Mutation Research 728 (2011) 23–34

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Review

Initial steps of metastasis: Cell invasion and endothelial transmigration

Franziska van Zijl a, Georg Krupitza b, Wolfgang Mikulits a,*
a
Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria
b
Institute of Clinical Pathology, Comprehensive Cancer Center Vienna, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria

A R T I C L E I N F O A B S T R A C T

Article history: Metastasis is the leading cause of cancer mortality. The metastatic cascade represents a multi-step
Received 15 December 2010 process which includes local tumor cell invasion, entry into the vasculature followed by the exit of
Received in revised form 4 May 2011 carcinoma cells from the circulation and colonization at the distal sites. At the earliest stage of successful
Accepted 5 May 2011
cancer cell dissemination, the primary cancer adapts the secondary site of tumor colonization involving
Available online 12 May 2011
the tumor–stroma crosstalk. The migration and plasticity of cancer cells as well as the surrounding
environment such as stromal and endothelial cells are mandatory. Consequently, the mechanisms of cell
Keywords:
movement are of utmost relevance for targeted intervention of which three different types have been
Epithelial cell plasticity
Cell invasion
reported. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of
Intravasation movement and intravasate the blood or lymph vasculature. Intravasation by the interaction of tumor
Metastasis cells with the vascular endothelium is mechanistically poorly understood. Changes in the epithelial
plasticity enable carcinoma cells to switch between these types of motility. The types of migration may
change depending on the intervention thereby increasing the velocity and aggressiveness of invading
cancer cells. Interference with collective or mesenchymal cell invasion by targeting integrin expression
or metalloproteinase activity, respectively, resulted in an amoeboid cell phenotype as the ultimate exit
strategy of cancer cells. There are little mechanistic details reported in vivo showing that the amoeboid
behavior can be either reversed or efficiently inhibited. Future concepts of metastasis intervention must
simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order
to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate.
Beyond the targeting of cell movements, the adhesion of cancer cells to the stroma in heterotypic
circulating tumor cell emboli is of paramount relevance for anti-metastatic therapy.
ß 2011 Elsevier B.V. Open access under CC BY-NC-ND license.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2. Mechanisms of cell invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.1. Collective cell invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2. Mesenchymal cell invasion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.3. Amoeboid cell invasion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3. Endothelial transmigration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4. Role of the tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
5. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1. Introduction dothelial migration of cancer cells into vessels known as

intravasation, (iii) survival in the circulatory system, (iv) extrava-
Metastasis is a multi-step process encompassing the (i) local sation and (v) subsequent proliferation in competent organs
infiltration of tumor cells into the adjacent tissue, (ii) transen- leading to colonization [1]. Metastasis is known as a very
inefficient process [2,3], because a coordinated choreography of
the multiple events is required to prevent failure of the complex
* Corresponding author. Tel.: +43 1 4277 65250; fax: +43 1 4277 65239. process that otherwise runs into the elimination of emigrating
E-mail address: [email protected] (W. Mikulits). cancer cells at any of the many steps on the way [2]. Although the

1383-5742 ß 2011 Elsevier B.V. Open access under CC BY-NC-ND license.

doi:10.1016/j.mrrev.2011.05.002
24 F. van Zijl et al. / Mutation Research 728 (2011) 23–34

