# Role of the Endothelium during Tumor Cell Metastasis: Is the Endothelium a Barrier or a Promoter for Cell Invasion  and Metastasis?

Authors: Claudia Tanja Mierke
Journal: Journal of Biophysics
 (2008)

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## Abstract

The malignancy of cancer disease depends on the ability of the primary tumor to metastasize to distant organs. The process of the metastasis formation has largely been analyzed, but still main pathways regarding the extravasation step at the end of the metastasis formation process are controversially discussed. An agreement has been reached about the importance of the endothelium to promote metastasis formation either by enhancing the growth of the primary tumor or by homing (targeting) the tumor cells to blood or lymph vessels. The mechanical properties of the invading tumor cells become the focus of several studies, but the endothelial cell mechanical properties are still elusive. This paper describes the different roles of the endothelium in the process of metastasis formation and focuses on a novel role of the endothelium in promoting tumor cell invasion. It discusses how novel biophysical tools and in vivo animal models help to determine the role of the endothelium in the process of tumor cell invasion. Evidence is provided that cell mechanical properties, for example, contractile force generation of tumor cells, are involved in the process of tumor cell invasion.


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## Body

## 1. Introduction

The coordinated regulation of cell adhesion and cell motility especially the
migration of cells in a three-dimensional (3D) environment (cell invasion) is
essential for the development and function of almost all multicellular
organisms. The transformation of cell-cell and cell-matrix adhesions as well as
the loss of contact responsiveness is an indicator for the malignancy of tumors
[1, 2]. Cell invasion is a driving force in
malignant tumor diseases, for example, in the progression of primary tumor
outgrowth and in the process of metastasis formation. The malignancy of
neoplasms is determined by the capability of single tumor cells to invade into
the surrounding tissue, intravasate into blood or lymph vessels, be
transported, possibly extravasate and invade into the connective tissue,
proliferate, and finally metastasize [3, 4]
(Figure 1).Metastasis formation involves several steps.
Two possible ways of metastasis formation: (a)
first, single tumor cells disseminate form the primary tumor and invade the
extracellular matrix, intravasate into blood or lymph vessels, and get
transported and adhere to the endothelium. Secondly, (a) invasive tumor cells
grow in vessels and do not transmigrate or (b) invasive tumor cells
transmigrate through the endothelium, invade and from a secondary tumor in the
targeted tissue.
(a)(b)Cell invasion is a central step in the process of metastasis formation. The
prerequisites for cell invasion involved in the metastasis formation process
are (1) cell adhesion (adhesion strength), (2) force generation, (3) detachment
(deadhesion and cytoskeletal remodeling), and (4) matrix composition and remodeling
via enzymes (Figure2). The balance of all these parameters determines the cell
invasiveness, for example, the migration speed in a 3D extracellular matrix. All
these considerations might play a more or less important role in cell invasion
depending on the cell type and tissue origin, gene, or protein expression
pattern of a cell, the environment (growth factors, matrix constituents, and
mechanical stiffness), and adjacent cells. In line with cell invasion, the interaction
of a cell with the neighboring cells underlies also mechanical properties of
the cytoskeleton of the two adjacent cells.Prerequisites for tumor cell migration on a 2D substrate and tumor cell
invasion in a 3D extracellular matrix. (a) Scanning electron microscopic image of an MDA-MB-231 breast
carcinoma cell on a planar glass and (b) transmission electron microscopic
image of an
MDA-MB-231 cell invaded in 3D collagen gel matrix. (a) For proper 2D motility,
a cell needs to adhere/deadhere fast, remodel its cytoskeleton fast, and possess
a high CSK-fluidity. (d) For proper 3D motility, a cell needs, in addition to
the 2D motility properties, to generate high contractile forces, and the cells
needs to overcome the viscous drag of the surrounding extracellular matrix to
move forward.
(a)(b)Alterations of cell-cell and
cell-matrix interactions are mirrored in the cytoskeleton and account for the
ability of tumor cells to cross tissue boundaries and to disseminate to distant organ
sites. Many adhesion molecules implicated in tumor metastasis have been
identified, but there exist only a few studies with a broad
designed approach. Several adhesion or cell surface receptors have been
identified to act either as negative, for example, E-Cadherin [5]
or positive factors, for example, αvβ3, CD24, and CXCR2 of tumor invasion and metastasis formation
[6–8]. The focus of this paper is on the processes
of cell invasion and transendothelial migration of tumor cells as well as on their
prerequisites including the presentation of novel biophysical tools and in vivo
metastasis models. The emphasis is on the function of the endothelium in the
process of metastasis formation, such that it promotes tumor cell invasion.


## 2. Tumor Cell Invasion

### 2.1. How Do Cell-Matrix Adhesion Receptors Influence Tumor Cell Invasion?

To generate tractions especially in a 3D extracellular matrix, the cells
need to have a tight connection between the extracellular matrix and the
intracellular cytoskeleton via focal adhesions and cell-matrix receptors. This
connection is commonly facilitated by transmembrane proteins like integrin cell
adhesion receptors. Integrins
are heterodimeric proteins that are involved in cell adhesion, migration, and
invasion processes and regulate the cell cycle. The protein family of human integrins
comprises 18α- and 8 β-subunits [9]. At least 24 different α-β-heterodimer combinations have been identified,
that are capable of binding to a large variety of ligands, among them were
components of the extracellular matrix (e.g., collagen, fibronectin, and laminin)
as well as binding partners on adjacent cells (e.g., VCAM-1 and PECAM-1) [10–13]. The
physical link between these adhesion receptors and the actin cytoskeleton via
intracellular focal adhesion proteins mediates the bidirectional force
transmission and the biochemical signal transduction across the plasma membrane
[14, 15].
The major signaling pathways activated by integrins through their α-subunits are different. The activation of α4 and α9 integrin subunits leads to decreased
cell spreading and increased cell migration on a planar substrate [16–18].
This is in contrast to the activation of other integrin α-subunits (e.g., α1, α5, αv, α3, α6, and αIIb) that lead to enhanced cell
spreading and decreased cell migration [19–23]
and show different bindings
to fibronectin via the canonical integrin recognition site
arginine-glycine-aspartic acid (RGD) [24–28].
Furthermore, the α5β1 integrin regulates the αvβ3-mediated adhesions and migration on the
extracellular matrix [29]. The formation, activation,
and regulation of focal adhesion proteins in the focal adhesions and their physiological
function are pivotal and have been the focus of intense research efforts and
may have opposite effects depending on the integrin subunit type [9],
but the mechano-regulating function, for example, in the process of cell
invasion is still elusive. The αvβ3, αvβ6, and α6β4 integrins have been described to act
as enhancers of tumor cell invasion and metastasis formation [30–32].
Their role in the process of metastasis formation and their impact on
transendothelial migration of tumor cells are still unclear.Theβ1 integrins have also been reported to promote tumor cell migration in a matrigel [33].
To make it more confusing, the role of the α5β1 in the process of 3D cell invasion,
tumor growth and metastasis formation is controversially discussed because several reports
described an invasion enhancing, tumor growth promoting, and metastasis
increasing role that is inversely correlated with E-cadherin expression [34, 35],
but others reported it vice versa [36–38].
The differences of the results might be explained by the use of different tumor
cell lines. The correlation of the expression of one gene alone might not be
helpful in determining the malignant potential of a tumor. The combination of
gene expression and biophysical measurements of the cell's mechanical properties
could be more sufficient because the expression a certain integrin by itself changes
the cytoskeletal structure of a cell [39]. Combined with gene
expression data, the overall cellular stiffness or the cytoskeletal dynamics of
a cell could explain cellular behavior, for example, cell invasion and help to
understand the general mechanisms important for the regulation of this process [6].


