer, and some potential targets for therapeutic intervention.

2. Primary Components of the Extracellular Matrix (ECM)

The ECM is composed of various proteins that give rise to different structures and properties
that exist within it. The main components of the ECM include collagen, proteoglycans, laminin, and
fibronectin. Even among these ECM components, there are subtypes that further specify their function
in the overall structure and properties of the ECM. As structure dictates function, different subtypes
and combinations of ECM molecules confer different functions that are essential for the whole body
to function.

2.1. Collagen as the Basis of ECM Architecture

Collagen is the most significant component of the ECM and the most abundant protein in
human tissue, with 28 unique subtypes discovered [12–15]. Each type is composed of homotrimers
or heterotrimers of left handed helical α chains that are twisted to form a right handed triple helix
structure [13,16]. The collagen superfamily is a large group of proteins that contain the Gly-X-Y motif,
where X and Y are usually either proline or hydroxyproline [16,17]. Despite the large amounts of
bulky proline, the right-hand helical structure is stabilized by the small glycine, interchained hydrogen
bonds, and electrostatic interactions involving lysine and aspartate [17,18]. Fibrillar collagens form
fibrous structures often found in tendons, cartilage, skin, and cornea [13,14]. Each collagen fibre is
made up of several subtypes of collagen in response to its tissue location. The most abundant type of
fibrillar collagen, type I collagen, and can be found in connective tissues ranging from skin and bone
to tendon and cornea [19]. Collagen I is involved heavily in processes such as a wound repair and
organ development.
All fibrillar collagens are first produced as precursors. The α chains are assembled together
in the rough endoplasmic reticulum to form the triple helical structure. Proline and lysine are
hydroxylated and the molecule is glycosylated to initiate the formation of the triple helical structure [20].
The procollagen is then brought to the Golgi apparatus where it is prepared for cellular export.
Processing of the procollagen happens either during or after secretion in the ECM [21–24]. The C
terminal propeptide is cleaved off by specific matrix metalloproteinases (MMPs) and if it is not
removed, it leads to high solubility of collagen that prevents it from forming fibrils [25]. For collagen
types I, II, and III, the N-propeptides are cleaved off, while for type V, XI, and other fibrillar collagens,
the N-propeptides remain (Figure 1A). This modifies the shape and diameter of the fibril without
affecting fibril formation [15,25–27]. The N-propeptides of type V and XI collagens protrude from
the gaps between collagen molecules to prevent lateral growth via steric hindrance and charge
interactions [25,26]. Type V and XI collagens are currently believed to be responsible for nucleating
and modulating the fibril formation of collagen [25,26]. It has been shown that the deletion of collagen
V in mice leads to failure of fibril assembly despite its low amounts in the total collagen content in
most tissues [28].
Int. J. Mol. Sci. 2018, 19, 3028 3 of 31

Once the microfibrils are formed, these may bind with other microfibrils so that they will grow
into larger fibres. This process is mediated by other ECM proteins (Figure 1C) [29]. Small leucine
rich proteoglycans (SLRPs) such as decorin and biglycan have collagen binding motifs allowing them
to modulate fibre growth, size, morphology, and content [15,29,30]. Another subfamily of collagen
are fibril-associated collagens with interrupted helices (FACIT) that do not form fibrils themselves
but are associated with the surface of collagen microfibrils [13]. Their primary function is to mediate
the formation of a higher-order structure via binding with other extracellular matrix proteins such as
SLRPs and proteoglycans [26,31]. The supramolecular assembly of collagen is further stabilized by
lysyl oxidase (LOX), which leads to overall enhanced mechanical properties. The N terminal and C
terminal ends of individual collagen molecules are covalently cross linked by LOX both within and
between microfibres, contributing to the great tensile strength of collagen [31,32].
In addition to fibrillar and FACIT collagens, there also exist network forming collagens such as
type IV, VIII, and X. These are found in the basal lamina of basement membranes (Figure 1B) [13].
Collagen IV forms a tetramer through their 7S N-terminal domain. Each of these collagen IV molecules
is bound to another collagen IV molecule via their C-terminal NC1 domain of each α-chain, forming
a hexamer [13]. These two domains of collagen IV allow it to form a stable collagen network that
separates the basal lamina from the interstitial stroma [33]. Other ECM proteins such as laminin,
nidogen, and perlecan can be found in the basal lamina that strengthens this barrier to effectively
maintain the organization of the cells in the body (Figure 1B) [33,34].
Although different types of collagen are able to build various types of supramolecular structures
that form the basis of the architecture of the ECM, the contribution of other ECM proteins such as
proteoglycans, laminins, and fibronectin cannot be ignored. They largely influence the chemical and
physical properties of the extracellular matrix such as through their growth factor binding motifs and
innate chemical properties. Furthermore, they also serve as connectors between the cells and the ECM.

2.2. Proteoglycans as Functional Modifiers of the ECM

Proteoglycans are characterized as proteins that have glycosaminoglycans (GAGs) covalently
bonded to them. These GAGs are long chains of negatively charged disaccharide repeats that can either
be heparin sulphate, chondroitin/dermatan sulphate, hyaluronan, or keratin sulphate. Due to the
negative charge of these GAGs, proteoglycans are able to sequester water and cations, which gives them
their space-filling and lubrication functions [35]. For the purpose of this review, only transmembrane
proteoglycans and those found in the pericellular and extracellular space will be discussed.
Of the 13 transmembrane proteoglycans, four of them are syndecans, proteins thought to act as
co-receptors [35]. Syndecans have an intracellular domain, transmembrane domain, and ectodomain
(Figure 1A). The GAGs, typically heparan sulphates, are found attached to the ectodomain, which can
be shed through the action of MMPs [35,36]. The ectodomain of syndecans is intrinsically disordered,
which allows it to interact with a wide variety of molecules to perform a broad range of biological
functions (Box 1) [35]. Some of its functions involve binding to growth factors and morphogens,
facilitating exosome uptake, and being co-receptors of receptor tyrosine kinases [36–39].
One of the proteoglycans found in the pericellular area of the basement membrane is perlecan.
As a large heparan sulfate proteoglycan (HSPG), perlecan has multiple domains, each with different
binding sites and functions (Figure 1A) [40]. These heparan sulfates can bind to a variety of molecules
such as growth factors, growth factor receptors, collagen, and other ECM proteins. In the basement
membrane, perlecan binds and links collagen IV, nidogen, and laminin in order to further strengthen
the basement lamina (Figure 1B) [33,34,41].
Proteoglycans found in the extracellular space are classified into hyalectans and SLRPs.
The structure of hyalectans are identical: the hyaluronic acid binding N terminal and lectin binding
C terminal with GAGs are attached between the N and C terminal ends (Figure 1A). Hyalectans are
encoded by 4 distinct genes: aggrecan, versican, neurocan, and brevican [35]. Aggrecan is found mostly
in bone cartilage and the brain while neurocan and brevican are found in the central nervous system.
Int. J. Mol. Sci. 2018, 19, 3028 4 of 31

On the other hand, versican is found in the ECM of almost all tissues and organs [42]. They can serve as
molecular bridges between the cell surface and the extracellular matrix [35]. Versican has been shown
to bind to collagen type I and fibronectin, which are both substrates of integrins [43]. The binding of
versican to fibronectin’s RGD motif leads to loss of cell adhesion as it sequesters fibronectin from the
cell’s integrins [42,43].
SLRPs make up the largest family of proteoglycans due to its 18 distinct gene products each with
multiple splice variants and processed forms [35]. These proteins have a relatively short protein core
with a central region dominated by leucine-rich repeats (LRRs). They are expressed in the ECM during
development of various tissue types, suggesting their critical involvement in directing organ size and
shape during embryonic development and homeostasis [44,45]. Decorin and biglycan are SLRPs that
have collagen-binding motifs and regulate collagen fibre assembly along with other proteoglycans
(Figure 1C) [46].
In summary, proteoglycans vary in form and structure that confer different functions in the ECM.
They are integral in the maintenance of a healthy ECM without which would lead to a non-functional
ECM and a collapse of its structure.

Box 1. Sensing the extracellular matrix’s (ECM) mechanical properties.

The ECM is sensed by the cell through transmembrane proteins such as integrins and syndecans
and other glycoproteins. Integrins are one of the most versatile transmembrane proteins as various
heterodimer combinations allows it to bind to fibronectin, laminin, and collagen [47]. Integrins themselves
are mechanosensors. Stretching has been shown to increase integrin binding to the ECM via conversion of
integrins to its high-affinity state in smooth muscle cells and fibroblasts [48]. Integrins also experience a
conformational change in their cytoplasmic domains, allowing it to activate several signalling pathways such
as mitogen-activated protein (MAP) kinases and Rho GTPases [49–51]. Syndecans can also bind to fibronectin,
resulting in a synergy between integrin and syndecan to activate signalling cascades through focal adhesion
kinase (FAK) and subsequent focal adhesion complex stabilization [52]. There are other receptors for other ECM
components such as CD44 for hyaluronan, 67 kDa laminin receptor for laminin, and discoidin domain receptors
(DDRs) for collagen [53–57].
Integrins and syndecans activate various pathways such as the MAPK and Rac1/RhoA pathways.
The selective activation of these pathways leads to context-dependent regulation of cell survival, growth,
proliferation, motility, spreading, or migration [52,58]. Integrins are connected to the actin cytoskeleton through
vinculins, talins, and other scaffold proteins while syndecans are connected to the microfilaments through
syntenin and through the actin cytoskeleton via a-actinin [52,58–61]. The adhesion complexes formed by
integrins and syndecans have been found to be mechanosensitive [9,62]. The intracellular signalling and
mechanotransduction through these receptors is still an active field of research. Much is still to be discovered
about the pathways that facilitate ECM mediated cellular responses.