dissemination from a primary tumor of 1 cm size (roughly favors the intrahepatic metastasis of HCC, physical barriers and
corresponding to 1  109 cancer cells) can infiltrate the circulatory systemic hurdles avoid colonization of gliomas because the
system with one million cancer cells per day [4], subsequent patients mostly die prior to the establishment of metastasis at
colonization is very limited due to incompatible distal sites. distal sites [21]. Besides the influence of the primary tumor on site-
Consequently, <0.1% of disseminated cancer cells successfully specific metastasis, a plethora of biological, chemical and
develop distal metastasis [5,6]. Albeit, dormant solitary tumor cells physiological constraints are governing metastasis [22]. Extrinsic
or micrometastases, which represent undetectable cancer cell chemical barriers are represented by e.g. pH, reactive oxygen
populations due to either a cell cycle arrest or a balance between species or hypoxia. The cellular microenvironment regulates
proliferation and apoptosis [6], might eventually outgrow to tumor dissemination by regulatory cytokine and chemokine
clinically detectable macrometastases many years post anti-cancer feedback loops and by the secretion of matrix metalloproteinases
treatment [6–8]. (MMPs). Physiological constraints include intratumoral tensional
The discussion whether metastasis is an early or late event in forces, the composition of the basement membrane and the
tumor progression is ongoing and still remains an open issue [9]. anatomy of capillary walls. Fenestrated bone marrow sinusoids are
On the one hand, the ‘‘linear progression model’’ suggests cancer rather easy to penetrate in comparison to the lung capillaries or the
cell dissemination after extensive expansion of primary tumors, blood brain barrier [18]. Thus, the anatomical differences and the
whereas on the other hand, the ‘‘parallel progression model’’ density of lymphatic or blood vessels have a huge impact on cancer
claims early tumor cell dissemination of small tumors with 1– cell spreading at distal sites of metastasis [2,23]. However, the
4 mm in size. In line with linear progression, an increased mutation metastatic process can be ambiguous and unpredictable as the
frequency was shown for the mutation of the tumor suppressor individual genetic disposition and constitution of the patient play a
TP53 at advanced stages (T3) of breast cancer as compared to pivotal role.
smaller tumors (T1 stage) [10]. In contrast, the parallel progression Prevention of cancer cell dissemination and secondary tumor
model accounts for distinct genetic alterations of tumor cells at formation must be a major goal of cancer therapy as about 90% of
primary and distal sites due to early separation and independent all cancer deaths are related to metastasis [24]. In this review, we
development. In fact, early tumor cell dissemination and formation focus on the initial steps of the metastatic process which is
of micrometastasis in the bone marrow and the lung were shown invasion of cancer cells and their entry into the circulatory system
for ERBB2 (HER2/neu) mutant breast cancer cells prior to the by endothelial transmigration. Elucidation of the cellular and
morphological invasion of the primary tumor [11]. Furthermore, molecular mechanisms underlying these critical steps in tumor
amplification of ERBB2 was observed in disseminated esophageal progression will facilitate new strategies to combat metastasis.
cancer cells and independent of the ERBB2 status in the primary
tumors [12,13]. In this line, numerous studies indicate that 2. Mechanisms of cell invasion
disseminated tumor cells (DTCs) can localize in lymph nodes or in
the bone marrow prior to the establishment of metastases [9,13]. Cancer cells invade other tissues either by moving collectively
The early spreading of DTCs to distant sites as described by the as epithelial sheets or detached clusters, or as single cells via
parallel progression model allows to develop concepts of cancer mesenchymal or amoeboid cell types [25]. During cancer
diagnosis and therapy by monitoring and molecularly character- progression, a variety of tumor cells show changes in their
izing DTCs that could not be attributed in linear cancer plasticity by morphological and phenotypical conversions (Fig. 1),
progression. Moreover, the dormancy of DTCs is of particular including the epithelial to mesenchymal transition (EMT) [26], the
relevance for tumor progression and manifestation of metastases collective to amoeboid transition (CAT) [27] and the mesenchymal
[14]. CD8+ T cells were recently found to provide cytostatic rather to amoeboid transition (MAT) [28].
than cytotoxic effects to DTCs which keep them in a dormant state, EMT has been increasingly recognized as a crucial event in cancer
whereas reduction of immunosurveillance by depletion of CD8+ T progression and metastasis during the last decade [26,29]. EMT
cells caused faster metastatic colonization [15]. This immune- represents a highly conserved process which occurs during
mediated dormancy of DTCs by the inhibition of cell cycle embryogenesis, chronic inflammation and fibrosis as well as upon
progression could be the reason for the comparable genetic cancer progression, referring to type I, type II and type III EMTs,
signature of early DTCs and established macrometastases at late respectively [30]. Type III EMT denotes a hallmark in the malignant
stage of tumor progression [16]. Interestingly, various studies progression to aggressive carcinoma [26,30,31], and has been
revealed that the dormancy of DTCs required interferon (IFN)-g described in the development of breast tumor [32], colorectal cancer
and tumor necrosis factor (TNF)-mediated signaling which was not [33], HCC [34,35], lung cancer [36], prostate carcinoma [37] and
correlated with the death of tumor cells [16]. Therefore, pancreatic cancer cells [38] (Table 1). Molecularly, EMT is
immunotherapies employing IFNs are suggested to reduce the characterized by loss of epithelial characteristics and the concomi-
metastatic burden. tant gain of a mesenchymal gene expression program [30]. One
An important issue is whether tumor cells prefer distinct organs hallmark of EMT is the downregulation or even loss of epithelial
for distant metastatic colonization. Stephen Paget described an (E-)cadherin, which is an essential component of adherence
organ-specific pattern of metastasis already in 1889 [17]. Employ- junctions. E-cadherin binds with its extracellular domain to an
ing his ‘‘seed and soil’’ theory, he proposed that secondary growth E-cadherin molecule of the neighbouring epithelial cell which
of cancer cells (the ‘‘seed’’) is dependent on the competence of the stabilizes cell-to-cell contacts. Intracellularly, E-cadherin binds to
distal organ (the ‘‘soil’’). Up to now, this theory has been approved b-catenin, a-catenin and p120-catenin which mediates intracellu-
as distinct cancer types metastasize at different and tumor-specific lar signaling and links adherence junctions to the actin cytoskeleton
sites [6,18]. For example, the major sites of metastasis of breast [39]. Downregulation of E-cadherin by the transcriptional repressors
carcinoma are the bone, the lung and the brain [2]. Colorectal and Snail/SNAI1, Slug/SNAI2, SIP1/ZEB2 or Twist leads to the disassem-
pancreatic carcinoma show preferential sites of distal colonization bly of adherence junctions and translocation of membrane-bound
in the liver and the lung. However, some cancer cells are highly b-catenin to the cell nucleus where it modulates transcription of
invasive but rarely seed other organs. For instance, hepatocellular numerous genes such as c-myc or cyclin D1 [26,40–42]. The
carcinoma (HCC) and gliomas most frequently show proximal subsequent upregulation of mesenchymal markers such as vimentin
metastasis leading to intrahepatic and intracerebral colonization, and neuronal (N-)cadherin is one of the hallmarks for mesenchymal
respectively [19,20]. While the dense hepatic vasculature mainly cells [43,44]. Expression of N-cadherin leads to the rearrangement of
 F. van Zijl et al. / Mutation Research 728 (2011) 23–34 25

Fig. 1. Plasticity of invading cancer cells. (A) In collectively invading cell strands or sheets, the tumor cell at the invasive front is designated as tip cell and executes the driving
force by promoting focal adhesions and ECM rearrangement. Cells following the tip cells maintain their epithelial characteristics such as desmosomes, adherence, tight and
gap junctions. (B) A single cancer cell can detach from the epithelial cluster by undergoing an EMT through e.g. TGF-b induced downregulation of E-cadherin and
rearrangement of the cytoskeleton. This mesenchymal-like cell is able to move freely by forming focal adhesions and rearranging the ECM by secretion of proteases. EMT-
transformed cells sustain a rear–front cell polarity. (C) The fastest way of cell invasion is the amoeboid cell movement, which leads to a total loss of cell polarity and paracrine
directed chemotaxis in a protease-independent fashion. Cancer cells were shown to acquire these characteristics in two ways: either by inhibition of b1-integrin leading to
CAT or by inhibition of proteases which subsequently induce MAT. CAT, collective to amoeboid transition; ECM, extracellular matrix; EMT, epithelial to mesenchymal
transition; MAT, mesenchymal to amoeboid transition; MMP, matrix metalloproteinase.

