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ORIGINAL ARTICLE 368

High Affinity Interaction of


Integrin a4b1 (VLA-4) and
Vascular Cell Adhesion Molecule
1 (VCAM-1) Enhances Migration
of Human Melanoma Cells Across
Activated Endothelial Cell Layers
MARTIN KLEMKE, TATJANA WESCHENFELDER,
MATHIAS H. KONSTANDIN, AND YVONNE SAMSTAG*
Institute for Immunology, University of Heidelberg, Heidelberg, Germany

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers.
This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular
nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin avb3 is involved in transendothelial
migration and its expression correlates with metastasis. However, many human melanoma cells do not express b3 integrins. Therefore, it
remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different
metastatic potency, which do not express b2 or b3 integrins, express the VCAM-1 receptor a4b1. VCAM-1 is up-regulated on activated
endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of
a4b1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with
high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate
that function-blocking antibodies against the integrin a4b1, as well as siRNA-mediated knock-down of the a4 subunit in these highly
metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the
integrin a4b1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human b2/b3-
negative melanoma cells.
J. Cell. Physiol. 212: 368374, 2007. 2007 Wiley-Liss.

Tumor cell metastasis is a complex process and consists of a the endothelial cell surface. Some non-hematopoetic human
series of sequential steps. Initially, tumor cells have to detach tumor cells express the avb3 integrin, which binds to the
from the primary tumor, migrate through the tissue and invade adhesion molecule L1 on endothelial cells (Danen et al., 1995;
the lymphatic system or blood vessels. As a next step, Saini et al., 1997; Varner and Cheresh, 1996; Mizejewski, 1999;
circulating tumor cells temporarily adhere to endothelial cells Voura et al., 2001). In certain human melanoma cells,
and then extravasate by infiltrating the underlying basement expression levels of this integrin correlate with the metastatic
membrane. Finally, cells migrate to a suitable location where potential of these cells (Albelda et al., 1990a; Saini et al., 1997;
they form metastases (Stetler-Stevenson et al., 1993; Nicolson, Hofmann et al., 2000), and this integrin is a critical component
1988; Engers and Gabbert, 2000; Orr et al., 2000). The for transendothelial migration of these cells (Voura et al., 2001).
capacity of tumor cells to form metastatic foci correlates Finally human melanoma cells exist which neither express b2
with their ability to interact with and migrate through the nor b3 integrins, but are still highly metastatic (Van Muijen et al.,
endothelial cell layer (transendothelial migration). Although 1991a,b). These cells should therefore also be able to efficiently
much is known about the adhesive interactions of undergo transendothelial migration. From leukocytes we know
non-hematopoetic tumor cells with components of the basal that, besides b2 integrins, the a4b1 integrin and its ligand
membrane, surprisingly little is known about the mechanisms by VCAM-1 are involved in the interaction of leukocytes with
which non-hematopoetic tumor cells adhere to and pass endothelial cells during transendothelial migration
through the endothelial cell layer. (Oppenheimer-Marks et al., 1991; Chan and Aruffo, 1993;
Most of the knowledge about molecular mechanisms involved Chuluyan and Issekutz, 1993; Hourihan et al., 1993; Weber and
in the process of transendothelial migration comes from studies Springer, 1998; Ding et al., 2001). The a4b1 integrin has a
performed with leukocytes (Ebnet et al., 1996; Worthylake and flexible molecular structure allowing initial capture, tethering,
Burridge, 2001; Alon and Feigelson, 2002; Luscinskas et al., rolling and firm attachment of hematopoetic cells to endothelial
2002). For migration of most leukocytes across endothelial cell
layers, interactions between the b2 integrins aLb2 (LFA-1,
CD11a/CD18) or aMb2 (Mac-1, CD11b/CD18) and their
ligand ICAM-1 are essential (Oppenheimer-Marks et al., 1991;
Reiss and Engelhardt, 1999; Wong et al., 1999; Lyck et al., 2003). *Correspondence to: Yvonne Samstag, Institute for Immunology,
These b2 integrins have also been implicated in transendothelial University of Heidelberg, Im Neuenheimer Feld 305, 69120
Heidelberg, Germany. E-mail: yvonne.samstag@urz.uni-heidelberg.de
migration of certain non-hematopetic human tumor cells
(Wang et al., 2005). However, most non-hematopoetic tumor Received 15 November 2006; Accepted 21 December 2006
cells do not express b2 integrins (Varner and Cheresh, 1996; DOI: 10.1002/jcp.21029
Mizejewski, 1999) and are therefore not able to bind ICAM-1 on