### 2.2. Role of The Extracellular Matrix Composition and Mechanical  Properties of The Tissue

The mechanical properties of the extracellular matrix determine the gene
expression patterns, the differentiation, proliferation, and cell mechanical
properties [40–42].
The ability to generate forces might also be influenced by the mechanical
properties of the 3D environment of the cell [43]. Furthermore, the
environmental properties of the primary tumor might also determine the
secondary tumor formation site maybe because of similar mechanical properties
of the primary tumor tissue and the new metastasis formation site. Adhesion
strength and cell stiffness determine the strength of the contractile forces [43] and might influence the
ability of cells to invade into the extracellular matrix and to transmigrate
through an endothelial cell monolayer and the basement membrane surrounding the
vessels. The ability to remodel their cytoskeleton to squeeze through the pores
of the extracellular matrix (amoeboid migration) with or without the generation
of contractile forces is important for the invasion speed, invasion depth, and
finally the ability to form metastasis. For the migration in a 3D matrix, the
CSK-fluidity may play also an important role, the higher the CSK-fluidity the
higher the invasiveness of the cells (Figure 2).


### 2.3. Enzymatic Modulation of The Mechanical Properties of A 3d Connective Tissue

Changes of the extracellular matrix composition and structure (e.g.,
matrix pore size) influence the mechanical parameters of the matrix that could impact
cell invasiveness via changing the mechanical properties inside the cell. These
changes of the extracellular matrix's mechanical properties could origin from
the invading cell itself. For example, cell shape changes such as protrusion
dynamics of mesenchymal tumor cells were monitored in combination with matrix
deformation due to enzymatic matrix remodeling using a 3D collagen matrix
embedded with polystyrene beads [44]. The cells themselves produce
enzymes either membrane-bound (e.g., MT-MMPs) or secreted to modulate the
adjacent extracellular matrix [33].
The expression of collagenases  MMP-14, MMP-12, and MMP-9  increase the
invasiveness of tumor cells migrating through a collagen fiber network or
through a Boyden-chamber (transwell membrane) [33].
MMP-9 cooperates with the αvβ3 integrin in supporting breast
carcinoma cell migration using transwell membrane assay [20]. The inhibition of these enzymes
using a cocktail of  inhibitors leads to reduced or impaired invasion of tumor cells and changes
the prior mesenchymal invasion to an amoeboid invasion type [33].
The production of sheddases leads to the cleavage (e.g., ADAMs = a disintegrin
and metalloprotease domain) of several adhesion receptors like α6 integrin and αvβ3 integrin, reduces cell adhesion, and
increases motility [22, 45, 46].
ADAMs are a family of type I transmembrane glycoproteins that have the ability
to bind to integrins
and disrupt their adhesion to the extracellular matrix [47–49].
The ADAM-15 sheddase contains an RGD-peptide motif and can hence act as a
natural binding partner for αvβ3 integrin and reduce cell adhesion, increasing migration and
invasion of tumor cells [50].
Despite the binding to αvβ3 integrin, the ADAM-15 interacts also
with the α5β1 integrin [51].
Hence, the expression of sheddases like ADAM-15 is associated with a poor
diagnosis of the cancer disease [52] and supports the interaction
of prostate cancer cells and endothelial cells in the process of metastasis
formation [53]. Confirming that the
overexpression of MMP-14 increased the invasiveness of the cells, more cells
were able to invade and they invade deeper into the gel via enhanced motility (or
a higher adhesion site turn over rate). The cutting of the extracellular part
of membrane-bound receptors reduced their cell surface expression that leads to
decreased adhesion strength and decreased traction generation. All these
enzymes alter either the extracellular matrix adjacent to the invading cell or
the adhesion receptor expression on the migrating cell itself. This might
influence, in turn, the adhesion strength (e.g., the adhesion process) and the
generation of contractile forces (Figure 2).


## 2.1. How Do Cell-Matrix Adhesion Receptors Influence Tumor Cell Invasion?

To generate tractions especially in a 3D extracellular matrix, the cells
need to have a tight connection between the extracellular matrix and the
intracellular cytoskeleton via focal adhesions and cell-matrix receptors. This
connection is commonly facilitated by transmembrane proteins like integrin cell
adhesion receptors. Integrins
are heterodimeric proteins that are involved in cell adhesion, migration, and
invasion processes and regulate the cell cycle. The protein family of human integrins
comprises 18α- and 8 β-subunits [9]. At least 24 different α-β-heterodimer combinations have been identified,
that are capable of binding to a large variety of ligands, among them were
components of the extracellular matrix (e.g., collagen, fibronectin, and laminin)
as well as binding partners on adjacent cells (e.g., VCAM-1 and PECAM-1) [10–13]. The
physical link between these adhesion receptors and the actin cytoskeleton via
intracellular focal adhesion proteins mediates the bidirectional force
transmission and the biochemical signal transduction across the plasma membrane
[14, 15].
The major signaling pathways activated by integrins through their α-subunits are different. The activation of α4 and α9 integrin subunits leads to decreased
cell spreading and increased cell migration on a planar substrate [16–18].
This is in contrast to the activation of other integrin α-subunits (e.g., α1, α5, αv, α3, α6, and αIIb) that lead to enhanced cell
spreading and decreased cell migration [19–23]
and show different bindings
to fibronectin via the canonical integrin recognition site
arginine-glycine-aspartic acid (RGD) [24–28].
Furthermore, the α5β1 integrin regulates the αvβ3-mediated adhesions and migration on the
extracellular matrix [29]. The formation, activation,
and regulation of focal adhesion proteins in the focal adhesions and their physiological
function are pivotal and have been the focus of intense research efforts and
may have opposite effects depending on the integrin subunit type [9],
but the mechano-regulating function, for example, in the process of cell
invasion is still elusive. The αvβ3, αvβ6, and α6β4 integrins have been described to act
as enhancers of tumor cell invasion and metastasis formation [30–32].
Their role in the process of metastasis formation and their impact on
transendothelial migration of tumor cells are still unclear.Theβ1 integrins have also been reported to promote tumor cell migration in a matrigel [33].
To make it more confusing, the role of the α5β1 in the process of 3D cell invasion,
tumor growth and metastasis formation is controversially discussed because several reports
described an invasion enhancing, tumor growth promoting, and metastasis
increasing role that is inversely correlated with E-cadherin expression [34, 35],
but others reported it vice versa [36–38].
The differences of the results might be explained by the use of different tumor
cell lines. The correlation of the expression of one gene alone might not be
helpful in determining the malignant potential of a tumor. The combination of
gene expression and biophysical measurements of the cell's mechanical properties
could be more sufficient because the expression a certain integrin by itself changes
the cytoskeletal structure of a cell [39]. Combined with gene
expression data, the overall cellular stiffness or the cytoskeletal dynamics of
a cell could explain cellular behavior, for example, cell invasion and help to
understand the general mechanisms important for the regulation of this process [6].