the cytoskeleton by mediating Rho-induced stress fibers and the Transforming growth factor (TGF)-b was shown to play a central role
formation of lamellopodia and filopodia by Rac1 and Cdc42 in tumor progression [47,48], leading to Smad activation and the
activation, respectively [45]. This change from E- to N-cadherin subsequent upregulation of transcriptional repressors of E-cadherin
expression, termed cadherin-switch, leads to enhanced motility of [49,50]. Recently, novel inducers of EMT in hepatocarcinogenesis
EMT-transformed cells [46]. Thus, cells that have undergone EMT have been described such as chromodomain helicase/ATPase DNA
lose their epithelial organization and gain the ability to detach from binding protein 1-like gene [51] and connective tissue growth factor
epithelial cell clusters in order to move as single cells in a [52].
mesenchymal fashion (Fig. 1A and B). Numerous factors have been EMT is not only associated with an increase in cell motility but
identified to induce EMT including integrins, hepatocyte growth also described as a trait to maintain stem cell properties, to prevent
factor (HGF), fibroblast growth factor (FGF), vascular endothelial apoptosis and senescence, to suppress immune reactions and to
growth factor (VEGF) and platelet derived growth factor (PDGF) [26]. acquire resistance against radiotherapy and chemotherapy [26].
The concomitant occurrence of stem cell characteristics and EMT
Table 1 features was suggested in colorectal cancer cells at the tumor–host
Cell invasion in cancers. border [53] and demonstrated in breast cancer [54]. Notably,
Type of cell invasion Cancer type Reference therapeutic compounds have been identified which specifically
target EMT-transformed cells that harbor cancer stem cell
Collective Oral squamous cell carcinoma [82,180]
Colorectal carcinoma [153,181] characteristics [55,56]. Recently, numerous microRNAs (miRs)
Melanoma [27] have been demonstrated to modulate EMT. miR-9 was shown to
Breast cancer [63,86,180] directly interact with CDH1 (E-cadherin), which leads on the one
Endometrial carcinoma [74] hand to increased cell motility and invasiveness, and on the other
Pancreatic cancer [182]
Single
hand to elevated VEGF levels resulting in enhanced angiogenesis
Mesenchymal Fibrosarcoma [28,91] [57]. Similarly, amplified miR-151 targeting RhoGDIA correlated
Glioblastoma [25,183] with aggravated intrahepatic metastasis of HCC by its synergistic
Melanoma [68,184] function with the focal adhesion kinase (FAK) and the subsequent
Amoeboid Breast cancer [65]
activation of Rac1, Cdc42 and Rho [58]. In contrast, the miR-200
Lymphoma [25]
Small-cell lung carcinoma [185] family including miR-200a, miR-200b, miR-200c, miR-429 and
Prostate carcinoma [25] miR-141 leads to epithelial differentiation. The zinc finger
Melanoma [68,184,186] enhanced binding (ZEB1 and ZEB2) transcription factors inhibit
Sarcoma [73] the miR-200 family at the transcriptional level, whereas these
EMT Hepatocellular cancer [31,122,187]
Breast cancer [32,188,189]
miRNAs themselves post-transcriptionally repress the EMT-
Colorectal cancer [33,60] inducers ZEB1 and ZEB2 [59]. This negative feedback loop between
Lung cancer [36,190] ZEBs and miR-200 is not only proposed to be the molecular driver
Prostate cancer [37,191,192] of cellular plasticity but additionally affects the balance between
Pancreatic cancer [38,193]
stemness and differentiation, longevity and senescence, cell cycle
MAT Fibrosarcoma [28,91,183]
Breast carcinoma [28,194] arrest and proliferation and between survival and apoptosis. It has
Melanoma [69,72] been suggested to occur in various human carcinomas including
CAT Melanoma [27] pancreatic and colorectal cancers. Interestingly, ZEB initiated EMT
EMT, epithelial to mesenchymal transition; MAT, mesenchymal to amoeboid and stem cell properties by upregulation of Klf4, Sox2 and Bmi1
transition; CAT, collective to amoeboid transition. which are normally repressed by the miR-200 family [60].
26 F. van Zijl et al. / Mutation Research 728 (2011) 23–34