2 0 0 7 W I L E Y - L I S S , I N C .
TRANSMIGRATION OF MELANOMA CELLS 369

cells (Chan and Aruffo, 1993; Masumoto and Hemler, 1993; 50 ml of FACS medium containing 2 mg/ml R-Phycoerythrin-conjugated
Alon et al., 1995). These properties result from affinity donkey anti-mouse IgG F(ab0 )2 fragments (Jackson ImmunoResearch).
regulation due to inside-out signaling (Shimaoka et al., 2002; After washing the melanoma cells in 200 ml of FACS medium and
Takagi and Springer, 2002). Endothelial cells express the ligand then in 200 ml of PBS to remove unbound antibodies, the stained
for the integrin a4b1, VCAM-1, on their surface only after melanoma cells were resuspended in 300 ml of PBS and analyzed by a
FACSCalibur cytometer using the CellQuest Pro software (Becton
inflammatory stimuli, as for example TNF-a (Xu et al., 1994; Dickinson, Heidelberg, Germany).
Haraldsen et al., 1996).
In this study, we demonstrate that only highly metastatic human Ligand-complex-based adhesion assay
melanoma cells expressed the a4b1 integrin in its high affinity
Assays were performed as described (Konstandin et al., 2006) with
conformation at the cell surface and adhered to and migrated minor modifications. For ligand-complex-based adhesion assays,
on VCAM-1. In contrast, human melanoma cells of low 270 ml of 20 mg/ml VCAM-1/Fc in PBS (PBS containing 1 mM CaCl2,
metastatic potential expressed the a4b1 integrin in its low 0.5 mM MgCl2 and 0.5% BSA) were incubated at 48C for 30 min with
affinity conformation. Moreover, transendothelial migration of 86.4 ml of R-PE conjugated anti-human Fcg fragment specific
the highly metastatic melanoma cells was supported through IgG F(ab0 )2 fragments to generate soluble multimeric VCAM-1/Fc
interaction of the integrin a4b1 with its ligand VCAM-1 on the F(ab0 )2-PE complexes. Melanoma cells were washed twice with PBS.
surface of activated endothelial cells. This suggests that highly The cell density was adjusted to 4  106 cells per ml in PBS. For each
metastatic melanoma cells preferentially leave the blood vessels sample 45 ml (1.8  105 cells) of this melanoma cell suspension were
at sites of inflammation. Therefore, blocking the a4b1 integrin mixed with 5 ml of the soluble multimeric VCAM-1/Fc F(ab0 )2-PE
complexes and incubated at 378C for the indicated time. Binding was
on metastatic melanoma cells or VCAM-1 on activated stopped by the addition of 1 ml 378C warm 4% PFA in PBS. Melanoma
endothelial cells may be a valuable approach to interfere with cells were fixed for 5 min at 378C and transferred into 1 ml of ice
tumor metastasis. cold PBS containing 5% FCS and 0.5% BSA. After pelleting the
melanoma cells, bound soluble multimeric VCAM-1/Fc F(ab0 )2-PE
Materials and Methods complexes were detected by flow cytometry using a FACSCalibur
Cell culture and the CellQuest Pro software (Becton Dickinson). The percentages
and mean channel fluorescence intensities (MFI) of labeled melanoma
The human melanoma cell lines 530, IF6, BLM and MV3 (a generous gift cells were determined.
of Dr. van Mujien, University Hospital, Nijmengen, The Netherlands)
were grown in RPMI 1640 medium supplemented with 10% fetal Haptotaxis and chemotaxis assay
calf serum (FCS), non essential amino acids, 2 mM glutamine, 25 mM
Transwell migration inserts (PET membrane, 8 mm pore-size, 6.4 mm
HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin at 378C in a
diameter, BD Biosciences) were left either untreated or the underside
humidified atmosphere containing 5% CO2. HMEC-1 cells (Ades
was coated in a total volume of 350 ml with 20 mg/ml of recombinant
et al., 1992) were grown in MCDB 131 medium (Invitrogen, Karlsruhe,
VCAM-1/Fc. After washing with migration medium (RPMI 1640
Germany) supplemented with 10% FCS, 1 mg/ml hydrocortisone,
medium supplemented with 0.1% (w/v) BSA), filters were placed into
10 ng/ml epidermal growth factor, 100 U/ml penicillin and 100 mg/ml
the chambers with the coated membrane side facing the lower
streptomycin at 378C in a humidified atmosphere containing 5% CO2.
compartment and 600 ml of migration medium (haptotaxis) or 600 ml of
Transfection of melanoma cells with siRNA migration medium supplemented with 10% FCS (chemotaxis) were