2.3. Connecting the Cell to the ECM through Laminin

Laminins are trimeric glycoproteins consisting of α, β, and γ chains that are often found in the
basal lamina or some mesenchymal compartments [15]. The 12 mammalian α, β, and γ chains can
theoretically create 60 unique laminins but only 16 combinations have been observed so far [34,63].
The α chains vary in size from 200 and 400 kDa while β and γ chains have sizes from 120 to 200 kDa.
A trimer can then have a size varying from 400 to 800 kDa [63]. During rotary shadowing electron
microscopy, laminins look like cross-shaped molecules [34,64,65]. The three chains form an α-helical
coiled coil structure that forms the long arm of the cross while the three short arms are composed of
one chain each (Figure 1A) [34]. At the end of the long arm are 5 laminin G-like (LG) domains from
the α chain that serve as attachment sites for the cell. Integrins, dystroglycan, Lutheran glycoprotein,
or sulfated glycolipids bind to these LG domains [63]. At the end of each short arm are laminin
N-terminal (LN) domains that are important for laminin polymerization and basement membrane
assembly (Figure 1B) [34].
Laminins have cell type-specific functions such as adhesion, differentiation, migration, phenotype
maintenance, and apoptotic resistance [63]. Through binding of integrins, laminins are able to create a
dynamic link between the cell and the ECM (Box 1) [63]. Unique heterotrimeric laminins will have
Int. J. Mol. Sci. 2018, 19, 3028 5 of 31

unique integrin heterodimers binding partners to allow the induction of signalling pathways and
organization of intracellular cytoskeleton [63,66]. Collagen IV deposition in the basement membrane
is seen as the maturation of the basement membrane that is essential for structural stability later in
development [34,67]. However, the exact mechanism by which laminins bind to collagen IV remains
unclear. Initial studies indicated that nidogen binds to laminin through the LE domains of the γ1 chain
and collagen IV, thus serving as an intermediary between the two networks found in the basement
membrane. However, recent research has indicated that nidogen might not be the major bridge in
connecting laminins and collagen IV [34]. It has been observed that the interaction between laminins
and collagen IV is directly mediated by heparan sulfates [68]. Perlecan was thought to mediate
this function, but genetic ablation of perlecan in mice did not result in collagen IV depletion [34,67].
It has since been postulated that agrin, another pericellular HSPG, serves as a compensating candidate.
In this model, both perlecan and agrin would bind to the nidogen containing laminin network and
to the collagen IV’s 7S and NC1 domains (Figure 1B) [69,70]. Laminins serve crucial roles in both
basement membrane assembly and ECM–cell interactions. Recent studies have indicated that basement
membrane assembly is initialized through laminin polymerization [53–58]. Indeed, genetic ablation of
either β1 or γ1 chains proved to be lethal due to the resultant failure of basement membrane assembly.
While collagen, proteoglycans, and hyaluronic acid comprise the major structural component of the
ECM, laminins are one of the molecules that bridge the interaction gap between the cells and the
ECM [15].

2.4. Fibronectin as the Mechanosensitive Connection Between the Cell and ECM
Fibronectin is a multi-domain protein that interacts with the various previously described
ECM components to connect the cell to the ECM [15]. It is encoded by a single gene, but it has
20 isoforms in humans as a result of alternative splicing of the mRNA [71,72]. Similar to collagen,
fibronectin forms a fibrillar network in the ECM (Figure 1C) [71]. Fibronectin naturally exists as
a dimer outside the cell, mediated by the two cysteine disulfide bonds, which is crucial for its
ability to assemble in a fibrillar fashion (Figure 1A) [71,73]. Fibronectin matrix assembly is mediated
by selective binding to α5β1 integrins through an RGD binding motif and a synergy site on the
fibronectin molecule [59,62]. Through these integrins, the compact and soluble secreted fibronectin
is unfolded revealing cryptic binding sites for other fibronectin molecules to form the fibronectin
fibrillar network (Figure 1C) [1,71,74]. Anti-integrin and anti-fibronectin antibodies have been shown
to prevent fibronectin fibril formation [71,75,76]. Fibronectin binding induces integrin clustering that
provides local high concentrations of fibronectin at the cell surface. This phenomenon promotes
fibronectin–fibronectin interactions through the N terminal assembly domains of each molecule [71].
Once fibronectin is tethered to the cell surface by integrins, the actin cytoskeleton can pull onto
fibronectin molecules to change its conformation [71,72]. This will affect the C terminal regions of
fibronectin, revealing cryptic binding sites for fibronectin, heparan sulfates, heparin, collagen, and
other ECM proteins [77–81]. It is through strong non-covalent protein-protein interactions that the
fibronectin network matures and becomes insoluble, although other ECM proteins may mediate
mature lateral interactions between fibrils [71]. These interactions stabilize the relatively weak binding
sites at individual sites. However, the turnover of the fibronectin matrix is still largely unexplored [71].
Due to fibronectin’s multiple binding sites for other ECM proteins, it has been implicated in various
functions, including a role in collagen type I assembly. It has been shown that in the absence of fibronectin,
collagen fibrils do not accumulate, suggesting a role for fibronectin in collagen assembly [82,83]. However,
this relationship may prove reciprocal as recent studies have also implicated that collagen has a role in
enhancing fibronectin assembly [71,84,85].
Int. J. Mol. Sci. 2018, 19, 3028 6 of 31
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 30

Figure 1. Unique ECM molecules and their organization in the basement membrane and interstitial
Figure 1. Unique ECM molecules and their organization in the basement membrane and interstitial
stroma. Panel A (top) shows the unique components of the extracellular matrix. Panel B and C
stroma. Panel A (top) shows the unique components of the extracellular matrix. Panel B and C (middle)
(middle)
shows how showsthesehow these collagens,
different different collagens, proteoglycans,
proteoglycans, laminins, andlaminins, and fibronectin
fibronectin are organized
are organized within the
within the basement membrane (B) and interstitial ECM (C). A breast acinus
basement membrane (B) and interstitial ECM (C). A breast acinus with epithelial cells is surrounded with epithelial cells is
surrounded by myoepithelial cells and the basement membrane. In the
by myoepithelial cells and the basement membrane. In the basement membrane, the laminin is bound basement membrane, the
laminin is bound to the cell and forms a network through its long arms. It is
to the cell and forms a network through its long arms. It is then connected to the collagen IV network then connected to the
collagen IV network
through nidogen through nidogen
and proteoglycans suchand proteoglycans
as perlecan such
and agrin. as perlecan
Outside and agrin.
the basement Outsideis the
membrane the
basement
interstitialmembrane
ECM where is fibroblasts
the interstitial
thatECM where
produce andfibroblasts
remodel the thatECM
produce
can be and remodel
found. theinterstitial
In the ECM can
be found.collagen
stroma, In the interstitial
fibres are stroma,
made up collagen fibres
of fibrils are made
composed of up of fibrils
collagen composed
I and collagenofV.collagen I and
The different
collagen V. The different proteoglycans, such as decorin, biglycan, and hyalectans,
proteoglycans, such as decorin, biglycan, and hyalectans, holds the fibrils together to form a collagen holds the fibrils
together to form a collagen
fibre. Fibronectin is boundfibre.
to the Fibronectin is boundand
cell via integrins to the cell via integrins
syndecans. and syndecans.
Once fibronectin Once
is unfolded,
fibronectin is unfolded,
it reveals cryptic binding it sites
reveals
forcryptic
heparan binding
sulfatesites for heparan(HSPGs)
proteoglycans sulfate proteoglycans (HSPGs) and
and collagen. Modified and
collagen.
combinedModified and Mouw
figures from combined figures
et al. fromand
2014 [15] Mouw et al. 2014
Hohenester and[15] and Hohenester
Yurchenco and Yurchenco
2013 [34].
2013 [34].
Int. J. Mol. Sci. 2018, 19, 3028 7 of 31

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 30

3. Function of ECM
3. Function of ECM
The plethora of unique ECM molecules serves several functions that influence biochemical and
biophysical processes
The plethora of unique in theECMcell simultaneously
molecules serves(Figure 2). Whilethat
several functions theinfluence
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for many years
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for
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the functions and phenotypes of cells has clearly emerged in the last two decades. The ECM
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and movement decades.
of cells [86].The ECM
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is emphasized
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the basement of cells [86]. Thisthat
membrane is emphasized
serves as ain the complex
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[6,87]. In addition to as a barrier between
structural integrityepithelial cells
and anchorage,
and the interstitial stroma [6,87]. In addition to structural integrity and anchorage, the ECM
the ECM components have several binding sites for growth factors, controlling their release and
componentstohave
presentation several
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This is especially factors,
important controlling theirasrelease
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gradients [88]. This is especially
Finally, the ECMimportant
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the cells, which gradients
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[88]. Finally, the ECM transmits mechanical signals to the cells, which
intracellular signalling pathways and cytoskeletal machinery [89]. Indeed, the ECM serves several activates several intracellular
signalling
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here weand cytoskeletal
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of the ECM the ECM
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of development functions and
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the westemreview the function of the ECM in the context of development and maintenance of the stem
cell niche.
cell The
niche.
function of the ECM is best described in the context of development. The development of a
mammalian The function
embryo of from
the ECM is best
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spatiotemporal
that involves carefully controlled mechanisms. In such a relatively rapid
composition, amount, and characteristics of the ECM must be tightly regulated. Several studies have process, the spatiotemporal
composition, amount, and characteristics of the ECM must be tightly regulated. Several studies have
shown that mutated ECM components lead to birth defects or embryonic lethality, which emphasizes
shown that mutated ECM components lead to birth defects or embryonic lethality, which emphasizes
its role in development [2,87]. The geometry, rigidity, and other physical properties of the ECM are
its role in development [2,87]. The geometry, rigidity, and other physical properties of the ECM are
sensed by the cells and ultimately direct their differentiation and the complex spatial and structural
sensed by the cells and ultimately direct their differentiation and the complex spatial and structural
arrangements they form in tissues (Box 1).
arrangements they form in tissues (Box 1).