The reversal of EMT, referred to as mesenchymal to epithelial in situ to invasive breast carcinoma [76]. Another example includes
transition (MET), is characterized by epithelial reorganization and the formation of leading endothelial tip cells showing filopodia
is also described as individual to collective transition [26,50]. This during sprouting angiogenesis that depends on VEGF-induced
process allows mesenchymal cells to regain the epithelial cell-to- Delta-like 4 expression, whereas trailing endothelial stalk cells
cell junctions for colonization at secondary sites [30,61]. In this generate the base of the emerging sprout showing activated Notch
context, EMT and MET have been discussed as frequent events signaling and decreased VEGF receptor II (VEGF-R2) and VEGF-R3
among many others in the metastatic cascade of epithelial cancers, levels [77].
where carcinoma cells initially start to invade collectively, Collectively invading cells maintain their cell-to-cell contacts
followed by incomplete or complete EMT, both of which are such as adherence, tight and gap junctions as well as desmosomes
reversible by MET for effective metastatic colonization at distal (Fig. 1A) [74,75]. However, leader or tip cells in the front and the
sites [62]. following cells at the back create a sort of front–rear asymmetry.
CAT known as collective to amoeboid transition has been Rho GTPases and myosin II mediate asymmetric stiffening of
described as the detachment of individual cells from cell clusters cortical actomyosin filaments that affect tip cells. Differences in
by the use of amoeboid migration, particularly in melanoma gene expression lead to distinct cell morphologies between tip and
(Fig. 1A and C; Table 1) [27,63]. Amoeboid cells show reduced cell– following cells. Whereas the tip cell exhibits a rather mesenchymal
ECM interaction which allows them to squeeze through gaps in the phenotype, the following cells are organized with intact epithelial
ECM barriers independently of ECM proteolysis [64]. Intravital cell-to-cell contacts (Fig. 1A). Signals at the leading edge include
imaging by multiphoton microscopy revealed that single breast chemokines and growth factors secreted in either paracrine,
carcinoma cells with amoeboid morphology migrated along ECM juxtacrine or autocrine fashion which affects the asymmetry of
fibers with high velocity [65]. CAT most frequently occurs after collectively invading cells. Collective migration can be induced by
inhibition with b1 integrin in multicellular clusters of melanoma the paracrine secretion of stromal-cell derived factor (SDF1/
cells that induces single cell movement in an amoeboid fashion CXCL12) or members of the FGF and the TGF-b family [74,78]. For
[27]. The escape of single cells from the collective cell organization example, the induction of the Wnt/b-catenin pathway by
is associated with the disassembly of E-cadherin-dependent cell members of the FGF family results in the differential expression
adhesion complexes and integrin-linked focal sites [66]. It is of membrane-bound CXCR4 at the front and CXCR7 at the rear of
currently an open issue whether CAT occurs directly or in an the migrating lateral line primordium in zebra fish which
indirect fashion by employing an intermediate mesenchymal cell maintains front–rear-asymmetry [79].
phenotype. To mediate collective cell movement, force generation is
MAT, referred to as the transition of mesenchymal cells to necessary for pulling cells from the front or pushing them from
amoeboid cells, was detected in breast cancer, melanoma and the rear. The generation of traction force is provided by substrate
fibrosarcoma (Table 1) and depends on Rac and Rho/ROCK binding integrins in leading cells (Fig. 1A). For this task, the
signaling and is independent on protease activities such as MMPs, leading edge expresses b1 and b3 integrins to mediate focal
serine proteases and cathepsins [25,67,68]. Indeed, blocking of adhesion complexes in order to connect to ECM components
extracellular proteolysis was shown to induce MAT (Fig. 1B and C). such as fibronectin [74]. Furthermore, the tip cell produces a2b1
Besides regulation by the tumor microenvironment, MAT can be and avb3 integrins to attach to collagen and fibrin-rich surfaces,
induced by regulatory proteins such as EphA2 kinase. Re- respectively. In two dimensional migrating cell clusters, follow-
expression of EphA2 in mesenchymal melanoma cells triggers ing cells even form basolateral lamelopodia which are protruded
changes in the plasticity to an amoeboid motility that is underneath the cell in front [80]. Hereby, a6b1 integrins help in
accompanied by the activation of a nonproteolytic invasive attaching to the basement membrane, which was produced by
program along with a Rho-mediated cell rounding [69,70]. the invading tip cells. Integrin-mediated attachment to the ECM
Furthermore, both the tumor-suppressor proteins p27kip1 and results in the activation of cytoskeletal adaptor proteins such as
p53 are involved in controlling the MAT of fibroblasts and cortactin, vinculin, paxilin and talin. For example, b1 integrin
melanoma cells in 3-dimensional matrices [71,72]. Loss of these can cooperate with discoid domain receptor 1 to activate FAK
tumor suppressors therefore enhances the aggressiveness of these and prolin rich tyrosin kinase (PYK)2/FAK2 [81], which
cancer cells by switching their mode of invasion. In addition, subsequently leads to upregulation of N-cadherin and thus to
overexpression of the microtubule destabilizing protein stathmin an increased cell motility [27]. Similarly, integrins a3 and a5 in
has been described to induce MAT of sarcoma cells [73]. fibroblasts were described to be important for force generation
and formation of tube-like tracks through which squamous cell
2.1. Collective cell invasion carcinoma cells follow collectively [82]. The rearrangement of
actin is crucial for both, the formation of the filopodia and the
The bulk invasion of cancer cells occurs in epithelial cancers maintenance of VASP/Mena-dependent cell–cell adhesion during
such as breast, endometrial and colorectal cancers and in collective migration [83,84]. Pseudopodia and filopodia are
melanoma (Table 1). Three hallmarks characterize the collective controlled by Rac and Cdc42 [85], respectively, whereas Rho
cell invasion [74]. First, cell–cell junctions remain intact during signaling is more important for single than collective cell
movement [75]. Second, the multicellular coordination of polarity movement [86].
and cytoskeletal activity generates the traction force required for Remodeling of the extracellular matrix (ECM) is essential in
collective cell movement [27]. Third, collective cell invasion initiating track formation and depends on (i) the invading cell
involves the remodeling of extracellular matrix (ECM) and itself and (ii) the ECM determinants including its dimension,
rearrangement of the basement membrane (Fig. 1A) [63]. The density and gap size, its stiffness and orientation [64]. Tip
types of collective cell invasion are manifold including formation of cells were shown to secrete MT1-MMP (membrane type 1
a monolayer which allows invading two dimensionally or building matrix metalloproteinase; MMP14) to initiate track formation
up cell strands for three dimensional invasion or even the [63,74,87,88] and following cells enlarge this track by further
detachment of a collectively invading cell cluster from the initial degradation of the ECM and subsequent deposition of laminins,
tumor. For instance, luminal epithelial cells exhibit reduced nidogen 1, perlecan and type IV collagen (Fig. 1A). Interestingly,
polarity by the loss of myoepithelial cells in mammary tumors MT1-MMP represents the rate limiting factor of multi-cellular
which causes their collective cell migration and the transition from invasion [89].
 F. van Zijl et al. / Mutation Research 728 (2011) 23–34 27