## 2.2. Role of The Extracellular Matrix Composition and Mechanical  Properties of The Tissue

The mechanical properties of the extracellular matrix determine the gene
expression patterns, the differentiation, proliferation, and cell mechanical
properties [40–42].
The ability to generate forces might also be influenced by the mechanical
properties of the 3D environment of the cell [43]. Furthermore, the
environmental properties of the primary tumor might also determine the
secondary tumor formation site maybe because of similar mechanical properties
of the primary tumor tissue and the new metastasis formation site. Adhesion
strength and cell stiffness determine the strength of the contractile forces [43] and might influence the
ability of cells to invade into the extracellular matrix and to transmigrate
through an endothelial cell monolayer and the basement membrane surrounding the
vessels. The ability to remodel their cytoskeleton to squeeze through the pores
of the extracellular matrix (amoeboid migration) with or without the generation
of contractile forces is important for the invasion speed, invasion depth, and
finally the ability to form metastasis. For the migration in a 3D matrix, the
CSK-fluidity may play also an important role, the higher the CSK-fluidity the
higher the invasiveness of the cells (Figure 2).


## 2.3. Enzymatic Modulation of The Mechanical Properties of A 3d Connective Tissue

Changes of the extracellular matrix composition and structure (e.g.,
matrix pore size) influence the mechanical parameters of the matrix that could impact
cell invasiveness via changing the mechanical properties inside the cell. These
changes of the extracellular matrix's mechanical properties could origin from
the invading cell itself. For example, cell shape changes such as protrusion
dynamics of mesenchymal tumor cells were monitored in combination with matrix
deformation due to enzymatic matrix remodeling using a 3D collagen matrix
embedded with polystyrene beads [44]. The cells themselves produce
enzymes either membrane-bound (e.g., MT-MMPs) or secreted to modulate the
adjacent extracellular matrix [33].
The expression of collagenases  MMP-14, MMP-12, and MMP-9  increase the
invasiveness of tumor cells migrating through a collagen fiber network or
through a Boyden-chamber (transwell membrane) [33].
MMP-9 cooperates with the αvβ3 integrin in supporting breast
carcinoma cell migration using transwell membrane assay [20]. The inhibition of these enzymes
using a cocktail of  inhibitors leads to reduced or impaired invasion of tumor cells and changes
the prior mesenchymal invasion to an amoeboid invasion type [33].
The production of sheddases leads to the cleavage (e.g., ADAMs = a disintegrin
and metalloprotease domain) of several adhesion receptors like α6 integrin and αvβ3 integrin, reduces cell adhesion, and
increases motility [22, 45, 46].
ADAMs are a family of type I transmembrane glycoproteins that have the ability
to bind to integrins
and disrupt their adhesion to the extracellular matrix [47–49].
The ADAM-15 sheddase contains an RGD-peptide motif and can hence act as a
natural binding partner for αvβ3 integrin and reduce cell adhesion, increasing migration and
invasion of tumor cells [50].
Despite the binding to αvβ3 integrin, the ADAM-15 interacts also
with the α5β1 integrin [51].
Hence, the expression of sheddases like ADAM-15 is associated with a poor
diagnosis of the cancer disease [52] and supports the interaction
of prostate cancer cells and endothelial cells in the process of metastasis
formation [53]. Confirming that the
overexpression of MMP-14 increased the invasiveness of the cells, more cells
were able to invade and they invade deeper into the gel via enhanced motility (or
a higher adhesion site turn over rate). The cutting of the extracellular part
of membrane-bound receptors reduced their cell surface expression that leads to
decreased adhesion strength and decreased traction generation. All these
enzymes alter either the extracellular matrix adjacent to the invading cell or
the adhesion receptor expression on the migrating cell itself. This might
influence, in turn, the adhesion strength (e.g., the adhesion process) and the
generation of contractile forces (Figure 2).


## 3. Interactions of Tumor Cells and  Endothelial Cells

### 3.1. Transendothelial Migration: Intravasation and Extravasation of Tumor Cells

The metastasis formation process begins
with the dissociation of single or clustered tumor cells from the primary tumor
and is followed by extracellular matrix invasion, entrance into blood or lymph
vessels (intravasation), and transport to other tissue sites of the body (Figure1). It has been reported that tumor cells sometimes escape from the
microvasculature (extravasation), invade into the tumor-type dependent
specified target tissue, and form a secondary tumor in distant organs [3, 54, 55].
The mechanisms of the tumor-type specific targeting of secondary tumor
formation are almost unknown. Some people speculate about  mechanical
properties of the tissues determining different target-organ selections for secondary
tumor formation [56, 57].
The extravasation process as well as the intravasation process involves
adhesion of tumor cells to endothelial cells and the transmigration through the
endothelium and underlying basement membrane [3, 58–60].
For several tumor cell types, it has been reported that they are able to pass
the endothelial barrier (perivascular metastasis) [60–65].
Nevertheless, the extravasation step is not the only mechanism for metastasis
formation, it is also possible that tumor cells intravasate into vessels, but they
never extravasate. These tumor cells grow inside the vessels on the endothelial
cell layer and form the metastasis at these sites (intravascular metastasis) [4].
Despite the extravasation step performed, the function of the endothelial
monolayer in the metastasis formation process is thought to be crucial in that
it can actively regulate metastasis formation by either allowing or blocking
the adhesion, and possibly transmigration, of tumor cells and thereby
determining the target organ of secondary tumor formation [62–64].
The endothelial cell function in the process of metastasis formation is still
unclear, and the effect of the endothelium even to promote tumor cell invasion
has recently been reported, but the mechanism is poorly understood [6]. More insights might bring
novel in vivo models on metastasis formation.