added to the lower compartment. Melanoma cells were labeled for
The siRNA against the human integrin subunit a4 (ON-TARGETplus 15 min at 378C/5% CO2 with 1 mM CFDA-SE (Molecular Probes,
SMARTpool L-005189-00-0005, Human ITG4) as well as non-targeting Eugene) in PBS. The labeling solution was removed and the melanoma
control siRNA (ON-TARGETplus siCONTROL Non-targeting pool cells were incubated in culture medium for an additional 30 min at
D-001810-10-05) were obtained from Dharmacon. Human melanoma 378C/5% CO2. Afterwards, melanoma cells were detached from the
cells were transfected using the Lipofectamine 2000 (LF2000) reagent cell culture dish by incubation with PBS containing 4 mM EDTA for 1
(Invitrogen) following the manufacturers instructions. Briefly, 0 min at 378C, pelleted, washed, and resuspended in migration
melanoma cells were seeded in a 24-well plate at 1  105 cells per well medium at a concentration of 2.5  105 cells/ml. To start the assay,
and transfected with 2.5 mg (100 nM) of the respective siRNA. 0.5  105 melanoma cells (200 ml) were added to the top of the filter.
Melanoma cells were incubated for 4872 h until they were analyzed Chambers were subsequently incubated at 378C/5% CO2 and
for protein knock-down. melanoma cells were allowed to migrate for 3 h. Then filters were
removed and all cells from the upper membrane surface were wiped
Antibodies off with a cotton swab. Filters were then washed, fixed and mounted
on glass slides. Melanoma cells, which had migrated to the coated
The function blocking monoclonal antibody against the integrin subunit underside of the filter were detected by confocal laser scanning
a4 and the monoclonal antibody against VCAM-1 were from microscopy, and cells in four defined optical fields were counted
CHEMICON. The monoclonal antibody against the integrin subunit for each filter.
aL and the monoclonal antibody against ICAM-1 were from DAKO.
The directly labeled monoclonal antibodies against the integrin Transendothelial migration assay
subunits b1, b2, and b3 were obtained from BD Biosciences
(Heidelberg, Germany). All monoclonal antibodies used are of the IgG1 HMEC-1 cells were seeded on the upper side of transwell migration
isotype. IgG1 isotype-matched control antibodies were from BD inserts (PET membrane, 8 mm pore-size, 6.4 mm diameter, BD
Biosciences. The R-Phycoerythrin-conjugated donkey anti-mouse Biosciences) and grown until a confluent monolayer was established.
IgG F(ab0 )2 fragments and the R-Phycoerythrin-conjugated goat The integrity of the monolayer was checked microscopically and by
anti-human Fcg fragment specific F(ab0 )2 fragments were purchased trypan blue exclusion. HMEC-1 cells were left either untreated or
from Jackson ImmunoResearch (Baltimore). stimulated overnight with 10 ng/ml TNF-a. After washing with
migration medium (RPMI 1640 medium supplemented with 0.1% (w/v)
Flow cytometry BSA), 200 ml of migration medium were added to the upper
compartment and 600 ml of RPMI 1640 medium supplemented with
For cell-surface staining, melanoma cells were released from the cell 10% FCS were added to the lower compartment. Melanoma cells were
culture dish by incubation with PBS containing 4 mM EDTA for 10 min labeled for 15 min at 378C/5% CO2 with 1 mM CFDA-SE (Molecular
at 378C and washed in 200 ml FACS medium consisting of PBS Probes) in PBS. The labeling solution was removed and the melanoma
containing 1 mM CaCl2, 0.5 mM MgCl2, 5 % FCS (w/v), 0.5% BSA (w/v) cells were incubated in culture medium for an additional 30 min at
and 0.1% sodium azide. Then 2  105 melanoma cells were incubated 378C/5% CO2. Afterwards, melanoma cells were detached from the
for 30 min at 48C in 50 ml of FACS medium containing the respective cell culture dish by incubation with PBS containing 4 mM EDTA for 10 min
monoclonal antibodies at the indicated concentrations. Melanoma cells at 378C, pelleted, washed, and resuspended in migration medium at
were washed twice in 200 ml FACS medium and, if the primary a concentration of 2.5  105 cells/ml. To start the assay, 0.5  105
antibodies were not directly labeled, incubated for 20 min at 48C in melanoma cells (200 ml) were added to the top of the filter. Chambers