2.2. Mesenchymal cell invasion inhibitors. For example, blocking of b1 integrin in cancer cell
clusters resulted in loss of cell–cell adhesion, cell detachment and
Mesenchymal cells employ a five-step migration cycle includ- transition to an amoeboid single-cell type of melanoma and
ing pseudopod protrusion, formation of focal contacts, focalized fibrosarcoma cells [27,91]. Similarly, MMP inhibitors failed to
proteolysis, actomyosin contraction, and finally, the detachment of prevent cancer progression in clinical trials showing the physio-
the trailing edge [25]. Invasion of single mesenchymal cells was logical relevance of amoeboid cell movement in vivo [89]. Yet,
detected in fibrosarcoma, glioblastoma and melanoma (Table 1). In most of the studies dealing with amoeboid cell invasion have been
carcinoma, mesenchymal cells mostly arise from epithelial cell performed in 3-dimensional ECM settings in vitro that do not
clusters through EMT (Fig. 1A and B; Table 1) [74]. The recapitulate the native collagen composition in vivo. Thus, the
rearrangement of F-actin leads to partially polarized cancer cells. protease-independent type of cancer cell movement is a current
Focal adhesions in the front tightly attach the cell to the ECM, matter of debate.
whereas the tail moves by contractions of the retraction fibers
(Fig. 1B) [65]. 3. Endothelial transmigration
Transient TGF-b signaling was shown to be responsible for the
dedifferentiation of cancer cells and the following detachment of The blood system has been considered as the main route of the
single cells from collectively moving cell strands [86]. Cells of metastatic spread, but there is increasing evidence that the
different motilities are heterogeneously distributed within the lymphatic system might be the key player in cancer cell
tumor [65,90], and actually mobile ones only account for 5% [61]. dissemination [92,93]. The lymphatic capillaries are thin-walled
After discrimination between collective clusters and single moving single layers of endothelial cells lacking inter-endothelial tight
cells, time-lapse studies revealed that single mesenchymal cells junctions (Fig. 2A). In contrast to blood capillaries, they are not
show an activated TGF-b signaling as indicated by nuclear covered by smooth muscle cells and lack a basement membrane
accumulation of Smad2 [86]. In contrast, collectively moving cells [94]. Interstitial fluids, macromolecules and even bacteria can
or non-motile cells retained Smad2 in the cytoplasm. Moreover, easily enter the lymphatic drain through valve-like openings.
disseminated cell clusters into lymph nodes and large pulmonary Anchoring filaments are connected to the lymphatic capillaries to
metastatic colonies were devoid of nuclear Smad2 accumulation. prevent vessel collapse in condition of high lymph flow. Whereas
Interestingly, intravasation of single mesenchymal cells into blood VEGF-A and the activation of VEGF-R1 and -R2 are widely known to
vessels was dependent on TGF-b/Smad2 signaling as interference induce blood vessel angiogenesis, signaling through VEGF receptor
with TGF-b type II receptor impeded hematogenous metastasis. III after activation by VEGF-C and VEGF-D is necessary for
However, collective invasion was not altered and cancer cell lymphangiogenesis (Fig. 2A) [95]. Tumoral expression of VEGF-C
clusters could be still detected in the lymph system [86]. This study and VEGF-D was not only correlated with lymphatic cell invasion
demonstrated that (i) TGF-b is essential to switch cancer cells from [96], distant metastasis and poor survival [97,98], but also
a cohesive to a single cell motility by activating Smad4, epidermal additionally thought to actively induce vessel sprouting towards
growth factor (EGF) receptor, Nedd4, M-RIP, FARP and RhoC and (ii) the tumor [99,100]. Moreover, tumor cells might be even attracted
the subsequent downregulation of TGF-b at secondary sites is by lymphatic vessels through secretion of chemokines such as
inevitable for distal metastasis. CCL21 (Fig. 2A) [94,101]. It is still an open discussion whether
tumor cells are actually passively trapped in the lymph nodes or
2.3. Amoeboid cell invasion whether lymph nodes promote spreading of cancer cells and their
distal metastasis [23]. Interestingly, a most recent study demon-
The hallmarks of amoeboid cell invasion are the loose strated that aggregates of breast cancer cells directly penetrate
attachment to the ECM, complete loss of cell polarity and the lymphatic vessels through ruptures in the vascular wall [102]. This
ability of chemotaxis (Fig. 1C) [65]. Invasion of single amoeboid process is mediated by the induction of the hypoxia-inducible
cells occurs in breast cancer, lymphoma, small-cell lung and enzyme ALOX12 or ALOX15 in mammary carcinoma cells which
prostate carcinomas as well as in melanoma and sarcoma (Table 1). metabolizes arachidonic acid to 12(S)-hydroxy-eicosatetraenoic
In contrast to mesenchymal cells, amoeboid cancer cells are acid (12(S)-HETE). Interestingly, exposure of lymphatic endothelial
roundish, grow in suspension, show no formation of stress fibers, cells to 12(S)-HETE transiently reduces their VE-cadherin expres-
do not remodel the ECM and are devoid of focalized integrins [25]. sion, resulting in the migration of these cells and subsequent
The amoeboid cell invasion is described as the fastest migratory formation of circular defects in the integrity of the lymphatic
phenotype by reaching a velocity of up to 20 mm/min as compared endothelial cell layer [102].
to the one of the mesenchymal cells showing a movement of 0.1– It is a matter of debate whether cancer cells actively migrate
1 mm/min [25]. Whereas mesenchymal and collective cell invasion through blood and lymph vessels in response to e.g. growth factor
depend on protease activities, amoeboid-like motility is protease- gradients or even passively crawl into the vasculature without the
independent as cells rather employ actomyosin-based mechanical involvement of an active cell migration machinery [103]. Such a
forces to physically displace matrix fibrils instead of degrading passive mechanism implicates that cancer cells impinge on fragile
them [89]. In particular, protease-independent amoeboid cell tumor endothelial cells that do not exhibit intact cell-to-cell
invasion is suggested to occur when the structural pores formed in structures. While physiological blood vessel formation is formed in
collagen networks fail to show stabilizing covalent crosslinks that a balanced process between pro- and anti-angiogenic factors,
determine ECM stiffness. Structural constraints limit the mobility tumor angiogenesis generates poorly organized and immature
of amoeboid cancer cells as collagen pores smaller than the size of blood vessels that might allow to pass the shedding cancer cells
the invading cell’s nucleus cannot be negotiated [89]. Naturally [104]. Evidence for a passive mechanism is provided by the
crosslinked collagen in vitro and in vivo even requires MT1-MMP observation that shed tumor cells form cell clumps in the lumen of
(MMP14) dependent proteolytic activity for amoeboid cell blood vessels due to intravascular proliferation of surviving cells
movement [44,89]. This provides evidence that the different types [105]. One major obstacle for the successful intravasation of
of movements are not mutually exclusive but can be entertained shedding cancer cells is provided by their ability to survive as most
simultaneously and cooperatively. of these cells undergo cell death [106]. Yet, phenotypical changes
Amoeboid cells most frequently develop after the treatment of of cancer cells such as the expression and secretion of growth
cancer with integrin-blocking antibodies or with protease receptors and its ligands strongly support the idea of active
28 F. van Zijl et al. / Mutation Research 728 (2011) 23–34