### 3.2. in Vivo Models: Vascular and Lymphatic Routes of Metastasis Formation

Metastasis
formation occurs via the vascular system as well via the lymphatic system [4, 66–68].
Tumor cells can form intravascular and perivascular metastases [3, 4, 69]
(Figure 1). Any type of cancer process, including tumor cell motility and
invasion, as well as metastasis formation can be measured using animal models
in combination with fluorescent proteins that stain the injected tumor cells as
well as the host tissue. Naturally fluorescent
proteins, especially those with long excitation wavelengths, visualize in real
time, aspects of tumor initiation, tumor growth, and metastasis formation in
living animals [66, 68, 70–73]. The benefit of these novel in vivo rodent metastasis models allows us to study
migration directly in live blood or lymph vessels and tumor-host interactions
in the presence or in the absence of anticancer drugs or chemotherapy drugs [67, 71, 74, 75]. Besides, the injection of tumor cells whole tissue explants (named
orthotropic model) can be transferred into nude mice and studied for response
to drugs or vascularization [71, 76]. These studies show that tumor cells metastasize via vascular and
lympathic vessels [4, 66–68]. The
lymphatic route (lymphatic system) of the tumor metastasis
formation is less well understood compared to the vascular route (circulatory
system) [77]. Using the real-time imaging of fluorescently labeled tumor cells that
were injected in the inguinal lymph node, tumor cell survival and migration to
the axillary lymph node were
measured. In addition, the effect of pressure on tumor cell shedding into lymph
vessels and migration to the lymph node was analyzed. Due to the clinical studies  indicating that high interstitial fluid pressure in the tumor is correlated
with a poor diagnosis [78–80], this lymphatic animal model shows that increased pressure on the
tumor increased the numbers of shedded tumor cells that may lead to higher
lymphatic metastasis formation [68]. The mechanical properties of the extracellular matrix (in this case of
the tumor) change the cellular properties toward  higher motility and
invasiveness. A  combination of these in vivo models with biophysical measurements will help to get
more insights and reveal the mechanisms why some tumors metastasize and others
do not and what role the endothelium plays.


### 3.3. Does The Endothelium Represent A Barrier for Tumor Cell Invasion?

It is still unclear whether tumor cells transmigrate through the
cytoplasm of  endothelial cells similar to leukocytes, or destroy
endothelial cells [81], or transmigrate between two
adjacent endothelial cells by Src-mediated disruption of their cell-cell
connections (VE-cadherin-β-catenin) [61].
It has been reported that transmigrating tumor cells are able to overcome the
endothelial barrier by inducing changes within the endothelial cell monolayer,
including the upregulation of adhesion molecule receptor expression [82],
the reorganization of the cytoskeleton [83],
Src-mediated disruption of endothelial cell-cell-adhesions [61],
the formation of “holes” within the endothelial layer [84], and the induction of
apoptosis [81] (Figure 3). Furthermore,
tumor cell invasion may use similar invasion strategies as leukocytes, for
which the endothelium acts as a barrier and greatly reduces invasion rates [85].Three currently discussed strategies for tumor cell transmigration through
an endothelial cell monolayer. (a) The tumor cell transmigrates through the
cytoplasm of the cell body. (b) The tumor cell induced apoptosis in the adjacent endothelial cell and
migrated through the hole in the endothelial cell layer. (c) The tumor cell
transmigrates through the endothelial cell-cell contacts without permanent
destroying the endothelial cell monolayer.
(a)(b)(c)


### 3.4. How Do Cell-Cell Adhesion Receptors Influence Tumor Cell Invasion?

Beside cell-matrix receptors, cell-cell adhesion receptors play a role in
cell invasion and especially facilitate transendothelial migration of tumor
cells as well as leukocytes. It has been reported thatαvβ3 integrin as well as glycosylphatidylinositol-anchored CD24
protein facilitates
tumor cell transmigration and enhances tumor cell invasion in a 3D collagen fiber network [6, 8, 86]
(Figure 4). Endothelial cells
expressed L1-CAM and PECAM-1 have been described as counter-receptors that
interact with cancer cell αvβ3 integrin [87, 88].
Endothelial cells
expressed L1-CAM, and P-selectin have been described as a ligands for CD24 [7, 89].
A loss of E-cadherin cell-cell contacts is important for single cell invasion
and has recently been discovered as a tumor suppressor gene [5]. The α4β1
integrin interacts either with VCAM-1
or CD14 to enhance tumor cell migration or invasion [11, 90].
It has to be ruled out how the interaction of tumor cells and endothelial cells
through these receptors might, in turn, alter the mechanical properties of both
cell types and change their invasive potential.Figure 4
Interactions between tumor and
endothelial cells during tumor cell adhesion and transmigration.