Figure2.2.Functions
Figure Functions ofof the
the ECM.
ECM. The
The ECM
ECMserves
servesas
asaapoint
pointofofanchorage
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cells is essential
that is essential
for maintaining tissue polarity and asymmetric stem cell division. Depending
for maintaining tissue polarity and asymmetric stem cell division. Depending on the context, on the context, it can
it can
impede or facilitate migration. It can sequester growth factors and prevent its free diffusion.
impede or facilitate migration. It can sequester growth factors and prevent its free diffusion. Other Other
ECMcomponents
ECM components can can bind
bind growth
growth factors
factorsandandcan
canserve
serveasasco-receptors
co-receptorsoror
signal
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which
help determine the direction of cell-cell communication. Through the action of metalloproteinases
help determine the direction of cell-cell communication. Through the action of metalloproteinases
(MMPs),fragments
(MMPs), fragmentsof of the
the ECM
ECM can
can also
also influence
influencecell
cellbehaviour.
behaviour.TheThephysical
physicalproperties
properties of of
thethe
ECM ECM
can be sensed by focal adhesion complexes, which lead to a variety of changes in cell phenotype such
can be sensed by focal adhesion complexes, which lead to a variety of changes in cell phenotype such as
as reorganization of the 3D genome. Figure modified and adopted from Lu, Weaver, and Werb 2012
reorganization of the 3D genome. Figure modified and adopted from Lu, Weaver, and Werb 2012 [90].
[90].
Int. J. Mol. Sci. 2018, 19, 3028 8 of 31

3.1. ECM as Tracks for Migration and Proliferation

Migration of cells is essential for tissue development and can be best illustrated by neural crest
cells, which migrate from the periphery of the neural tube to different parts of the embryo to form
parts of the heart, spinal nerve, skin, and cranium [91]. How these cells direct their migration and final
destination is a complex question that has been extensively studied.
The ECM influences the migration track and speed of migrating cells through its topography,
composition, and physical properties. The alignment of the underlying ECM has been shown to
direct cell migration and proliferation. Sharma et al. [92] previously used aligned fibres to direct
cell migration in the context of wound healing in vitro. Moreover, self-aligning materials have been
recently used to create ECM constructs for brain tissue regeneration in vivo [93].
Cells migrate from regions with lower ECM concentration to higher ECM concentration due to an
adhesion gradient in a type of cell migration known as haptotaxis [87]. However, this relationship is
nuanced. If the concentration of the ECM is too high, the adhesion force experienced by the cell is too
large from them to continue to migrate. Accordingly, the speed of migration is dependent on the ECM
concentration as well. As migration is a coordinated interplay between adhesion and deadhesion of
the cell onto the ECM, the speed of migration is characterized by a bell-shape function with respect to
ECM concentration [94].
The speed of migration is also influenced by the composition of the ECM. Hartman et al. [95] have
shown that fibroblasts cultured in rigidity gradients composed of fibronectin exhibited cell migration
while those cultured on matrices covered with either laminin or a fibronectin/laminin mixture did not
exhibit any migration. These results indicate that the ECM not only serves as a track for migration, but
also dictates cell migration due to its mechanical properties. It was previously demonstrated by Wang
et al. [96] that matrix stiffness and cell contractility also control RNA localization of genes responsible
for cellular organization and signalling to cellular protrusions. Proteases that degrade the ECM also
facilitate migration of cells through a process involving the interplay of MMPs, adamlysins, meprins,
metalloproteinase inhibitors (MMPIs), and other enzymes [2]. It is important to note that constant
ECM remodelling is occurring in development. The ECM components and their concentration are
continuously modified to dictate the developmental program.

3.2. ECM as the Dynamic Blueprint for Development

The role of ECM in structural organization is best studied in branching morphogenesis, which
involves epithelial buds and tubes invading the surrounding mesenchyme rich ECM [87]. This
process can be seen in various parts of the body, including mammary glands, salivary glands, and
kidneys [87]. Structural organization emphasizes the key functions played by the ECM and different
ECM components such as glycosaminoglycans (GAGs), collagen, and proteoglycans. Furthermore, the
ECM is constantly changing as this process occurs, highlighting the spatiotemporal control needed to
facilitate the development of these organs.
Branching in the mammary gland occurs at the terminal end buds that have a thin hyaluronic
acid-rich ECM and an accumulation of thick ECM composed of collagen IV, laminins 1 and 5,
and HSPGs at the flanks [97,98]. Thick ECM provides a structure to maintain the tubular organization
by serving as anchors for the cells while the reduced ECM at the end bud facilitates the migration
of epithelial cells, specifically the cell-budding process [87,99,100]. However, fibrillar collagen does
not only serve as a physical barrier. As it binds to discoidin domain receptors (DDRs) expressed by
mammary gland cells, it prevents hyper-proliferation possibly to maintain the tubular structure [87,101].
The architecture of the ECM serves as a guide to control how branching occurs through local
anisotropies in terms of tension as well [99]. Topographical variation in structure and elasticity of the ECM
provides a blueprint for where cells can bend, twist, and break off to form complex morphologies that are
essential for the different organs. Using micro-patterned organotypic cultures, Nelson et al. [102] showed
that tissue geometry dictates the position of the branches. Furthermore, Gjorevski and Nelson [103]
have shown that endogenous patterns of mechanical stress in the surrounding ECM specify the
Int. J. Mol. Sci. 2018, 19, 3028 9 of 31

branching pattern. There are several techniques to study ECM architecture in 3D such as atomic
force microscopy (AFM) combined with second-harmonic generation (SHG) [104]. Robinson et al.
used image analyses algorithms to analyse AFM and SHG micrographs to monitor and analyse ECM
remodelling of pancreatic stellate cells. This technique could similarly be used to study how the
mechanical forces and ECM architecture continuously evolve during development.
As the end bud of the mammary gland grows, it continuously degrades the ECM, which in turn
releases factors dictating the branching direction of the growing buds [2]. ECM degradation releases
collagen fragments tumstatin and endostatin that regulate the migration, survival, and proliferation
of these cells [105]. Furthermore, the ECM’s binding sites for morphogens and growth factors, such
as Wnt glycoproteins, epidermal growth factors (EGFs) and fibroblast growth factors (FGFs), allow
them to sequester and control the release of these factors [35,106]. Through this, the ECM facilitates
the formation of a morphogen gradient, which is required for diverse types of cells and structures to
develop [88]. The growth of the bud is finally terminated through the deposition of inelastic ECM
composed of sulphated GAGs (SGAGs) and collagen I [99].
Overall, the ECM modulates the growth of tissues to form complex structures that are required
for these organs to function. The ECM provides structural organization not only through its action as a
physical barrier to growing cells, but also by activating intracellular signalling in a time and context
dependent manner. The ECM does this through growth factor distribution modulation, physical
anisotropies, and anchorage.

3.3. ECM as the Driver for Cell Fate

The ECM influences cell fate through the previously discussed morphogenetic gradient in
development. However, this is not the only mechanism by which ECM affects cell fate, as the physical
properties of the ECM also play a critical role.
The role of ECM composition on cell fate is exemplified in mammary gland differentiation.
Even with hormonal stimulation in vitro, mammary gland cells do not secrete milk proteins. However,
upon exposure of these cells to laminin-1, they begin secreting milk proteins [107]. This activation is
due to integrin binding by laminin-1 leading to phosphorylation of the prolactin receptor, an upstream
regulator of STAT5. STAT5 then activates the transcription of milk proteins β-lactoglobulin and
β-casein, indicating that an appropriate 3-D ECM microenvironment is critical for cells to function
properly [102,107].
Using a simple yet elegant approach, Engler et al. [10] demonstrated that when mesenchymal
stem cells (MSCs) are cultured on collagen matrices with various elasticities, the MSCs differentiated
into osteocytes, myocytes, and neurons on the substrates that resembled their respective native
tissue. The process by which these MSCs are able to sense the elasticity and stiffness mechanical
environment before initiating an intracellular signalling cascade to dictate cell fate is known as
mechanotransduction. This phenomenon allows the cells to sense the physical properties of the
underlying matrix and activate appropriate intracellular pathways [89]. When myosin II, a key
molecule in various mechanotransduction-signalling pathways, was blocked, MSCs became insensitive
to the matrix elasticity mediated cell differentiation [10]. This study emphasized that the ECM’s
physical properties themselves have differentiating capability.
Engler’s results indicate that environmental cues can be relayed to the nucleus biochemically
or biophysically [89]. Indeed, often biochemical and biophysical relays work in conjunction
with one another to transmit environmental information [89,108]. One prominent example of a
mechanosensitive genetic regulator is the pair of the transcription co-activators, yes-associated protein
(YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). These functionally redundant
transcriptional coactivators are known to play crucial roles in critical cellular processes, such as
proliferation, wound healing, fibrosis, and other physiological processes that involve changes in
biomechanical properties [109]. Importantly, these transcription co-activators are known to be sensitive
to both biochemical and biophysical cues. The nuclear localization of YAP/TAZ is regulated by cell
Int. J. Mol. Sci. 2018, 19, 3028 10 of 31