Fig. 2. The tumor microenvironment and its impact on transendothelial migration. (A) Passive migration of epithelial cancer cells into lymph vessels may occur through
intercellular gaps and active attraction of lymph endothelial cells upon secretion of VEGF-C, VEGF-D and CCL21. (B) (Transient) TGF-b induced EMT and the secretion of VEGF-
A mediates hematogeneous dissemination. (C) Once transmigrated, tumor cells can use platelets as a shield against shear forces and natural killer (NK) cell attacks. (D) Two
prominent cell types of the tumor microenvironment are tumor associated macrophages (TAMs, green color) and cancer associated fibroblasts (CAFs, red color). Both TAMs
and CAFs secrete components which stimulate cancer progression. (E) CAFs, also referred to as myofibroblasts, rearrange the ECM and are able to form a track through which
epithelial tumor cells can migrate. (F) In close contact with tumor cells, macrophages are able to promote hematogenous transmigration. CCL, chemokine (C–C motif) ligand;
CSF, colony stimulating factor; CXCL12 (SDF1), stromal cell derived factor; ECM, extracellular matrix; EGF, epidermal growth factor; MMP, matrix metalloproteinase; NF-kB,
nuclear factor-kappa B; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

intravasation during metastasis. In this scenario, the microenvi- Once disseminated, cancer cells have to survive the challenges of
ronment-controlled migration of breast carcinoma cells towards the blood stream including physical constraints and the immune
blood vessels has been reported to depend on the secretion of EGF system. Surface shielding by platelets was shown to protect cancer
by colony stimulating factor (CSF) receptor-expressing macro- cells from (i) shear forces (Fig. 2C), (ii) NK-cell mediated lysis [110]
phages and the CSF1 release of EGF-R expressing mammary tumor and (iii) facilitate extravasation at the distal site [111]. Remarkably,
cells [107,108] (see also Section 4). Thus, we might assume that cancer cells express membrane bound tissue factor (Tf) which is the
both active and passive mechanisms exist that contribute to the receptor for coagulation factors VIIa and X, which is the main
entry of cancer cells into the vasculature [103]. initiator of blood coagulation and tumor-associated microthrombi
The ability for hematogenous intravasation of metastatic and [112,113]. The Tf-VIIa complex is capable of stimulating proteinase
non-metastatic mammary carcinoma cells showed no correlation activated receptor (PAR)2, leading to immune regulation, angiogenic
with the size of the primary tumor [90]. However, metastatic cells stimulation and anti-apoptotic signaling in cancer cells by activating
were oriented towards the blood vessels and showed protrusions G-protein-coupled signaling [114]. Furthermore, PAR2 recruits the
(Fig. 2B), whereas non-metastatic cells rather underwent frag- intracellular adaptor protein b-arrestin to mediate cancer cell
mentation when interacting with blood vessels [65]. Interestingly, migration. In clinics, high concentrations of platelets have been
TGF-b type II receptor/Smad4 activation has been shown to cause associated with decreased survival in a variety of cancers including
hematogenous dissemination of breast carcinoma (Fig. 2B) [86], breast, colorectal and lung cancer. Noteworthy, treatment with anti-
although prolonged TGF-b activation hampered lung metastasis coagulants was shown to reduce metastasis in patients [115].
formation suggesting a transient TGF-b activation for successful
metastasis. Interestingly, highest TGF-b activation was monitored 4. Role of the tumor microenvironment
at blood vessels and tumor margins including 30–50% of cancer
cells. Disseminated cells found in the lymphatic vasculature were The microenvironment comprises various cell types including
not dependent on TGF-b signaling and exclusively organized in endothelial cells forming lymph and blood vessels, pericytes,
clusters (Fig. 2A). An alternative model proposes metastasis being a stromal fibroblasts and bone marrow derived cells such as
cooperation between EMT cells and non-EMT cells [109]. It is macrophages, neutrophils, mast cells and mesenchymal stem
hypothesized that hematogenous intravasation is only feasible in cells [107,116]. In general, the tumor microenvironment contrib-
cooperation between those two cell types, whereas subsequent utes to tumor progression by secretion of growth factors, cytokines
metastasis at distal sites is solely provided by non-EMT cells. The and chemokines, and by the rearrangement of ECM (Fig. 2D).
latter phenotype was also observed by extravasation of tumor cells TGF-b, HGF, FGF, EGF and insulin-like growth factor (IGF) are
from lymphatics [86]. potent inducers of EMT [26,44,46,50,117]. These factors including
 F. van Zijl et al. / Mutation Research 728 (2011) 23–34 29