### 3.5. Role of Cytokines, Chemokines, or Other Stimuli in Tumor Cell Invasion and Transmigration

The scatter factor/hepatocyte growth factor (HGF) has largely been analyzed to
enhance tumor cell migration on  2D substrates in in vitro cell culture assays [91].
Consistent to in vitro experiments, in vivo experiments have shown that this factor
increased tumor cell spreading, dissemination, and metastasis formation in mice [91].
Recently, it has been shown that HGF influences the expression of integrin receptors,
which in turn mediates the secretion of MMPs [92].
Despite this interaction, HGF ligand binding to the Met receptor as well as the
α6β4 integrin can independently enhance tumor cell invasion [32].Cytokines
affect the barrier function of an endothelial cell monolayer (Figure5). For
example, the function of the endothelial cell barrier against both leukocyte
trafficking and tumor cell transmigration is reduced in the presence of inflammatory
cytokines like such as necrosis factor-α and interleukin-1β [62, 82, 93, 94].
These cytokines induce an upregulation of the cell-cell adhesion molecule
E-selectin [82].
The subsequent adhesion of tumor cells expressing the E-selectin
counter-receptor sialyl Lewis x (CD15s) to endothelial E-selectin leads to an
upregulation of stress-activated protein kinase-2 (SAPK2/p38) in endothelial
cells [82]
and triggers actin polymerization and actin reorganization into stress fibers [83]
(Figure 4).Figure 5
Prerequisites for tumor cell transendothelial migration. Single tumor cells adhere to the endothelium
via cell adhesion receptors like integrins, muccins, or immunoglobulins and
their counter-receptors on endothelial cells. Many other factors like enzymes,
cytokines/chemokines influence these interaction as well as biomechanical
properties of both cells (CSK-dynamics, CSK-fluidity, cell stiffness, and
contractile force generation).Chemokines
and their receptors have an impact on leukocyte trafficking [95, 96]
and tumor cell invasion [97]. The superfamily of chemokines
consists of small cytokine-like proteins that induce cytoskeletal
rearrangements in endothelial cells and leukocytes, the firm adhesion of
leukocytes to endothelial cells, and the directional migration (chemotaxis) of
leukocytes [95]. The involvement of
chemokines in tumor-endothelial interactions and their effect on tumor cell
mechanical properties during invasion are considerably less well understood.We recently found that the expression of the chemokine receptor CXCR2 enhanced
tumor cell motility in 3D extracellular collagen matrix. The expression of the
CXCR2 influences other mechanical properties of the receptor bearing cells [6]. High CXCR2 expressing
subcell lines derived from the human breast carcinoma cells MDA-MB-231 produced
enhanced traction forces on fibronectin-coated 2D substrates [6]. CSK-dynamics, cell stiffness,
and CSK-fluidity are
enhanced [6].Another
impact of the CXCR2 receptor in the metastasis formation process could be the
interaction with endothelial cells producing the CXCR2 ligands Gro-β and IL-8. Both chemokines enhance the
transmigration and the followed invasiveness in vitro 3-D collagen fiber matrix
assays [6]. Recently, it has been
reported that Gro-β and IL-8 receptor (CXCR2) expressions on tumor cells
are the key mediators responsible for
the breakdown of the endothelial barrier function by enhancing tumor cell force
generation and cytoskeletal remodeling dynamics (Figure 6). These results have
to be expanded on other tumor cell types and further improved by using animal
metastases models. All these different stimulations of the cells might underlie
a common change of the cell's mechanical properties.New role of the endothelium
in the process of tumor cell invasion. (a) A human endothelial cell monolayer (e.g., HUVEC)
cultured on top of a collagen fiber network-induced tumor cell invasion of
cocultured human tumor cell lines. (b) The endothelium decreased the invasion
of several invasive tumor cell lines (barrier for cell invasion), did not alter
the invasion of tumor cells, or even enhanced tumor cell invasion into a
collagen gel fiber matrix (enhancer for cell invasion).
(a)(b)


## 3.1. Transendothelial Migration: Intravasation and Extravasation of Tumor Cells

The metastasis formation process begins
with the dissociation of single or clustered tumor cells from the primary tumor
and is followed by extracellular matrix invasion, entrance into blood or lymph
vessels (intravasation), and transport to other tissue sites of the body (Figure1). It has been reported that tumor cells sometimes escape from the
microvasculature (extravasation), invade into the tumor-type dependent
specified target tissue, and form a secondary tumor in distant organs [3, 54, 55].
The mechanisms of the tumor-type specific targeting of secondary tumor
formation are almost unknown. Some people speculate about  mechanical
properties of the tissues determining different target-organ selections for secondary
tumor formation [56, 57].
The extravasation process as well as the intravasation process involves
adhesion of tumor cells to endothelial cells and the transmigration through the
endothelium and underlying basement membrane [3, 58–60].
For several tumor cell types, it has been reported that they are able to pass
the endothelial barrier (perivascular metastasis) [60–65].
Nevertheless, the extravasation step is not the only mechanism for metastasis
formation, it is also possible that tumor cells intravasate into vessels, but they
never extravasate. These tumor cells grow inside the vessels on the endothelial
cell layer and form the metastasis at these sites (intravascular metastasis) [4].
Despite the extravasation step performed, the function of the endothelial
monolayer in the metastasis formation process is thought to be crucial in that
it can actively regulate metastasis formation by either allowing or blocking
the adhesion, and possibly transmigration, of tumor cells and thereby
determining the target organ of secondary tumor formation [62–64].
The endothelial cell function in the process of metastasis formation is still
unclear, and the effect of the endothelium even to promote tumor cell invasion
has recently been reported, but the mechanism is poorly understood [6]. More insights might bring
novel in vivo models on metastasis formation.


## 3.2. in Vivo Models: Vascular and Lymphatic Routes of Metastasis Formation

Metastasis
formation occurs via the vascular system as well via the lymphatic system [4, 66–68].
Tumor cells can form intravascular and perivascular metastases [3, 4, 69]
(Figure 1). Any type of cancer process, including tumor cell motility and
invasion, as well as metastasis formation can be measured using animal models
in combination with fluorescent proteins that stain the injected tumor cells as
well as the host tissue. Naturally fluorescent
proteins, especially those with long excitation wavelengths, visualize in real
time, aspects of tumor initiation, tumor growth, and metastasis formation in
living animals [66, 68, 70–73]. The benefit of these novel in vivo rodent metastasis models allows us to study
migration directly in live blood or lymph vessels and tumor-host interactions
in the presence or in the absence of anticancer drugs or chemotherapy drugs [67, 71, 74, 75]. Besides, the injection of tumor cells whole tissue explants (named
orthotropic model) can be transferred into nude mice and studied for response
to drugs or vascularization [71, 76]. These studies show that tumor cells metastasize via vascular and
lympathic vessels [4, 66–68]. The
lymphatic route (lymphatic system) of the tumor metastasis
formation is less well understood compared to the vascular route (circulatory
system) [77]. Using the real-time imaging of fluorescently labeled tumor cells that
were injected in the inguinal lymph node, tumor cell survival and migration to
the axillary lymph node were
measured. In addition, the effect of pressure on tumor cell shedding into lymph
vessels and migration to the lymph node was analyzed. Due to the clinical studies  indicating that high interstitial fluid pressure in the tumor is correlated
with a poor diagnosis [78–80], this lymphatic animal model shows that increased pressure on the
tumor increased the numbers of shedded tumor cells that may lead to higher
lymphatic metastasis formation [68]. The mechanical properties of the extracellular matrix (in this case of
the tumor) change the cellular properties toward  higher motility and
invasiveness. A  combination of these in vivo models with biophysical measurements will help to get
more insights and reveal the mechanisms why some tumors metastasize and others
do not and what role the endothelium plays.