shape, stiffness, ECM topology, and shear stress [109–115]. The integrin complexes that sense this
matrix stiffness are linked to the cytoskeleton, which in turn is linked to the nucleus through the
linker of nucleoskeleton and cytoskeleton (LINC) complex, which is composed of nesprins, sun, and
lamin proteins [116,117]. This allows direct transmission of mechanical cues from the extracellular
matrix to the nucleus [117]. The extensive role of YAP/TAZ in cell processes elucidates the role of
mechanotransduction in development and diseases, such as cancer [108].
In addition to matrix stiffness, the response of the cell to the ECM’s physical properties has also
been shown to be dependent on the ligand tether length. It has been shown that the focal adhesion
sizes and cell-adhesion strengths were affected when the tether length of the ECM coating’s ligand was
varied [118]. Coating stiff ECM with RGD ligands with longer tether lengths lead to the cell sensing
a softer ECM thereby controlling mechanotransduction mediated YAP/TAZ nuclear localization in
cancer. This might be a novel effective treatment against cancer.

4. Tissue Homeostasis
The ECM is a highly dynamic structure. Even after development, ECM is constantly being
deposited, degraded and modified to maintain tissue homeostasis. This is especially important in
maintaining the phenotype of cells and in physiological processes such as wound healing, angiogenesis,
and bone remodelling [6,119,120].
To maintain tissue homeostasis, the cells in contact with the ECM sense the properties of the
ECM through receptors and focal adhesion complexes. In turn, the cell regulates the expression of
ECM components and enzymes based on the signals of the ECM. This creates a feedback mechanism
wherein the cell also influences the ECM, which results in a balance of deposition and degradation of
ECM components [116].
The response of the cells to other stimuli, including shear stresses exerted by blood flow,
are ultimately influenced by the ECM component. Chen and Tzima [121] showed that platelet
endothelial cell adhesion molecule-1 (PECAM-1), a mechanosensitive molecule, is essential for vascular
remodelling, which occurs in response to long-term changes in hemodynamic conditions. Furthermore,
Collins et al. [122] recently demonstrated that the ability of platelet endothelial cell adhesion molecule-1
(PECAM-1) to respond to mechanical forces is influenced by the type of ECM they are adhered to. This
exemplifies the complexity and importance of the feedback mechanism that exists between the ECM
and the cell to maintain tissue homeostasis.
The importance of the ECM in maintaining tissue homeostasis is exemplified by the study
performed by Weaver et al. [123] where they were able to revert the malignant breast cancer cell
phenotype to the normal phenotype. They did this by culturing breast cancer cells onto basement
membrane based 3-D substrates coated in integrin β1 blocking antibodies [123]. This study confirmed
that the ECM is able to override the mutations causing the cancer phenotype, emphasizing the ECM’s
role in maintaining the correct cell phenotype. The role of dysregulated ECM in cancer progression
will be discussed further in the next section.
That an imbalance in the deposition and degradation of the ECM leads to diseases is a hallmark
not just of cancer but also other prominent diseases including fibrosis [124–126]. Overall, the ECM’s
role in tissue homeostasis is to direct proper cell response and phenotype to maintain the tissue’s
mechanical integrity and function.

5. ECM in Cancer

5.1. Dysregulation of ECM Molecules in Cancer Progression

Traditional perspectives of cancer have shifted to reflect the important role of the ECM in
regulating cell proliferation, migration, and apoptosis. On a microscopic level, the particular
arrangement and orientation of ECM constituents form a tissue-specific microenvironment that plays a
critical role in tumour progression [11,127,128]. It is now understood that the ECM not only undergoes
Int. J. Mol. Sci. 2018, 19, 3028 11 of 31

continuous active remodelling, but also elicits biochemical and biophysical cues to influence cell
adhesion and migration [129]. As such, small changes in microenvironment homeostasis can have
significant effects on the proliferation of cancer cells. As the most significant ECM component,
collagenInt.dictates
J. Mol. Sci. the
2018,primary
19, x FOR PEER REVIEW properties of the matrix. Indeed, changes in the deposition
functional 11 of 30 or

degradation of collagen can lead to the loss of ECM homeostasis [130,131].

ECM component, collagen dictates the primary functional properties of the matrix. Indeed, changes
As tumour cells proliferate, the surrounding ECM undergoes significant architectural changes
in the deposition or degradation of collagen can lead to the loss of ECM homeostasis [130,131].
in a dynamic As interplay
tumour cells between the microenvironment
proliferate, the surrounding ECMand residentsignificant
undergoes cells. These changes,changes
architectural including
increased
in asecretion
dynamic of fibronectin
interplay andthe
between collagens I, III, and IV
microenvironment andillustrate
residentthat tumour
cells. progression
These changes, demand
including
increased
a continuous secretion of
interaction fibronectin
between and collagens
the ECM and tumour I, III, cells
and IV illustrate
(Figure that tumour
3) [132]. progression
Increased deposition
demand
of matrix proteinsa continuous
promotesinteraction between theby
tumour progression ECM and tumour
interfering withcells (Figure
cell–cell 3) [132]. cell
adhesion, Increased
polarity,
deposition of matrix proteins promotes tumour progression by interfering
and ultimately amplifying growth factor signalling [133]. However, the exact role of collagen depositionwith cell–cell adhesion,
cell polarity,
in tumour progression and ultimately
is nuanced. amplifying growth have
Recent studies factorshown
signalling
that[133]. However,
increased the exact
collagen role of
cross-linking
collagen deposition in tumour progression is nuanced. Recent studies have
and deposition leads to tumour progression via increased integrin signalling [134,135]. Interestingly, shown that increased
collagen cross-linking and deposition leads to tumour progression via increased integrin signalling
however, depletion of fibrillar collagens I and III can also promote malignant behaviour, indicating
[134,135]. Interestingly, however, depletion of fibrillar collagens I and III can also promote malignant
that biomechanical forces produced by collagen deposition can have both beneficial and deleterious
behaviour, indicating that biomechanical forces produced by collagen deposition can have both
effects beneficial
on tumour and progression [136,137].
deleterious effects on tumour progression [136,137].

1. Regulation of Healthy Tissue Homeostasis

Cancer - Associated Fibroblast
Fibroblast

Epithelial Cell Neoplastic Cell

2. ECM Remodeling During Tumor Progression

Collagen MMPs

Stromal- Tumor-Derived
Derived LOX LOX

3. Collagen Alignment Guides Cell Motility

Figure Figure
3. ECM 3. remodelling
ECM remodelling during
during cancer
cancer progressionand
progression andinitiation.
initiation. (1)
(1) Epithelial
Epithelialneoplastic
neoplasticcells
cells
proliferate rapidly, inducing strain on the basement membrane. (2) Basement membrane bulges due
proliferate rapidly, inducing strain on the basement membrane. (2) Basement membrane bulges
to mechanical strain. Adjacent cancer-associated fibroblasts increase deposition of collagen. Stromal-
due to mechanical strain. Adjacent cancer-associated fibroblasts increase deposition of collagen.
derived lysyl oxidase (LOX) aligns collagen. (3) Neoplastic cells breach membrane and migrate along
Stromal-derived lysyl oxidase (LOX) aligns collagen. (3) Neoplastic cells breach membrane and migrate
aligned collagen. (Adapted from Lu et al. [6].)
along aligned collagen. (Adapted from Lu et al. [6].)
Collagen cross-linking can occur in both an enzyme-mediated and non-enzyme-mediated
Collagen
fashion. cross-linking can occur
Regulated collagen in both an
cross-linking enzyme-mediated
is coordinated and
primarily bynon-enzyme-mediated
LOX and the LOX family fashion.
of
amine oxidase enzymes [1]. LOX, secreted by primary tumour cells, is responsible for catalysing the
Regulated collagen cross-linking is coordinated primarily by LOX and the LOX family of amine oxidase
enzymescross-linking
[1]. LOX,ofsecreted
both collagen and elastin,
by primary which
tumour in turn
cells, increases matrix
is responsible stiffness andthe
for catalysing total adjacent
cross-linking
of bothECM volume.
collagen andIncreased ECM stiffness
elastin, which in turnactivates
increasesintegrins
matrixand augments
stiffness and Rho-generated
total adjacentcytoskeletal
ECM volume.
tension
Increased ECM to promote
stiffnessfocal adhesion
activates formation
integrins andaugments
and cell motility [138]. Elevated LOX
Rho-generated activity has
cytoskeletal been to
tension
clinically associated with increased collagen cross-linking, fibrosis, and elevated risk of cancer
metastasis [139]. Moreover, elevated LOX activity found on invasive edges of tumours has been noted
Int. J. Mol. Sci. 2018, 19, 3028 12 of 31