interleukin (IL)-1a also play a role in the stimulation of collective TNF and RANKL induced by NF-kB has been shown to cause
cell invasion by the particular influence on the tip cell. TGF-b has inflammation and to induce tumor cell dissemination [140,141]. In
an exceptional role in the tumor–stroma interaction. The tumor- a multidrug resistance (Mdr)2 knock out model, TNF-a induced
promoting effects of TGF-b are numerous [118], and include the (i) NF-kB activation is responsible for HCC development [142].
induction of EMT [119], (ii) generation of myofibroblasts Interestingly, sex steroidal hormones such as estrogens can
[120,121], (iii) production of autocrine mitogens such as PDGF interfere with NF-kB activity and IL-6 production and thus can
[122] and (iv) evasion of immunity by targeting CD8+ T cells [123]. protect against liver cancer development in female mice
Even more, TGF-b in cooperation with HGF/c-met signaling can [141,143,144].
induce hypoxia-inducible factor (HIF)-1A driven VEGF-A secretion Tumor associated macrophages (TAMs) produce growth factors
which stimulates angiogenesis. Furthermore, TGF-b affects distal and matrix-degrading enzymes and thus promote angiogenesis,
metastasis of breast carcinoma cells by priming pulmonary cell invasion and hematogenous intravasation (Fig. 2D) [145–148].
metastasis through upregulation of angiopoietin-like protein The interaction between tumor cells and TAMs can be stimulated
(ANGPL)4 [124]. Interestingly, ANGPL4 dissociates endothelial by EGF produced by macrophages and subsequent EGF receptor
cell–cell junctions which allows to infiltrate ANGPL4-secreting signaling in tumor cells which promotes migration and invasion.
tumor cells into the lung, thus facilitating pulmonary metastasis of Cancer cells themselves secrete CSF1, which is a potent chemoat-
breast cancer [18]. In the bone marrow, mammary carcinoma cells tractant for CSF1 receptor-expressing macrophages [149,150].
do not benefit from ANGPL4 for extravasation as vascular Using this paracrine cytokine loop, TAMs were shown to directly
capillaries are fenestrated and TGF-b works via a different assist hematogenous dissemination of mammary cancer cells
mechanism to cause organ-specific metastasis. Notably, TGF-b [145], which is further supported by the secretion of proteases
stored in the bone matrix stimulates the production of parathyroid from both cancer cells and macrophages at the site of entry (Fig. 2F)
hormone-related protein, IL-11, and connective tissue growth [108]. Moreover, TAMs contribute to ECM rearrangement by MMP
factor of circulating cancer cells [125]. These factors stimulate secretion [129] and the production of cysteine cathepsins [151]
osteoblasts to release receptor activator of NF-kB ligand (RANKL) and serine proteases [152]. For example, the production of MMP2
that mobilizes osteoclasts for inducing and maintaining an and MMP9 of immature myeloid cells at the invasive front
osteolytic cycle and outgrowth of bone macrometastasis. Recently, promotes collective invasion of colorectal cancer cells [153].
it has been proposed that an additional potential mechanism of The impact of the tumor microenvironment is crucial for cancer
switching TGF-b from anti- to pro-oncogenic functions is the progression and even goes beyond the location of the primary
attraction of host immune cells [126,127]. Interestingly, attraction tumor. The preparation of the ‘‘soil’’ at the putative distal site of
of myeloid Gr-1+CD11b+ progenitor cells which express high levels metastasis, referred to as pre-metastatic niche, has been shown to
of TGF-b1 and MMPs facilitate tumor invasion and cancer cell be under the control of the tumor microenvironment [154].
dissemination into the circulation [128]. Importantly, metastasizing cancer cells bring their own soil such as
Besides providing growth factors and cytokines, the tumor activated fibroblasts, endothelial cells and macrophages from the
microenvironment promotes tumor progression by ECM rear- primary tumor to the distant site i.e. the lung or the brain which
rangement (Fig. 2D). MMPs are upregulated in almost every type of protects traveling cancer cells from apoptosis [155]. Stromal cell
human cancer and were shown to associate with enhanced cell passengers dramatically increase the metastatic colonization and
proliferation, migration, angiogenesis, metastasis and poor surviv- interestingly, the metastatic capacity is lost when heterotypic cell
al [129]. MMPs can be secreted from all cancer cells or tip cells of clumps are dissociated. Studying the relevant adherence junction
collective cell clusters, myofibroblasts and by almost all immune molecules is therefore of utmost importance.
cells [129]. The functions of MMPs are (i) cleaving cell adhesion Secreted growth factors from the primary tumor are suggested
molecules such as E-cadherin, (ii) the degradation of ECM proteins, to affect bone marrow derived hematopoietic progenitor cells
and (iii) the processing and activation of cytokines and growth (BMDCs), which can form cellular clusters at the tumor-specific
factors [107]. Their functions are even more far-reaching as MMPs pre-metastatic site. Notably, VEGF-R1+VLA-4(integrin a4b1)+
co-regulate inflammation and contribute to the generation of the BMDCs interacting with fibronectin-secreting fibroblasts were
metastatic niche [130]. shown to play an essential role in the adherence and growth of
Cancer associated fibroblasts (CAFs) represent essential cellular disseminating cancer cells at the distal site [156]. Yet, the
components of the tumor microenvironment. CAFs are able to sufficiency of blocking VEGF-R1 in this context is a matter of
secrete MMPs, cytokines (e.g. IL-8 and VEGF) and chemokines (e.g. considerable debate [157]. Although inhibition of VEGF-R1 was
CXCL12) and thus promote proliferation, cancer cell invasion, and effective against metastasis formation, the effect on lung
neoangiogenesis (Fig. 2D) [118]. They were shown to communicate metastasis was lost after primary tumor resection [158]. Numer-
with cancer cells by a CXCL12/CXCR4 loop contributing to a ous studies revealed many more regulatory components which
controlled cancer cell migration [107]. Moreover, CAFs are able to have an important role in forming the pre-metastatic niche. For
form invading channels through which carcinoma cells can follow example, tissue inhibitor of metalloproteinase 1 (TIMP1) was
by maintaining their epithelial characteristics (Fig. 2E) [82]. shown to promote colon cancer metastasis into the liver by
Previous studies demonstrated that immune cells of the tumor inducing HGF signaling [159]. Prostate cancer is known to prepare
microenvironment execute a tumor-promoting rather than a a metastatic niche by the crosstalk with bone cells [160]. In
tumor-suppressing role [116,131–134]. It is well established that particular, prostate cancer cells bind to cell surface expressed
chronic infections and inflammation frequently lead to cancer annexin II of osteoblasts in the bone marrow which induce
development and enhancement of tumor progression [134,135]. expression of the growth arrest-specific (Gas6) receptor Axl in
For example, chronic infections with hepatitis B or hepatitis C virus tumor cells [161]. Gas6 produced by osteoblasts induces metas-
are well described etiological factors for the generation of liver tastic tumor cell dormancy of prostate cancer cells and protects
inflammation and HCC [20]. Thus, the impact of the inflammatory them from chemotherapy-induced apoptosis.
microenvironment has been proposed for inclusion as an
additional hallmark in a renowned model of cancer [136,137]. 5. Concluding remarks and future perspectives
Interestingly, NF-kB has been identified as a key activating
signaling pathway in both cancer cells and tumor-associated Local cancer cell invasion and endothelial transmigration
immune cells [138,139]. Expression of cytokines such as IL-1, IL-6, represent the initial steps of metastasis that dramatically worsen
30 F. van Zijl et al. / Mutation Research 728 (2011) 23–34