## 3.3. Does The Endothelium Represent A Barrier for Tumor Cell Invasion?

It is still unclear whether tumor cells transmigrate through the
cytoplasm of  endothelial cells similar to leukocytes, or destroy
endothelial cells [81], or transmigrate between two
adjacent endothelial cells by Src-mediated disruption of their cell-cell
connections (VE-cadherin-β-catenin) [61].
It has been reported that transmigrating tumor cells are able to overcome the
endothelial barrier by inducing changes within the endothelial cell monolayer,
including the upregulation of adhesion molecule receptor expression [82],
the reorganization of the cytoskeleton [83],
Src-mediated disruption of endothelial cell-cell-adhesions [61],
the formation of “holes” within the endothelial layer [84], and the induction of
apoptosis [81] (Figure 3). Furthermore,
tumor cell invasion may use similar invasion strategies as leukocytes, for
which the endothelium acts as a barrier and greatly reduces invasion rates [85].Three currently discussed strategies for tumor cell transmigration through
an endothelial cell monolayer. (a) The tumor cell transmigrates through the
cytoplasm of the cell body. (b) The tumor cell induced apoptosis in the adjacent endothelial cell and
migrated through the hole in the endothelial cell layer. (c) The tumor cell
transmigrates through the endothelial cell-cell contacts without permanent
destroying the endothelial cell monolayer.
(a)(b)(c)


## 3.4. How Do Cell-Cell Adhesion Receptors Influence Tumor Cell Invasion?

Beside cell-matrix receptors, cell-cell adhesion receptors play a role in
cell invasion and especially facilitate transendothelial migration of tumor
cells as well as leukocytes. It has been reported thatαvβ3 integrin as well as glycosylphatidylinositol-anchored CD24
protein facilitates
tumor cell transmigration and enhances tumor cell invasion in a 3D collagen fiber network [6, 8, 86]
(Figure 4). Endothelial cells
expressed L1-CAM and PECAM-1 have been described as counter-receptors that
interact with cancer cell αvβ3 integrin [87, 88].
Endothelial cells
expressed L1-CAM, and P-selectin have been described as a ligands for CD24 [7, 89].
A loss of E-cadherin cell-cell contacts is important for single cell invasion
and has recently been discovered as a tumor suppressor gene [5]. The α4β1
integrin interacts either with VCAM-1
or CD14 to enhance tumor cell migration or invasion [11, 90].
It has to be ruled out how the interaction of tumor cells and endothelial cells
through these receptors might, in turn, alter the mechanical properties of both
cell types and change their invasive potential.Figure 4
Interactions between tumor and
endothelial cells during tumor cell adhesion and transmigration.


## 3.5. Role of Cytokines, Chemokines, or Other Stimuli in Tumor Cell Invasion and Transmigration

The scatter factor/hepatocyte growth factor (HGF) has largely been analyzed to
enhance tumor cell migration on  2D substrates in in vitro cell culture assays [91].
Consistent to in vitro experiments, in vivo experiments have shown that this factor
increased tumor cell spreading, dissemination, and metastasis formation in mice [91].
Recently, it has been shown that HGF influences the expression of integrin receptors,
which in turn mediates the secretion of MMPs [92].
Despite this interaction, HGF ligand binding to the Met receptor as well as the
α6β4 integrin can independently enhance tumor cell invasion [32].Cytokines
affect the barrier function of an endothelial cell monolayer (Figure5). For
example, the function of the endothelial cell barrier against both leukocyte
trafficking and tumor cell transmigration is reduced in the presence of inflammatory
cytokines like such as necrosis factor-α and interleukin-1β [62, 82, 93, 94].
These cytokines induce an upregulation of the cell-cell adhesion molecule
E-selectin [82].
The subsequent adhesion of tumor cells expressing the E-selectin
counter-receptor sialyl Lewis x (CD15s) to endothelial E-selectin leads to an
upregulation of stress-activated protein kinase-2 (SAPK2/p38) in endothelial
cells [82]
and triggers actin polymerization and actin reorganization into stress fibers [83]
(Figure 4).Figure 5
Prerequisites for tumor cell transendothelial migration. Single tumor cells adhere to the endothelium
via cell adhesion receptors like integrins, muccins, or immunoglobulins and
their counter-receptors on endothelial cells. Many other factors like enzymes,
cytokines/chemokines influence these interaction as well as biomechanical
properties of both cells (CSK-dynamics, CSK-fluidity, cell stiffness, and
contractile force generation).Chemokines
and their receptors have an impact on leukocyte trafficking [95, 96]
and tumor cell invasion [97]. The superfamily of chemokines
consists of small cytokine-like proteins that induce cytoskeletal
rearrangements in endothelial cells and leukocytes, the firm adhesion of
leukocytes to endothelial cells, and the directional migration (chemotaxis) of
leukocytes [95]. The involvement of
chemokines in tumor-endothelial interactions and their effect on tumor cell
mechanical properties during invasion are considerably less well understood.We recently found that the expression of the chemokine receptor CXCR2 enhanced
tumor cell motility in 3D extracellular collagen matrix. The expression of the
CXCR2 influences other mechanical properties of the receptor bearing cells [6]. High CXCR2 expressing
subcell lines derived from the human breast carcinoma cells MDA-MB-231 produced
enhanced traction forces on fibronectin-coated 2D substrates [6]. CSK-dynamics, cell stiffness,
and CSK-fluidity are
enhanced [6].Another
impact of the CXCR2 receptor in the metastasis formation process could be the
interaction with endothelial cells producing the CXCR2 ligands Gro-β and IL-8. Both chemokines enhance the
transmigration and the followed invasiveness in vitro 3-D collagen fiber matrix
assays [6]. Recently, it has been
reported that Gro-β and IL-8 receptor (CXCR2) expressions on tumor cells
are the key mediators responsible for
the breakdown of the endothelial barrier function by enhancing tumor cell force
generation and cytoskeletal remodeling dynamics (Figure 6). These results have
to be expanded on other tumor cell types and further improved by using animal
metastases models. All these different stimulations of the cells might underlie
a common change of the cell's mechanical properties.New role of the endothelium
in the process of tumor cell invasion. (a) A human endothelial cell monolayer (e.g., HUVEC)
cultured on top of a collagen fiber network-induced tumor cell invasion of
cocultured human tumor cell lines. (b) The endothelium decreased the invasion
of several invasive tumor cell lines (barrier for cell invasion), did not alter
the invasion of tumor cells, or even enhanced tumor cell invasion into a
collagen gel fiber matrix (enhancer for cell invasion).
(a)(b)