promote focal adhesion formation and cell motility [138]. Elevated LOX activity has been clinically
associated with increased collagen cross-linking, fibrosis, and elevated risk of cancer metastasis [139].
Moreover, elevated LOX activity found on invasive edges of tumours has been noted to drive actin
polymerization, cell contractility, and migration, providing a pathway for successive tumour cells to
follow [130].
Visualization of surrounding epithelial tissue during tumour metastasis has revealed localized
matrix organization and alignment along the leading edge of invasive tumours [131,140]. Indeed,
local cell invasion of these tumours has been observed to be oriented along aligned collagen fibres,
suggesting that the linearization of collagen fibres facilitates tumour invasion [141]. It is believed that
these densely aligned collagen fibres act as tracks for proliferating neoplastic cells to migrate out of the
tumour. Breast cancer serves as an important example of collagen alignment during tumour metastasis.
Although collagen within epithelial structures is typically tangled and disorganized, collagenous tissue
surrounding mammary tumours is frequently thickened, stiffened, and aligned perpendicularly to
the tumour boundary [142]. Recent studies indicate that the topography of matrix fibres increases the
efficiency of tumour migration by reducing the protrusions along the collagen fibre, and hence the
distance travelled by the migrating cell [143].
Much like collagen and LOX, elevated levels of the glycosaminoglycan hyaluronic acid in the
ECM correlates to increased likelihood of malignancy and poor prognosis [144]. As a naturally
occurring omnipresent linear polysaccharide, hyaluronic acid is critical in determining the compressive
properties of most biological tissues. The combination of tensile resistance due to collagen and
compression compliance due to hyaluronic acid creates the ideal biophysical properties for tissue
homeostasis [145]. In addition, it has been found that hyaluronic acid is both an induction signal for
mesenchymal transition and a migration substrate [146]. Accordingly, hyaluronic acid is frequently
used as a biomarker for prostate and breast cancer. While augmented levels of collagen and LOX
directly promote ECM stiffness and mechanically drive cell motility and proliferation, the exact role of
hyaluronic acid in cancer metastasis remains unclear. However, its dysregulation can serve as a key
biomarker for metastasis and cancer invasion.

5.2. Protein Unfolding Mediates Mechanotransduction

ECM signalling is a crucial cellular process that drives cell proliferation, differentiation, and
defers apoptosis [147]. In brief, if a cell cannot sense its mechanical environment, it cannot survive.
Many studies have reported that cells are capable of sensing their microenvironment through chemical
signalling, such as growth factors and metabolic precursors [148–150]. In order to detect ECM rigidity,
it is believed that cells mechanically probe their microenvironment via lamellipodia and sense the
mechanical feedback and resistance of their environment through integrin-based focal adhesions,
triggering an intracellular signalling cascade [151]. The ability for cells to probe their microenvironment
is attributed to the actin cytoskeleton, as inhibition of F-actin polymerization limits the ability of
cells to generate force, which induces a biological effect similar to plating cells on a soft substrate.
Specifically, the ability for cells to produce internal forces is derived from contractile actin bundles
and their upstream regulators, such as Rho-associated protein kinase (ROCK), which are necessary to
mechanically sense their environment [109]. While mechanical rigidity clearly has profound effects
on cell behaviour, the mechanism that translates mechanical force into gene transcription is not
fully understood.
Our recent work has illustrated the importance of protein unfolding in the transduction of
mechanical force exerted by the ECM. Indeed, talin, a prominent molecule in focal adhesion complexes
that couples focal adhesions to the actin cytoskeleton, has been shown to mechanically unfold during
force transmission [152]. Deleted in liver cancer 1 (DLC1) is a negative regulator of RhoA and cell
contractility that regulates cell behaviour when concentrated to focal-adhesion complexes bound
to talin [153]. Mechanical clamping of the R8 domain of talin prevented mechanical unfolding of
the molecule, interrupting downstream signalling of DLC1 and, consequently, cell behaviour [153].
Int. J. Mol. Sci. 2018, 19, 3028 13 of 31

Moreover, single molecule force microscopy revealed that every talin rod subdomain is susceptible to
unfolding over a physiologically relevant range of forces between 10 and 40 pN [152]. Because the
observed range of talin subdomain stabilities within the focal adhesion complex depend on small
structural differences, it is possible the mechanical stability of talin rod bundles could be influenced
by a few single point mutations. These mutations could lead to misinterpretation of ECM signals,
altering cellular response. Incorrect interpretations of ECM information could influence the behaviour
of cancer cells in the tumour microenvironment, potentially triggering DLC1 deactivation, increased
cell contractility, and cell migration [152,153].

5.3. YAP & TAZ Mechanotransduction in Cancer Progression

As robust regulators of cell proliferation and survival, YAP and TAZ play critical roles in
regulating organ development, cell differentiation, and progenitor cell self-renewal [109]. During these
processes, the YAP/TAZ proteins actively shuttle between the nucleus and the cytoplasm. While in the
cytoplasm, the YAP/TAZ proteins play a relatively passive role, regulating specific signalling cascades,
such as the Wnt signalling pathway. Meanwhile, when in the nucleus, they readily interact with
DNA-binding transcription factors, particularly TEA domain family members (TEAD), to regulate
genetic expression associated with proliferation, a key hallmark of cancer [154,155]. Upon biochemical
inhibition, YAP/TAZ accumulate in the cytoplasm, suggesting that the main functionality of YAP/TAZ
is gene transcription regulation in the nucleus. Importantly, upon cell detachment from a substrate,
YAP/TAZ activity is inhibited; suggesting that YAP/TAZ translocation to the nucleus can be regulated
by the F-actin cytoskeleton and mechanical force [156]. Moreover, in mammalian systems, matrix
elasticity and cell-spreading geometry are noted to heavily regulate YAP/TAZ nuclear transport and
their corresponding physiological processes [157]. Taken together, these results suggest that focal
adhesion and cytoskeleton-mediated cell signalling of mechanical rigidity is coupled to the YAP/TAZ
pathway to induce metastasis and tumour invasion, indicating a direct chemical pathway linking
mechanical force with malignant cellular behaviour.
Although cytoskeletal tension is sufficient for YAP/TAZ nuclear translocation, there exist multiple
potential pathways and proteins that mediate YAP/TAZ nuclear translocation. For example, the
heparan sulfate proteoglycan, agrin, is most commonly known for its role in the formation of
neuromuscular junctions during embryogenesis. However, recent advances in the field have suggested
that agrin may also serve as an ECM sensor that stabilizes focal adhesions and facilitates YAP/TAZ
nuclear translocation through the lipoprotein-related receptor-4 (Lrp4) and muscle-specific kinase
(MuSK) pathway [158,159]. Activation of Lrp4 and MuSK by agrin inhibits the Hippo tumour
suppressor pathway, ultimately leading to elevated YAP/TAZ nuclear translocation [158,160]. Agrin
depletion was shown to promote the inhibitory phosphorylation of YAP, which forced nuclear YAP
to remain in the cytosol [161]. Contrarily, supplementary introduction of agrin into cells cultured on
compliant matrices was sufficient for YAP activation [161]. Multiple junctional proteins, including the
Angiomotin (AMOT) family of proteins regulate YAP/TAZ in combination with changes in actomyosin
contractility [161,162]. AMOT proteins have been shown to directly bind to YAP, inhibiting its function.
F-actin competitively binds with AMOT to disrupt YAP:AMOT complexes, releasing YAP from its
inhibitory state to translocate into the nucleus [161]. Interestingly, agrin depletion elevated YAP:AMOT
binding, which ultimately led to decreased YAP activity [159]. Moreover, recent work demonstrates
that the Ras-related GTPase, Rap2, is also a key intracellular mediator that transduces ECM rigidity
signals to influence YAP/TAZ nuclear translocation [163,164]. At low ECM stiffness, Rap2 is known to
bind and activate MAP4K4, MAP4K6, MAP4K7, and ARHGAP29, which stimulate LATS1a and LATS2
while inhibiting YAP and TAZ nuclear translocation [163]. These findings demonstrate that ostensibly
unrelated proteins, such as Rap2 and agrin, play significant roles in ECM sensing and regulation of
YAP/TAZ activity.
Int. J. Mol. Sci. 2018, 19, 3028 14 of 31

5.4. ECM-Mediated Tumour Initiation and Migration

A crucial hallmark of carcinoma and other cancer cells is their ability to migrate through
surrounding tissues, penetrating the adjacent basement membrane. This dense, highly cross-linked
membrane of ECM serves not only as an anchor for epithelial cells to surrounding connective tissue,
but also as a significant barrier to epithelial cell migration [165]. However, due to the need for
cells to migrate within the body during healthy tissue homeostasis, cancer cells have adopted a few
methods of traversing the collagenous barrier [166]. One such method is the use of mechanical force.
Mechanical force has increasingly been seen as a compelling factor in triggering the breaching of
the basement membrane. As epithelial cancer cells proliferate, they are spatially constrained by the
bordering basement membrane. This burgeoning population of cancer cells significantly increases the
mechanical stress along the membrane, ultimately causing rupture and allowing cells to escape their
microenvironment (Figure 3) [167].
Another method of membrane navigation is anchor cell invasion, in which anchor cells breach the
basement membrane using protrusive, F-actin rich subcellular structures called invadopodia [168,169].
Indeed, electron micrographs of invasive tumours have demonstrated that leading invasive cells extend
a single protrusive arm into the basement membrane [170]. After initial breach by the invadopodia,
the membrane fissure widens, allowing for subsequent cells to traverse the collagen boundary [171].
However, along these breaching sites, elevated levels of collagen IV degrading products have also been
found, indicating a possible third factor in the migration of cancer cells [172]. Increased accumulation
of MMPs along the basement membrane has led to the commonly held assumption that proteases were
solely responsible for degradation of the basement membrane. However, staining of the membrane
during invasion reveal that laminin and collagen IV are in fact pushed aside by the invadopodia
rather than fully degraded [165,167]. These results indicate that MMPs may, rather, play a role in the
initial breaching of the basement membrane or in softening the matrix while anchor cell invadopodia
facilitate direct invasion [166].