prognosis and patient’s survival after successful completion. ECM, showing that this intervention fails to block cell invasion [130].
Numerous attempts have tried to interfere with these early events Clinical studies employing MMP inhibitors are still in the process of
and to eradicate metastasis at its initiation. Interference with recruiting patients (http://clinicaltrials.gov) and others are still
VEGF-A using the anti-VEGF monoclonal antibody Bevacizumab running. The outcome of these trials has to be awaited. It seems that
(Avastin) aimed to hinder blood supply for the tumor and was MMPs are too ubiquitous and hence unspecific targets, which may
found to be effective in clinical trials of many cancers such as non- cause intolerable side effects particularly in combination with
small-cell lung and breast cancer [162]. This led to its approval by standard therapies [130]. Yet, the specific inhibition of MT1-MMP
the FDA (Food and Drug Administration) for first-line treatment of might be auspicious in targeting cancer cell invasion as this protease
metastatic colorectal cancer in combination with chemotherapy exhibits a dominant role in tumor cell dissemination [89].
[163]. Similarly, interference with VEGF-R3 or blocking VEGF-C Refinement of the specificity of MMP inhibitors for individual
signaling reduced lymphatic tumor cell dissemination of breast MMPs and the development of selective inhibitors of proteolytic and
carcinoma cells in mice [99,164]. non-proteolytic MMP functions may provide further advance in the
VEGF-R2 is targeted in clinical trials to inhibit angiogenesis but pharmacological interference with cancer progression and metas-
indirectly also tumor cell intravasation into the vasculature. tasis [130].
However, most of the clinical trials that intend to target Another approach of novel anticancer treatment employs histone
angiogenesis or metastasis are combinations of a multitude of deacetylase (HDAC) inhibitors. A few are currently in development
clinically used drugs or antibodies, which are not specifically for clinical use (entinostat, belinostat, mocetinostat, panobinostat)
designed against pro-metastatic targets. Interestingly, two clinical and vorinostat (suberoylanilide hydroxamic acid, SAHA) is already
trials target semaphorin-4D (Clinical Trials.gov Identifier: FDA-approved for the treatment of relapsed cutaneous T-cell
NCT01313065; phase II) and neuropilin-1 (NCT00747734; phase lymphoma [173]. SAHA changes the behavior of highly invasive
I) with monoclonal antibodies. Semaphorins and their receptors MDA-MB231 breast cancer cells, which build up extensive actin-rich
that are expressed on endothelial cells trigger cell motility of cell extensions [174] and inhibits brain metastasis of a derivative of
cancer cells and of the microenvironment including the blood- and this cell line in immuno-suppressed mice [175]. Also the invasion of
lymphendothelium. Blocking the contact between cancer cells and tumor spheroids derived from the pleura effusion of patients with
the microenvironment and the resulting signaling along this axis inflammatory breast cancer and locally advanced breast cancer was
could provide specificity for anti-metastatic intervention. Sema- blocked by SAHA, which was accompanied by a relocation of E-
phorins-3E and -3F are repellent molecules and could destroy the cadherin from the membrane to the cytoplasm [176]. TNF-a and
integrity of endothelial vascular walls and generate gates for TGF-b treated esophageal carcinoma cell spheroids that were
invading tumor cells as observed with 12(S)-hydroxy-eicosate- exposed to SAHA plus the proteasome inhibitor bortezomib
traenoic acid that is secreted by tumor cells [102,165–168]. In decreased in vitro cell invasion due to restoration of E-cadherin
addition, this could make the metabolizing enzymes i.e. ALOX12/ expression and attenuation of TGF-b induced EMT [177]. Moreover,
15 and the migratory machinery involving cell adhesion molecules SAHA increases the expression of paxillin in human leukemic cell
such as E-selectin to potential targets in clinical trials for lines and facilitates their attachment to fibronectin [178]. In our
combating early metastatic processes. The timing of this anti- hands, upregulation of paxillin and attachment of MCF-7 mammary
metastatic therapy is of course crucial as it aims to block the very tumor spheroids to endothelial cells correlated with the increased
early steps of tumor intravasation into the vasculature. Therefore, formation of gates into the lymphatic vasculature, termed circular
it has a more preventive than curing character and attempts chemorepellent-induced defects (CCID), which facilitates the
disease management. Of note, this intervention fails to work as transmigration of cancer cells. This is considered as a measure for
soon as sentinel/post-sentinel lymph node colonization or even potential tumor cell intravasation into the lymphatic vasculature
distant metastases have formed. ([168]; our unpublished data). SAHA is tested in 194 clinical trials
Alternatively, inhibition of integrins was considered as a (status April 2011) with 29 completed but with only a handful
promising attempt to block invasion. Unfortunately, these efforts reporting data (NCT00486702, NCT00106626 and NCT00373490),
resulted in the development of amoeboid cell types and thus and some were terminated for various reasons. The other trials are
caused an increase of invasion velocity and tumor progression still running or recruiting.
rather than an inhibition of tumor dissemination [27,91,169]. Nowadays, researchers aim to cope with the complex influence
Nevertheless, targeting adherence molecules, particularly those of the immune system on tumor progression by developing novel
facilitating the hitch hike of tumor stroma cells such as activated anti-inflammatory drugs including antibodies, small RNAs and
fibroblasts that protect cancer cells from apoptosis and prepare a peptides. These strategies raise new hopes to design novel and
niche at the distant metastatic site [155,170], will be a very effective anti-cancer therapies [179]. Intravital imaging to monitor
promising approach to control tumor spreading in the blood or disseminating cancer cells [65] and its combination with
lymph circulation. Another possible intervention at this stage functional genomics is a promising attempt to elucidate pathways
seems to be the specific modulation of gene expression of which enable the early steps of metastasis in vivo. There is still an
passenger fibroblasts, as they apparently can express anti- urgent need for intense research of the initial steps of metastasis in
metastatic gene products such as fibulin-5, which downregulates order to investigate the underlying cellular and molecular
MMP9, and that itself becomes downregulated by cancer cell- mechanisms. In particular, the amoeboid type of cancer cell
secreted factors [171]. invasion, which seems to be an ultimate exit strategy of cancer
Furthermore, MMPs that are abundantly secreted by cancer cells cells, needs to be studied in more detail. Furthermore, future
and the tumor microenvironment were also targeted for interven- concepts of metastasis intervention must simultaneously address
tion of tumor cell invasion and metastasis. However, the outcome of the collective, mesenchymal and amoeboid mechanisms of cell
clinical trials was disappointing as the treatment with MMP invasion. Undoubtedly, more insights into the complexity of the
inhibitors is particularly delicate and depends on the tumor stage regulatory networks will allow the development of novel
[172]. For instance, the inhibition of MMP2 or MMP9 (NCT00001683, strategies to efficiently combat cancer cell dissemination.
NCT00064142 and NCT00695851), even with monoclonal anti-
bodies, does not seem to have the anticipated effect. The Conflict of interest statement
employment of MMP inhibitors resulted in the protease-indepen-
dent mode of amoeboid cell invasion which squeezes through the None.
 F. van Zijl et al. / Mutation Research 728 (2011) 23–34 31

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