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP


370 KLEMKE ET AL.

were subsequently incubated at 378C/5% CO2 and melanoma cells TABLE 1. Cell surface expression of integrin subunits
were allowed to migrate for 5 h. Then filters were removed and all cells
from the upper membrane surface were wiped off with a cotton swab. Control a4 b1 b2 b3
Filters were then washed, fixed and mounted on glass slides. Melanoma
cells that had transmigrated to the underside of the filter were 530 2W1 39 W 9 147 W 10 2W1 2W0
IF6 3W1 22 W 1 143 W 23 3W1 4W1
detected by confocal laser scanning microscopy and cells in four BLM 3W1 36 W 14 113 W 15 2W1 2W1
defined optical fields were counted for each filter. MV3 3W1 34 W 10 184 W 18 3W1 3W1
a
Results Shown are the mean MFI (mean fluorescence intensity) values  SD of three independent
measurements. Control: secondary antibody only.
b2/b3 integrin-negative human melanoma cells with
different metastatic capacity express comparable
levels of the integrin a4b1 on their cell surface
Expression of b3 integrins is important for melanoma cell differentially adhere to VCAM-1. To this end, a novel assay that
migration and metastasis (Albelda et al., 1990a; Hofmann et al., we have recently developed (ligand-complex-based adhesion
2000). However, none of the human melanoma cell lines assay, LC-AA; Konstandin et al., 2006) was employed. This
investigated here (530, IF6, MV3, and BLM) expressed b2 or b3 method allows to measure differences in integrin affinity and
integrins (Fig. 1 and Table 1). Nevertheless, two of them (MV3 avidity at the single cell level. Melanoma cells were incubated
and BLM) are highly metastatic upon injection into nude mice with soluble multimeric VCAM-1/Fc F(ab0 )2 complexes and the
(Van Muijen et al., 1991a; van Muijen et al., 1991b) and should percentages and mean fluorescence intensities of VCAM-1/Fc-
therefore be able to efficiently undergo transendothelial complex-binding cells were determined by flow cytometry
migration. We found that all b2/b3 integrin negative melanoma (Fig. 2A). Indeed, binding of a given cell line to multimeric
cell lines analyzed in this study express the a4b1 integrin with VCAM-1 correlated with the metastatic potential of the
comparable levels on their surface (Fig. 1 and Table 1). This respective cell line. Thus, melanoma cells of low metastatic
integrin recognizes, besides fibronectin, VCAM-1 (Chan and potential (IF6 and 530) bound to VCAM-1/Fc-complexes to a
Aruffo, 1993), a cell surface protein expressed by activated
endothelial cells (Xu et al., 1994; Haraldsen et al., 1996; Lee
et al., 2001; Park et al., 2001). Since VCAM-1 is known to be
important for transendothelial migration of hematopoetic
cells (Oppenheimer-Marks et al., 1991; Chan and Aruffo, 1993;
Chuluyan and Issekutz, 1993; Hourihan et al., 1993; Weber
and Springer, 1998; Ding et al., 2001), we investigated, whether
the integrin a4b1 is involved in transendothelial migration of
these human melanoma cell lines of different metastatic
potential.