5.5. Metalloproteinases (MMPs) in Tumour Progression

The role of MMPs in cancer cell invasion is multi-pronged: they not only assist in the
degradation of surrounding ECM barriers, but also release active growth factors and promote tumour
angiogenesis [148]. The ECM is known to promote cell proliferation primarily through contact with
the integrin family of cell surface receptors. However, certain ECM binding sites responsible for cell
proliferation and survival have been shown to be “cryptic” or partially hidden within the ECM. MMPs
simply unmask these hidden binding sites by degrading and loosening surrounding collagen, allowing
for integrins along the cell membrane to interact directly with the matrix [148,173].
In addition to removing physical barriers and revealing cryptic binding sites, MMP-mediated
degradation of collagen also exposes signalling components embedded within the ECM [173]. Stored
in an inactive state when embedded within collagen, various growth factors are activated upon ECM
degradation and allowed to bind with their target receptor. For example, MMP-2 mediated ECM
degradation is known to release the active form of transforming growth factor-β (TGF-β). Upon its
release, TGF-β is able to modulate cell invasion, immune response, and cell proliferation [174–176].
In effect, MMPs not only physically manipulate the surrounding ECM to allow for cell migration, but
also create a microenvironment conducive to tumour development through growth factor release and
cryptic binding site exposure. Thus, targeting MMPs could serve as a promising therapeutic approach,
despite a previous lack of success (Box 2).
Despite MMP-induced angiogenesis, vasculature networks in the tumour are frequently
disorganized with inter-capillary regions often exceeding the diffusion distance of oxygen. As such,
hypoxia, or the state in which cells are devoid of oxygen, is a hallmark of cancer. In fact, measurements
of the partial pressure of oxygen in tumours reveal that poorly oxygenated tumours strongly correlate
to increased malignancy [173]. It is believed that cancer cells are able to withstand oxygen-derived
regions by altering the transcription of various genes associated with angiogenesis. Hypoxia-inducible
Int. J. Mol. Sci. 2018, 19, 3028 15 of 31

factors (HIF) are known play a crucial part in the regulating this intracellular cancer cell response
to hypoxia [177–179]. Recent studies have indicated that HIF-1α, a member of the HIF family of
transcription factors, has been associated with increased MMP and collagen production [177–179].
Importantly, HIF-1α is known to increase LOX deposition, ultimately stiffening the surrounding
matrix [180]. Finally, HIF-1α has also been shown to activate transcription factors associated with
epithelial–mesenchymal transition (EMT), the process by which cells lose their polarity and adhesion
with adjacent cells, augmenting the invasive behaviour of cancer cells [180].

Box 2. MMPs as a therapeutic target.

As the role of the ECM in tumour progression becomes more apparent, cancer therapeutic interventions have
begun to target key elements of the ECM in an attempt to limit metastasis. One key ECM component that has
been targeted is the MMP family of enzymes. Due to the significant role of MMPs during cancer progression,
the pharmaceutical industry has worked to develop safe therapies to inhibit MMP activity [181,182]. Several
groups of synthetic MMP inhibitors, such as Marimastat, Minocycline, and Matimastat, have been developed to
target broad groups of MMPs and tested in stage III clinical trials in late stage cancer patients [183].

Unfortunately, most therapies specifically targeting MMP activity demonstrated poor outcomes
during clinical trials [184]. A few possible explanations exist for the poor clinical outcomes. Firstly,
patients selected to receive the MMP-inhibiting therapies were late-stage cancer patients. As previously
discussed, MMPs are known to play a role in tumour initiation and progression. It is possible that
MMP-inhibiting agents could be more effective in early stage patients. Moreover, it is known that
specific MMPs play different roles during cancer progression. It is likely that synthetic inhibitors need
to be developed to target unique MMP subgroups at specific timeframes during cancer progression.

5.6. Role of Mechanical Stress in Tumour Growth and Treatment

As cancer progresses, tumours rapidly grow in size and stiffen due to the increased appearance of
structural components, such as ECM, cancer-associated fibroblasts (CAFs), and cancer cells. This rapid
rise in the rigidity of tumours is indeed one of the only easily detectable mechanical features of tumours
that aid physicians in predicting malignancy and prognosis [185–188]. As the tumour progresses
and stiffens, internally generated forces allow the tumour to disarrange adjacent healthy tissue and
migrate into surrounding spaces. Accordingly, tumour progression is directly facilitated by these
intratumour-generated forces and forces arising from interactions with its microenvironment [189].
These mechanical forces induce two unique types of stress on tumour cells: fluid and solid stress [190].
Generally, solid stress is created by the non-fluid components of the tumour. Initial evidence
for the existence of solid stress within tumours came from the realization that blood and lymphatic
vessels are mechanically compressed during tumour formation [191]. Growth-induced solid stress
accumulates within tumours as the cancer cells rapidly proliferate. During this rapid reproduction
process, cells grow into one another and strain the tumour microenvironment, which ultimately strains
the surrounding healthy tissue [192]. In addition to intratumour-generated solid stress, externally
generated solid stress accrues due to the adjacent tissue, which attempts to resist tumour expansion.
In brief, solid stresses directly influence tumour progression in two manners: they first apply direct
mechanical stress on cancer cells to alter genetic expression and, therefore, increase malignancy and
invasion [193]. Secondly, solid stress deforms blood and lymphatic vessels to induce hypoxia [194].
As the name suggests, fluid stresses stem from forces generated by the fluid elements of
the tumour. This includes shear stresses created by blood and lymphatic flow within the vessels,
microvasculature (capillaries), and interstitial fluid flow [195,196]. In fact, fluid stress and solid stress
are highly intertwined, as compression of blood/lymphatic vessels by solid stress greatly influences
the fluid stress exerted on the surrounding epithelial tissue [190,197,198]. Vessel constriction reduces
the cross-sectional area of the vessel to increase resistance to lymphatic flow, which in turn increases
shear stress, interstitial fluid volume, and decreases perfusion rates [199]. This decrease of perfusion
rates and flow significantly limits the ability for lymphatic vessels to remove excess fluid from the
Int. J. Mol. Sci. 2018, 19, 3028 16 of 31

tumour, which ultimately increases interstitial fluid pressure in adjacent tumour tissue. Moreover,
compression of blood and lymphatic vessels has significant negative ramifications for the effectiveness
of chemo and
Int. J. Mol. Sci.immunotherapies [200].
2018, 19, x FOR PEER REVIEW 16 of 30

Solid Stress and Stiffness as a Function of Tumour Diameter

Solid Stress
Stiffness

Tumour Diameter (mm)

Figure
Figure 4. Solid
4. Solid stress
stress andand stiffnessas
stiffness asaa function
function of
oftumour
tumourdiameter (adapted
diameter (adaptedfromfrom
Nia et al.)et[201].
Nia al.) [201].
As rigidity
As rigidity of the
of the ECMECM remainsconstant,
remains constant, anan increase
increase in
intumour
tumourdiameter is associated
diameter withwith
is associated increased
increased
solidsolid stress
stress within
within thethe tumour.
tumour.

Elevated
Elevated solidsolid and fluid
and fluid stressstress
withinwithin
tumours tumours place cancer
place cancer cells incells in an unique
an entirely entirelyphysiological
unique
physiologicalIncreases
environment. environment. Increasesand
in tension in tension and compression
compression acting onacting on themechanically
the cells cells mechanicallyactivates
activates tumourigenic pathways, increases proliferation rates, and promotes
tumourigenic pathways, increases proliferation rates, and promotes collective migration [146,148]. collective migration
[146,148]. In addition to elevated rigidity, cancer cells produce and therefore are exposed to an
In addition to elevated rigidity, cancer cells produce and therefore are exposed to an elevated level of
elevated level of force than adjacent tissues [202]. While the bulk rigidity of tumours is relatively
force than adjacent tissues [202]. While the bulk rigidity of tumours is relatively simple to quantify,
simple to quantify, measuring solid stress within tumours has proven to be a much more elusive task.
measuring solidhave
Researchers stress withinbegun
recently tumours has proven
to quantify to be ainmuch
solid stress moretumour
individual elusivecells.
task.NiaResearchers
et al. [202] have
recently begun to quantify solid stress in individual tumour cells. Nia et al. [202]
has recently provided the experimental framework for creating in-situ two-dimensional mapping has recently provided
of
solid stress. framework for creating in-situ two-dimensional mapping of solid stress.
the experimental
Researchers
Researchers accomplish
accomplish thismapping
this mapping by by releasing
releasingthethesolid stress
solid within
stress withintissues in a controlled
tissues in a controlled
method
method using
using predefinedgeometry
predefined geometry thatthat encapsulates
encapsulates the thetumour
tumour in inagarose
agarosegel gel
and and
recordsrecords
deformation after a precise incision is made. Combination of mathematical
deformation after a precise incision is made. Combination of mathematical modelling and experimental modelling and
experimental
analysis analysis
has revealed hasimportant
a few revealed afindings:
few important findings:
solid stress solid stress
increases increases
linearly linearly size
with tumour withwhile
tumour size while rigidity remains constant, and adjacent healthy tissue contributes significantly to
rigidity remains constant, and adjacent healthy tissue contributes significantly to the solid stress within
the solid stress within the tumour. These findings suggest that rigidity of the tumour is decoupled
the tumour. These findings suggest that rigidity of the tumour is decoupled from the solid stress
from the solid stress implemented on tumour cells (Figure 4).
implemented on tumour cells (Figure 4).
5.7. Quantification of Tumour Cell Mechanical Stress in vivo
5.7. Quantification of Tumour Cell Mechanical Stress in vivo
Tissue development, growth, and regeneration are crucially dependent on spatiotemporal
variations in microenvironment
Tissue development, mechanics.
growth, and However,
regeneration most current
are crucially techniques
dependent for stress
on spatiotemporal
quantification
variations utilize two-dimensional
in microenvironment mechanics.analysis that most
However, can only be performed
current techniquesinfor
vitro, limiting
stress its
quantification
application for determining microenvironment mechanics during tumour progression.
utilize two-dimensional analysis that can only be performed in vitro, limiting its application for To address
this issue, microenvironment
determining researchers in the Campàs group recently
mechanics during developed a novel oil microTo
tumour progression. droplet technique
address this issue,
to quantify local cell-generated mechanical stresses in tumours in a spatiotemporal
researchers in the Campàs group recently developed a novel oil micro droplet technique manner [201,203–
to quantify
205].
local cell-generated mechanical stresses in tumours in a spatiotemporal manner [201,203–205].
Int. J. Mol. Sci. 2018, 19, 3028 17 of 31
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 17 of 30