## 4. Role of Other Cell Types during  Metastasis Formation

Several recent reports discussed the importance of platelets in adhering
to single tumor cells and promote or induce their transendothelial migration [98–100],
but in contrast to them, many other reports show clearly that single tumor
cells are by themselves able to transmigrate through the endothelium [6–8, 62].
The platelets might be able to decorate single tumor cells and enhance their
transmigration and invasion capabilities. But the question of do other cell types promote
or induce tumor cell invasion and metastasis formation is still not answered
fully. Do platelets guide tumor cells through the endothelium? For example, the
role of mast cells either in solid tumors or in close neighborhood to tumors is
poorly investigated. Did mast cells also promote or induce tumor cell
transmigration or invasion? Do mast cells stimulate tumor cells to contract
after histamine release? It has been reported that mast cells survive in close
contact to endothelial cells and
even proliferate there [101].
This might explain the increased numbers of mast cells in vascularized solid
tumors [102–104].
Of course other cell types like eosinophilic granulocytes, lymphocytes, or
monocytes/macrophages might also influence the mechanical properties of tumor
cells either by secretion of contractile force stimulating (enhancing) or by
relaxing the substances. The role of mast cells in tumor metastasis formation
has to be ruled out by using biophysical tools as well as dual-color imaging
animal metastasis models.The presence of macrophages in a tumor has been correlated with a poor
prognosis, but how macrophages are involved in hematogenous metastasis was
unclear. Do macrophages change the mechanical properties of the tumor cells
either by direct binding or by secretion of cytokines or by growth factors? Direct
visualization of macrophages that assisted tumor cell intravasation in
hematogenous metastasis of mammary tumors has been reported by using
multiphoton microscopy [105]. Macrophages supported tumor
cell motility toward blood vessels and tumor cell intravasation [105]. How this affects the
mechanical properties of the tumor cells and their ability to generate
contractile forces to move forward remain unclear and should be further
investigated using biophysical tools, for example, traction microscopy. The dual-color
fluorescence imaging technology distinguishes injected and fluorescently
labeled tumor cells from the host stroma cells of transgenic mice and enables
further insights in tumor-host interactions [106, 107].


## 5. Contractile Forces and the Role of Vinculin

### 5.1. Measurement of Contractile Forces

To move forward in a 3D matrix, the invading tumor cells need to generate
contractile forces to overcome the viscous drag. Despite the composition and
the enzymatic digestion of the matrix, the coordinated regulation of cell adhesion
(adhesion strength) and deadhesion (including cytoskeletal remodeling)  together
with the generation of contractile forces determine   the invasion speed in a 3D matrix. Our
knowledge of cell migration, mechanical tensions, and forces is derived from
studies of cells cultured on planar substrates (e.g., glass or polyacrylamide
hydrogels). Previous reports showed how to measure traction forces during cell
migration in 2D culture systems [108, 109]
and were further improved to quantitative tools [110–113].
All the methods depend on the measurement of the deformations of an elastic
substrate with known elastic modulus on which the cells adhered and spread. During
this cell-matrix contact, the cell generates tractions and deforms the
substrate. The tractions were computed from the substrate displacements
visualized by embedded markers (fluorescent 0.5 μm beads) using continuum
mechanics theory. Measurement of the displacement field is accomplished by
tracking these beads that are near the surface of the substrate. The spatial
resolution of the traction map obtained with traction microscopy approaches is 1 μm, which is sufficient to resolve the forces from individual focal adhesions [112]. Traction microscopy has
brought new insights into tumor cell migration in particular [6, 86, 113].
The cells feel and respond to the stiffness of the extracellular matrix through
dynamic regulation of integrin clustering, altered focal adhesion formation,
and remodeling of the cell's cytoskeleton [40]. To conclude, contractile
force generation and cell invasion are strongly influenced by the mechanical
properties of the extracellular matrix [109].


### 5.2. Cell-Matrix Adhesions Protein Vinculin

For the generation of contractile forces, a cell needs to adhere to the
3D matrix via cell-matrix adhesions.  The focal adhesion
protein vinculin has been described to be structurally important connecting
the extracellular matrix via cell-matrix adhesions and adjacent cells via
cell-cell adhesions with the actin cytoskeleton by providing an intracellular
linkage between the integrin receptor and the actin cytoskeleton via the focal
adhesion formation [114–117].
The vinculin protein is encoded by 1066 amino acids, has a molecular weight of
~117 kDa, and binds to many other focal adhesion and cytoskeletal proteins
including talin, paxillin, tensin, zyxin, VASP,  α-actinin and actin  [118].The function of vinculin has been largely analyzed in cells cultured in 2D systems.
Vinculin knock out (k.o.) mouse embryonic fibroblasts (MEFs) as well as
vinculin k.o. mouse embryonic carcinoma cells (F9 cells) exhibit a more round cell
shape, show fewer and less stable lamellipodia, but similar filopodia compared
to vinculin-expressing MEFs (wild type) [116, 119, 120].
Focal adhesion complexes of vin-/- cells are smaller and consist of increased
tyrosine-phosphorylated proteins like talin, α-actinin, FAK, and paxillin without increasing
total protein content [116, 121].
Vinculin-deficient fibroblasts (MEFvin-/-)
and carcinoma cells (vin-/-) show reduced adhesion to extracellular matrix
proteins like fibronectin, vitronectin, laminin, and collagen as well as a two-fold
increase in motility and three-fold increase in focal adhesion kinase activity
compared to wild-type cells [121, 122].
All the described studies were performed in 2D migration or 2D adhesion assays
and did not properly mirror the in
vivo situation of cells invading and contracting the 3D tissue environment
during the malignant progression of tumor disease involving tumor outgrowth and
metastasis formation.Vinculin's
function in tumor disease has so far hardly been investigated. We recently reported
that vinculin functions as a mechano-coupling protein, and that its tail-domain
acts as a regulator for contractile force generation [86].In a recent study, we examined whether vinculin is involved in cell invasion into
a 3D extracellular matrix [123]. We found that vinculin enhanced
the generation of tractions, and that this mechanical parameter despite all
other mechanical parameters (e.g., cell adhesion, detachment, and matrix
degradation) plays a prominent role in cell invasion in a 3D matrix to overcome
the steric hindrances, but not on a 2D substrate, where the viscous drag is
negligible. Future analyzes of the transmission of forces in cells via focal
adhesions might help to determine the prerequisites of the invasive potential
of a tumor cell.


## 5.1. Measurement of Contractile Forces

To move forward in a 3D matrix, the invading tumor cells need to generate
contractile forces to overcome the viscous drag. Despite the composition and
the enzymatic digestion of the matrix, the coordinated regulation of cell adhesion
(adhesion strength) and deadhesion (including cytoskeletal remodeling)  together
with the generation of contractile forces determine   the invasion speed in a 3D matrix. Our
knowledge of cell migration, mechanical tensions, and forces is derived from
studies of cells cultured on planar substrates (e.g., glass or polyacrylamide
hydrogels). Previous reports showed how to measure traction forces during cell
migration in 2D culture systems [108, 109]
and were further improved to quantitative tools [110–113].
All the methods depend on the measurement of the deformations of an elastic
substrate with known elastic modulus on which the cells adhered and spread. During
this cell-matrix contact, the cell generates tractions and deforms the
substrate. The tractions were computed from the substrate displacements
visualized by embedded markers (fluorescent 0.5 μm beads) using continuum
mechanics theory. Measurement of the displacement field is accomplished by
tracking these beads that are near the surface of the substrate. The spatial
resolution of the traction map obtained with traction microscopy approaches is 1 μm, which is sufficient to resolve the forces from individual focal adhesions [112]. Traction microscopy has
brought new insights into tumor cell migration in particular [6, 86, 113].
The cells feel and respond to the stiffness of the extracellular matrix through
dynamic regulation of integrin clustering, altered focal adhesion formation,
and remodeling of the cell's cytoskeleton [40]. To conclude, contractile
force generation and cell invasion are strongly influenced by the mechanical
properties of the extracellular matrix [109].