Adhesiveness of a4b1 expressing human melanoma cells


to VCAM-1 correlates with their metastatic potential
To undergo transendothelial migration, tumor cells first have to
stably adhere to the endothelial cells. As the integrin a4b1 is
subject to affinity regulation due to inside-out-signaling, we
investigated, whether the a4b1 expressing melanoma cells

Fig. 2. Adhesiveness of a4b1 expressing melanoma cells to VCAM-1


correlates with their metastatic potential. A: Melanoma cells of either
low metastatic capacity (530 and IF6), or high metastatic capacity
(MV3 and BLM) were incubated at 37-C with 50 mg/ml of soluble
multimeric VCAM-1/Fc F(ab()2-PE complexes for the indicated time
points. Melanoma cells were fixed, and bound PE-labeled VCAM-1/Fc
Fig. 1. b2/b3 integrin-negative human melanoma cells express complexes were detected by flow cytometry. B: 530, IF6, MV3, and
comparable levels of the integrin a4b1 on their cell surface. Cell BLM melanoma cells were preincubated for 30 min at 4-C with 20 mg/
surface levels of the integrin subunits a4, b1, b2, and b3 were ml of function blocking antibodies against the integrin subunits a4 and
determined by flow cytometry. Open histogram, dotted line: control aL as indicated and allowed to bind soluble multimeric VCAM-1/Fc
(cells stained with secondary antibody only). Open histogram, solid F(ab()2-PE complexes for 20 min at 37-C. Melanoma cells were fixed,
line: staining with an isotype-matched control antibody. Filled and bound PE-labeled VCAM-1/Fc complexes were detected by flow
histogram: staining with the respective monoclonal antibody. Shown cytometry. Shown is one representative experiment out of two. Mean
is one representative experiment out of three. fluorescence intensity (MFI).

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TRANSMIGRATION OF MELANOMA CELLS 371

much lower extent and with slower kinetics than the highly The interaction between the integrin a4b1 and
metastatic melanoma cell lines (BLM and MV3). As expected, VCAM-1 enhances transendothelial migration of
adhesion to multimeric VCAM-1/Fc F(ab0 )2 complexes could be human melanoma cells
completely blocked by pre-incubation of the cells with a
function blocking antibody to the integrin subunit a4 but not To investigate, whether the high affinity interaction between
with an antibody directed against aL (Fig. 2B). Moreover, all the integrin a4b1 on melanoma cells and VCAM-1 on
melanoma cell lines used did not bind to ICAM-1/Fc F(ab0 )2 endothelial cells supports transendothelial migration of human
complexes due to lack of b2-integrin expression (data not melanoma cells, immortalized human dermal microvascular
shown). Together, these data show, thatdespite similar endothelial cells (HMEC-1; Kielbassa et al., 1998; Lidington
a4b1surface expression levelsonly the highly metastatic et al., 1999; Kielbassa-Schnepp et al., 2001; Eum et al., 2004)
melanoma cell lines MV3 and BLM, but not the low metastatic were used. As shown by flow cytometry, unstimulated HMEC-1
melanoma cell lines 530 and IF6, can use multimeric VCAM-1 as cells expressed integrin b1 and ICAM-1, but not VCAM-1, on
a ligand for stable adhesion. their cell surface (Fig. 4A, upper panels). In accordance with data
published by others (Xu et al., 1994; Haraldsen et al., 1996),
Haptotactic and chemotactic migration of the highly upon treatment with TNF-a for 18 h, HMEC-1 cells also
metastatic a4b1 expressing human melanoma cells is expressed VCAM-1 (Fig. 4A, lower panels). In addition, the
enhanced in the presence of VCAM-1 expression level of ICAM-1 increased while the level of the
integrin b1 subunit remained unchanged.
Adhesion to endothelial cells is the first step in transendothelial
migration. Then cells need to migrate on and finally through the
endothelial cell layer. For monocytes it is known that the
interaction of a4b1 with VCAM-1 facilitates lateral migration
on endothelial cells and thereby supports transendothelial
migration (Weber and Springer, 1998). We therefore
determined, whether the presence of high affinity VCAM-1
would enhance the haptotactic and chemotactic migration of
a4b1 expressing melanoma cells. Cell migration was assayed
using transwell inserts which were either left uncoated or
coated with VCAM-1/Fc before the assay was performed. For
haptotaxis experiments, no chemoattractant was added to the
lower compartment, whereas for chemotaxis assays, FCS was
added to attract the cells. The presence of VCAM-1/Fc
significantly increased the haptotactic (Fig. 3A) as well as
chemotactic (Fig. 3B) migration of the two metastatic
melanoma cell lines MV3 and BLM, which display high affinity
a4b1 on their surface. In marked contrast, the low metastatic
melanoma cell lines IF6 and 530 showed almost no migration
even in the presence of VCAM-1/Fc. Therefore, the migratory
behavior on immobilized VCAM-1 correlated with the
metastatic potential of the melanoma cell lines.