Figure5.
Figure 5. Schematic
Schematic of
of oil
oil micro
micro droplet
droplet inin vivo
vivo stress
stress quantification
quantification described
described byby Campàs
Campàs et
et al.
al. [203].
[203].
An oil droplet with calibrated surface tension is injected into living embryonic or cancerous
An oil droplet with calibrated surface tension is injected into living embryonic or cancerous tissue. tissue. As
cells proliferate, they exert force onto the micro droplet and deform it. Deformations in
As cells proliferate, they exert force onto the micro droplet and deform it. Deformations in curvature curvature
(red)of
(red) ofthe
theoil
oildroplet
dropletare
areused
usedto
tocalculate
calculateanisotropic
anisotropicstress
stresswithin
withinthe
thetissue.
tissue.

Fluorescentoil
Fluorescent oilmicro
microdroplets
dropletswith
withcalibrated
calibratedsurface
surfacetensions
tensionsare areinjected
injectedbetween
betweentumour
tumour cells
cells
in living
in living tissue
tissue while
while fluorescence
fluorescence microscopy
microscopy is is used
used to to image
image localized
localized oil
oil droplet
droplet deformation.
deformation.
Provided droplet
Provided droplet surface
surfacetension,
tension,measurements
measurements of of
curvature
curvature deformation
deformation along the oil
along thedroplet yield
oil droplet
precise
yield information
precise regarding
information localized
regarding anisotropic
localized mechanical
anisotropic mechanicalstresses exerted
stresses by adjacent
exerted cells
by adjacent
[201,203].
cells This technique
[201,203]. of localized
This technique stress quantification
of localized stress quantificationshownshownabove in Figure
above in 5Figure
revealed that the
5 revealed
magnitude
that of cell-generated
the magnitude stress varies
of cell-generated only weakly
stress varies only weakly spatially during
spatially tumour
during progression,
tumour progression,but
increases
but increasesdramatically
dramaticallyover time
over [205].
time Campàs
[205]. Campàs et etal.al.[203]
[203]further
furtheradapted
adaptedthethe oil micro droplet
oil micro droplet
technique to
technique to incorporate
incorporate aa biocompatible
biocompatible ferrofluid
ferrofluid magnetic
magnetic micro micro droplets
droplets to
to serve
serve as
as mechanical
mechanical
actuators [206,207].
actuators [206,207]. Using
Using this
this technique,
technique, researchers
researchers are are able
able toto actively
actively apply
apply localized
localized stress
stress on
on
tissueswhile
tissues whileobserving
observingtissue
tissuemechanical
mechanicalresponse.
response. Indeed,
Indeed, this this novel
novel ferrofluid
ferrofluid micro
micro droplet
droplet allows
allows
forthe
for thesimultaneous
simultaneous measurement
measurement of tissue
of tissue mechanical
mechanical properties properties and local cell-generated
and local cell-generated mechanical
mechanical
stress [207]. stress [207].

5.8. Role
5.8. Role of
of ECM
ECM Mechanics
Mechanics in
in Behaviour
Behaviour of
of Myofibroblastic
MyofibroblasticCells
Cells
Many aggressive
Many aggressive malignancies,
malignancies, such suchasaspancreatic
pancreatic ductal
ductaladenocarcinoma
adenocarcinoma (PDAC),
(PDAC),are
characterized by extensive desmoplasia and collagen deposition, which ultimately increases the
are characterized by extensive desmoplasia and collagen deposition, which ultimately increases
rigidity
the of the
rigidity tumour.
of the tumour. Myofibroblast-like
Myofibroblast-like cells,
cells,such
suchasaspancreatic
pancreaticstellate
stellate cells
cells (PSCs), are crucial
crucial
mediatorsin
mediators inthe
the production
productionof ofthis
thisfibrotic
fibroticECM
ECM[208].
[208]. When
When quiescent,
quiescent, PSCs
PSCs are
are responsible
responsiblefor for ECM
ECM
turnover and remodelling through the production of MMPs. During wound repair, PSCs become
turnover and remodelling through the production of MMPs. During wound repair, PSCs become
activated by numerous
activated numeroussolublesolublefactors,
factors,including
including IL-1,
IL-1,IL-6, andand
IL-6, TGF-
TGF-β[141,142].
[141,142].Alternatively, PSCs
Alternatively,
in theintumour
PSCs the tumourdesmoplasia
desmoplasia of human
of human pancreatic
pancreatic cancers
cancersbehave
behaveerratically,
erratically, become chronically
chronically
activated, and
activated, and create
create aa microenvironment
microenvironmentconduciveconduciveto totumour
tumourgrowth
growth[144].
[144]. ItIt has
has been
been shown
shown that
that
pancreatic tumour
pancreatic tumour cells
cells are
are able
ableto toinduce
induceactivation
activationof ofPSCs
PSCsthrough
throughincreased
increasedsecretion
secretionofofTGF-β1
TGF-1
and PDGF
and PDGF [141].
[141]. However,
However, recent
recentstudies
studieshave
have indicated
indicated that that PSCs
PSCs may
may be be able
able to to sustain
sustain activation
activation
due to
due to the
the mechanical
mechanical properties
propertiesof ofthe
themicroenvironment
microenvironmentalone alone[209,210].
[209,210].
Ourrecent
Our recentwork
workhashas shown
shown that that
matrixmatrix stiffness
stiffness is sufficient
is sufficient for activation
for activation of PSCs. Upon of PSCs. Upon
activation,
activation, PSCs were found to mechanically sense the increased rigidity of the environment as they
PSCs were found to mechanically sense the increased rigidity of the environment as they produce
produce
excess excess [210,211].
collagen collagen [210,211]. This mechanosensing
This mechanosensing of tissueactivates
of tissue stiffness stiffnessintracellular-signalling
activates intracellular-
signallingwithin
pathways pathways within
the PSC, the PSC,the
encouraging encouraging the myofibroblast-like
myofibroblast-like cell todeposits
cell to produce excess produce excess
collagen.
deposits collagen. This process of stiffness mechanosensing forms a positive-feedback loop, in which
PSCs continue to secrete collagen as the matrix becomes stiffer and stiffer [210,212]. Moreover, we
Int. J. Mol. Sci. 2018, 19, 3028 18 of 31

This process of stiffness mechanosensing forms a positive-feedback loop, in which PSCs continue to
secrete collagen as the matrix becomes stiffer and stiffer [210,212]. Moreover, we also found that matrix
rigidity influences PSC migration, as PSCs migrate from adjacent soft tissue towards the stiff tumour
microenvironment [212]. Thus, as the matrix is stiffened, distant PSCs are recruited towards the stiff
tissue and become activated, further enhancing the positive feedback loop.
Inactivating the mechanosensing and remodelling capability of PSCs may be an effective
therapeutic strategy. All-trans-retinoic acid (ATRA) has been shown to suppress PSC mechanosensing
by downregulating MLC-2 actomyosin contractility. This leads to PSC inactivation and turns off the
positive feedback loop of increased matrix rigidity and PSC activation [213]. By inactivating PSC’s
ability to sense the mechanical environment, ATRA reduces fibrosis and suppresses cancer invasion.
Furthermore, inactivation of PSC’s ECM remodelling capability prevents its ability to mechanically
liberate TGFβ from Latent TGFβ Binding Protein (LTBP) [214].
These results indicate that the mechanical environment is a powerful regulator of PDAC
progression via PSC activation and ECM remodelling, suggesting that reprogramming of PSCs and
other resident cells may be a viable therapeutic target to alleviate tumour growth. A review of the role
of mechanical rigidity and mechanical stress in tumour proliferation can be seen in Box 3.

Box 3. Rigidity and stress influence cell behaviour.