## 5.2. Cell-Matrix Adhesions Protein Vinculin

For the generation of contractile forces, a cell needs to adhere to the
3D matrix via cell-matrix adhesions.  The focal adhesion
protein vinculin has been described to be structurally important connecting
the extracellular matrix via cell-matrix adhesions and adjacent cells via
cell-cell adhesions with the actin cytoskeleton by providing an intracellular
linkage between the integrin receptor and the actin cytoskeleton via the focal
adhesion formation [114–117].
The vinculin protein is encoded by 1066 amino acids, has a molecular weight of
~117 kDa, and binds to many other focal adhesion and cytoskeletal proteins
including talin, paxillin, tensin, zyxin, VASP,  α-actinin and actin  [118].The function of vinculin has been largely analyzed in cells cultured in 2D systems.
Vinculin knock out (k.o.) mouse embryonic fibroblasts (MEFs) as well as
vinculin k.o. mouse embryonic carcinoma cells (F9 cells) exhibit a more round cell
shape, show fewer and less stable lamellipodia, but similar filopodia compared
to vinculin-expressing MEFs (wild type) [116, 119, 120].
Focal adhesion complexes of vin-/- cells are smaller and consist of increased
tyrosine-phosphorylated proteins like talin, α-actinin, FAK, and paxillin without increasing
total protein content [116, 121].
Vinculin-deficient fibroblasts (MEFvin-/-)
and carcinoma cells (vin-/-) show reduced adhesion to extracellular matrix
proteins like fibronectin, vitronectin, laminin, and collagen as well as a two-fold
increase in motility and three-fold increase in focal adhesion kinase activity
compared to wild-type cells [121, 122].
All the described studies were performed in 2D migration or 2D adhesion assays
and did not properly mirror the in
vivo situation of cells invading and contracting the 3D tissue environment
during the malignant progression of tumor disease involving tumor outgrowth and
metastasis formation.Vinculin's
function in tumor disease has so far hardly been investigated. We recently reported
that vinculin functions as a mechano-coupling protein, and that its tail-domain
acts as a regulator for contractile force generation [86].In a recent study, we examined whether vinculin is involved in cell invasion into
a 3D extracellular matrix [123]. We found that vinculin enhanced
the generation of tractions, and that this mechanical parameter despite all
other mechanical parameters (e.g., cell adhesion, detachment, and matrix
degradation) plays a prominent role in cell invasion in a 3D matrix to overcome
the steric hindrances, but not on a 2D substrate, where the viscous drag is
negligible. Future analyzes of the transmission of forces in cells via focal
adhesions might help to determine the prerequisites of the invasive potential
of a tumor cell.


## 6. How Can Mechanical Parameters of the Cells Help to Improve Tumor Diagnosis?

Several reports describe a relationship between tumor cell mechanical
properties and their malignant potential in promoting metastasis formation [6, 43, 124].
The measurement of only a single cell mechanical parameter might not be
sufficient to predict the invasiveness of tumor cells nor their metastasis formation
potential. Many factors may be involved in promoting tumor cell invasion (Figure 2) and
tumor cell transendothelial formation (Figure 5). The mechanical
properties of the tumor cells as well as their adhesion receptor, sheddases, or
enzyme expressions
together determine the ability to invade a tissue and to transmigrate the
endothelium (Figures 2 and 5). Only small fraction of tumor cell lines is able to invade a 3D extracellular
matrix [6]. To provide a proper analysis tool to determine the invasive or
metastasis potential of a tumor, it is first important to isolate subcell
fractions of isolated primary tumor cells using well-defined mechanical
parameters (contractile force generation). These obtained primary tumor cells
can be analyzed for their metastatic potential by using metastatic animal
models.


## 7. Role of Clinically Relevant Metastatic  Animal Models

Orthotropic metastatic animal models can be used to discover
anticancer drugs and to determine drugs' efficiency [71]. These models are established for a wide variety of tumors including
spontaneous metastatic bone models of prostate cancer, breast cancer, and lung
cancer as well as spontaneous
liver and lymph node metastastic models of colon, pancreatic, stomach, ovarian, bladder, and
kidney cancer [71]. These models present a new generation of rodent tumor models and
differ from transgenic tumor models or subcuntanenously-growing human tumors in
immunodeficient mice, because the latter models did not represent sufficient
models in regard to metastasis formation or drug sensitivity [71]. The advantage of novel orthotropic metastatic models is that
histologically intact fragments of human cancer—directly taken
from the patient—were implanted to
the corresponding organ of immunodeficient mice. This allows researchers to
determine tumor growth and metastatic potential of transplanted tumors [125, 126]. These orthotropic metastatic animal models can be combined with
measurements of the cells mechanical properties and the mechanical properties of
the transplanted tissue. To understand the diverse roles of the
endothelium in the tumor metastasis, a transgenic nude mouse model visualizing
tumor angiogenesis could be helpful. It is based on the nestin-promotor-linked
green fluorescent protein because many of the newly formed blood vessels
originate from hair-follicle stem cells that express nestin. The transcription
of GFP indicates newly formed blood vessels in the animal [127].


## 8. Conclusions and Future Directions

In summary, this paper
presented a new function of the endothelium in inducing or enhancing tumor cell
invasion. It discussed the two described pathways for tumor cell metastasis
formation and pointed out the importance of cell-matrix and cell-cell adhesion receptors as well as
chemokine receptors for tumor-endothelial cell interactions. It focused the
view on the mechanical properties of tumor cells as well as that of endothelial cells
during the process of metastasis formation. It discussed in vivo animal models to visualize vascular
and lymphoid metastases
formation of single injected tumor cells or implanted orthotropic tissue
resections.The main new direction of tumor cell invasion and metastasis research is
to measure the ability of tumor cells to generate contractile forces in a 3D
environment and to determine the malignancy of a tumor using in vivo animal models. The mechanical
measurements of tumor cell subpopulations in patient's liquids or tissue
samples might help to determine whether a tumor is able to form metastases. A
future direction of tumor metastasis research is to focus on the diverse roles
of the endothelium. The combination of biophysical tools for mechanical
properties analysis of tumor and endothelial cells, the generation of
mechanical diverse tumor cells, and with their injection into nude mice using
the dual-color in vivo imaging
method will be extremely helpful in developing new diagnosis tools and drugs
reducing tumor metastases.



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*Source: 183516-2009-03-05.xml*