As discussed in the previous three sections, mechanical stiffness and stress of the tumour both play extremely
important yet distinct roles in influencing cancer cell behaviour. Although there is significant overlap in effects
of rigidity and mechanical stress, the points below highlight certain differences:
Tumour Rigidity

v Caused by elevated ECM deposition

v Mechanically activates pancreatic stellate cells (PSCs) to produce ECM
v Induces EMT in epithelial cells
v Amplifies growth-factor signalling
Tumour Stress (Solid and Fluid)

v Caused by increased cell proliferation and blood flow

v Induces hypoxia in tumours
v Augments cell proliferation
v Increases chemo resistance

5.9. Seed and Soil

For many years, the scope of study regarding cancer metastasis had primarily focused on cancer
cells and their migration through the vasculature. However, as the complex interplay between the
ECM and cancer progression became apparent, the perspective on cancer progression to distant
organs evolved. In 1889, surgeon Stephen Paget posited that cancer metastasis is dependent on
complex interactions between the migrating tumour cells (the “seed”) and its microenvironment
(the “soil”) [208,209]. Despite doubt over the course of the twentieth century, Paget’s hypothesis
was strengthened in the 1970s when Isaiah Fidler’s research demonstrated that successful tumour
migration could only occur at certain organ sites [215].
Further research demonstrated that primary tumours have the ability to induce the formation of a
microenvironment at distant organ sites that are conducive to tumour growth [209,213]. This newly
formed distant microenvironment, known as the pre-metastatic niche (PMN), promotes tumour
growth through a variety of methods, including increased inflammation, vascular permeability,
immunosuppression, and tissue stiffness [215–217]. Studies have indicated a variety of molecules
and mechanisms involved with the generation of the PMN, such as tumour-derived secreted factors
(TDSFs) and tumour-derived extracellular vesicles (EVs) [218,219].
Int.
Int.J.J.Mol.
Mol.Sci. 2018,19,
Sci.2018, 19,3028
x FOR PEER REVIEW 19 of
19 of 31
30

1. Primary Tumor Secretes TDSFs and EVs

3. TDSFs and EVs induce ECM

remodeling

2. TDSFs and EVs

travel to pre-
metastatic organ via
bone marrow and
vasculature • Immune Response
• ECM stiffening
• Hypoxia

TDSF Immune Cell BMDSC

EV Fibroblast Collagen

Figure6.6.Illustration
Figure Illustrationof
ofpre-metastatic
pre-metastaticniche
nicheformation
formationin inthe
theliver.
liver.(1)
(1)The
Theprimary
primarytumour
tumourlocated
locatedinin
thepancreas
the pancreasemitsemits tumour-derived
tumour-derived secreted
secreted factors
factors (TDSFs)
(TDSFs) and extracellular
and extracellular vesiclesvesicles (EVs).
(EVs). (2) TDSFs(2)
TDSFs
and EVsand EVs migrate
migrate throughthrough the vasculature
the vasculature and boneandmarrow
bone marrow
to the to the secondary
secondary organ.organ.
WhileWhile
in thein
the bone marrow, TDSFs and EVs recruit bone marrow-derived stem cells (BDSCs), such as
bone marrow, TDSFs and EVs recruit bone marrow-derived stem cells (BDSCs), such as hematopoietic
hematopoietic
stem cells, to thestem cells, organ
secondary to thesite.
secondary
(3) TDSFsorgan site. induce
and EVs (3) TDSFs
immuneand cell
EVsrecruitment
induce immune
and ECM cell
recruitment through
remodelling and ECMLOXremodelling through LOX
and cancer-associated and cancer-associated
fibroblasts at the pre-metastaticfibroblasts
site. [220]at the pre-
metastatic site. [220]
One primary form of tumour-derived EV is the exosome, which has a diameter ranging from
30–100 One
nmprimary form of tumour-derived
[218]. Exosomes containing proteins, EV mRNAs,
is the exosome,
and unique which has a diameter
ECM-binding ranging
integrins are from
first
30–100 nm [218]. Exosomes containing proteins, mRNAs, and unique ECM-binding integrins are first
secreted from the primary tumour and travel through the vasculature, promoting vessel leakiness in
secreted
distant fromsites.
organ the primary
EVs and tumour
TDSFs, and travel
such through
as TGF-β andtheMMP-9,
vasculature, then promoting vessel leakiness
alter local resident cells and in
distant organ sites. EVs and TDSFs, such as TGF-β and MMP-9, then alter local resident cells and
fibroblasts [219,221]. Fibroblasts altered by exosomes recruit more inflammatory cytokines, such as
fibroblasts
TGF-β, and[219,221].
dramatically Fibroblasts
increasealtered
collagen by deposition
exosomes recruit more
to stiffen inflammatory
tissue in the PMNcytokines,
[222]. Just such
as inas
TGF-β, tumour
primary and dramatically increase
tissue, elevated collagen
collagen deposition
deposition to stiffen tissue
and inflammation in theinterstitial
increase PMN [222]. Just
stress as in
within
primary
the tissuetumour
to induce tissue, elevated
hypoxia [220].collagen deposition and inflammation
Finally, TDSF-mediated recruitment ofincrease interstitial
non-resident cells, stress
such
within
as bone the tissue to induce
marrow-derived hypoxia
cells (BMDCs) [220].
andFinally,
immune TDSF-mediated
suppressor cells, recruitment
ultimatelyofattracts
non-resident cells,
circulating
such as bone marrow-derived cells (BMDCs) and immune suppressor cells, ultimately attracts
tumour cells (CTCs) to the site (Figure 6) [223]. CTCs traveling through the vasculature are able to
circulating tumour cells (CTCs) to the site (Figure 6) [223]. CTCs traveling through the vasculature
easily permeate into the PMN due elevated vasculature leakiness and collectively migrate through the
are able
organ to easily
toward the permeate intointhe
stiffer tissue, PMN due
a process elevated
known vasculature
as durotaxis leakiness and collectively migrate
[224,225].
through the organ toward the stiffer tissue, in a process known as durotaxis
Interestingly, the notion of the PMN proposes that critical changes [224,225].
to the ECM, typically associated
Interestingly, the notion of the PMN proposes that critical changes to the ECM,The
with primary tumour formation, can occur prior to the arrival of tumour cells [226,227]. typically
Seed
associated
and with primary
Soil process tumourthe
closely mirrors formation,
process of can occur prior
primary tumour to the
ECM arrival of tumour
remodelling, cells
with one[226,227].
critical
The Seed and Soil process closely mirrors the process of primary tumour ECM remodelling, with one
difference: it occurs in the absence of cancer cells. Furthermore, it specifies that TDSF and EV-mediated
critical
ECM difference:isitcrucial
remodelling occursin infacilitating
the absence of cancer
cancer cells. Furthermore,
metastasis. However, many it specifies
aspectsthat TDSF
of the and EV-
mechanism
mediated ECM remodelling is crucial in facilitating cancer metastasis. However, many aspects of the
regarding PNM formation remain elusive.
mechanism regarding PNM formation remain elusive.
Although exosomes have been known to play a crucial role in PMN development, why cancer
cells only metastasize to specific organs, a process known as organotropism, has remained unclear.
Int. J. Mol. Sci. 2018, 19, 3028 20 of 31

Although exosomes have been known to play a crucial role in PMN development, why cancer
cells only metastasize to specific organs, a process known as organotropism, has remained unclear.
Recent research in the Lyden laboratory has demonstrated that exosomes secreted by primary
tumour cells have specific integrin expression patterns that dictate organotropism. Indeed, exosome
proteomics revealed that exosomal integrins α6β4 and α6β1 are closely linked with lung metastasis,
while exosomal integrin αvβ5 is associated with liver metastasis [220]. Targeting of these specific
integrins decreased exosomal uptake by resident cells and decreased lung and liver metastasis,
respectively. These results suggest that exosomal integrin expression could be used to predict
organ-specific metastatic sites. Moreover, there is an implication that cancer therapies may be most
beneficial if tailored to distinct metastatic sites (lung, liver, etc.) and each stages of cancer metastasis:
pre-metastatic and post-metastatic [215].

6. Challenges and Future Perspectives

In this review we have discussed the complex and nuanced role of the ECM in tissue development
and cancer progression. Over the past 20 years, studies have revealed the importance of the ECM
in regulating crucial physiological processes such as stem cell lineage specification, cell migration,
and proliferation [138–140]. Accordingly, perspectives have shifted to address cancer not only as
a disease of uncontrolled cell proliferation, but also of dysregulation of the microenvironment.
The ostensibly static ECM actively undergoes dynamic remodelling during all stages of cancer
progression in a complex interplay between cancer cells, resident cells, and non-cellular components.
Advances in understanding of the role of ECM in cancer progression have provided hope and revealed
promising therapeutic targets for mitigating cancer’s ability to metastasize. However, a lack of success
in targeting broad ranges of proteins, such as MMPs and collagen, reveals the temporal sensitivity and
specificity needed to effectively limit the spread of the tumour cells.
As neoplastic cells proliferate rapidly in tumours, they experience an increase in mechanical
stress, which mechanically activates tumourigenic pathways, increases migration, and induces hypoxia.
An essential region of future cancer research will be to ascertain the mechanism by which increased
mechanical stress in tumours relates to malignant behaviour and angiogenesis. These signalling
pathways relating to exogenous mechanical stress and malignant behaviour serve as an auspicious
therapeutic target to resist cancer progression. Moreover, understanding the relationship between
increased solid stress and angiogenesis mechanisms would elucidate possible advancements for drug
delivery. Recent developments in in vivo cell stress quantification techniques may provide novel
insights into the relationship between ECM-generated stress and hypoxia [213–216].

Funding: This work was supported by the ERC grant 282051 “ForceRegulation”.
Conflicts of Interest: The authors declare no conflict of interest.

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