Fig. 4. The interaction between a4b1 and VCAM-1 enhances


transendothelial migration of human melanoma cells. A: Inducible
VCAM-1 expression on HMEC-1 endothelial cells. HMEC-1 cells were
either left untreated (upper row) or stimulated with 10 ng/ml TNF-a
for 18 h (lower row). Cell surface levels of the integrin subunit b1,
VCAM-1 and ICAM-1 were determined by flow cytometry. Open
histogram: control (secondary antibody only), filled histogram:
staining with the respective monoclonal antibody. Shown is one
Fig. 3. Haptotactic and chemotactic migration of the highly representative experiment out of two. B: Transendothelial migration
metastatic a4b1 expressing human melanoma cells is enhanced in the through a HMEC-1 endothelial cell layer. CFDA-SE labeled 530, IF6,
presence of VCAM-1. The underside of cell culture transwell inserts BLM, and MV3 melanoma cells were allowed to transmigrate for 5 h at
was either left uncoated (gray bars) or coated with 20mg/ml VCAM-1/ 37-C across a confluent monolayer of HMEC-1 cells grown on the
Fc (black bars) before the assay. To analyze haptotaxis (A), CFDA-SE upper side of a transwell cell culture insert (8 mm pore size). HMEC-1
labeled 530, IF6, BLM, and MV3 melanoma cells were allowed to cells were either left untreated or stimulated with 10 ng/ml TNF-a
migrate across cell culture inserts (8 mm pore size) for 3 h at 37-C. For for 18 h as indicated. For function blocking of integrins, melanoma
chemotactic migration (B), FCS was added to the lower cells were preincubated with either 20 mg/ml of anti-a4 or anti-aL
compartment of the transwell chamber before the assay was started. antibodies for 30 min at room temperature as indicated. The
The numbers of melanoma cells that migrated to the underside of the numbers of melanoma cells that migrated to the underside of the
cell culture insert were determined by confocal laser scanning cell culture insert were determined by confocal laser scanning
microscopy. Cells in four defined optical fields were counted. Shown microscopy. Cells in four defined optical fields were counted.
are the mean values W SD of one representative experiment out of Shown are the mean values W SD of one representative experiment
three. out of three.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP


372 KLEMKE ET AL.

For the analysis of transendothelial migration, HMEC-1 cells


were grown to confluency on transwell inserts and VCAM-1
expression was induced by TNF-a as described (Xu et al., 1994;
Haraldsen et al., 1996). Melanoma cells were allowed to
transmigrate for 5 h across the endothelial cell layer. As shown
in Figure 4B, only the highly metastatic melanoma cell lines MV3
and BLM displayed considerable transendothelial migration.
Transmigration already occurred through unstimulated,
VCAM-1 negative HMEC-1 cells. It could be significantly
enhanced by induction of VCAM-1 expression on the surface of
the HMEC-1 cells through TNF-a treatment (Fig. 4B). To
confirm the role of integrin a4b1/VCAM-1 interaction in this
increase in transendothelial migration, melanoma cells were in
parallel incubated with a function blocking antibody against the
integrin a4 subunit. This treatment indeed reduced
transmigration to the level observed with unstimulated HMEC-
1 cells. Note that an unrelated antibody against integrin aL had
no effect (Fig. 4B). These data demonstrate that the enhanced
transmigration upon TNF-a treatment relies solely on the
interaction of high affinity a4b1 with VCAM-1.
siRNA mediated knock-down of the integrin subunit a4
reduces transendothelial migration of a4b1 expressing
human melanoma cells
To confirm the role of the a4b1 integrin in enhanced
transendothelial migration across TNF-a activated endothelial
cells by an independent experimental approach, we performed
an siRNA-mediated knock-down of the integrin subunit a4 on Fig. 5. siRNA mediated knock-down of the integrin subunit a4
the two highly metastatic melanoma cell lines MV3 and BLM. reduces transendothelial migration of a4b1 expressing melanoma
Treatment with an integrin a4 specific siRNA reduced the cell cells. A: Knock-down of the integrin subunit a4. MV3 and BLM
surface levels of the integrin subunit a4 by around 70% as melanoma cells were transfected with 100 nM of either a non-
targeting control siRNA (open histogram with black line), or an
compared to a non-targeting control siRNA, whereas the cell a4-specific siRNA (filled histogram). The cell surface levels of the
surface levels of the integrin subunit b1 did not change (Fig. 5A). integrin subunits a4 and b1 were determined by flow cytometry 72 h
In accordance with the experiments performed with function post-transfection. Open histogram with dotted line: secondary
blocking antibodies, treatment of MV3 and BLM cells with the antibody only. B: Transendothelial migration of siRNA-treated
melanoma cells. CFDA-SE-labeled BLM and MV3 melanoma cells
a4 specific siRNA reduced transmigration across TNF-a pretreated with either a non-targeting control siRNA or an
stimulated HMEC-1 cells down to the level observed with a4-specific siRNA were allowed to transmigrate for 5 h at 37-C across
unstimulated HMEC-1 cells (Fig. 5B). These data clearly a confluent monolayer of HMEC-1 cells grown on the upper side of a
demonstrate that the interaction of a4b1 and VCAM-1 transwell cell culture insert (8 mm pore size). Before the
transmigration assay was performed, HMEC-1 cells were either left
enhances transendothelial migration of human melanoma cells untreated or stimulated with 10 ng/ml TNF-a for 18 h as indicated to
which express the a4b1 integrin its high affinity state. induce VCAM-1 expression. The numbers of melanoma cells that
migrated to the underside of the cell culture insert were determined
by confocal laser scanning microscopy. Cells in four defined optical
Discussion fields were counted. Shown are the mean values W SD of one
representative experiment out of two.
In human melanoma, increased expression of the a4b1 integrin
correlates with tumor progression and metastasis (Hart et al.,
1991; Moretti et al., 1993; Schadendorf et al., 1993; Schadendorf
et al., 1995). However, the underlying mechanism has not been
investigated. In this study we demonstrate, that the interaction melanoma cells remain to be investigated. Haptotactic as well as
of the integrin a4b1 with the adhesion molecule VCAM-1 chemotactic migration on VCAM-1 also correlated strictly with
enhances transendothelial migration of human melanoma cells the metastatic potential of the melanoma cells. The two low
across activated endothelial cells. metastatic melanoma cell lines 530 and IF6 displayed hardly any
All melanoma cells used in this study are devoid of b2 and b3 haptotactic or chemotactic migration even in the presence of
integrins, but express the a4b1 integrin at comparable levels. VCAM-1. In contrast, the two highly metastatic melanoma cell
As this integrin is subject to affinity regulation by inside-out lines MV3 and BLM displayed considerable haptotactic and
signaling (Shimaoka et al., 2002; Takagi and Springer, 2002), we chemotactic migration in the presence of VCAM-1.
investigated whether the melanoma cell lines with different Interestingly, enhanced migration upon presence of VCAM-1
metastatic capacity used in this study would bind soluble was most prominent in the haptotaxis migration assay.
VCAM-1 with similar affinities. Interestingly, the two highly Therefore, especially in the absence of chemotactic signals, the
metastatic melanoma cell lines MV3 and BLM bound VCAM-1 presence of a4b1 in the high affinity conformation may decide
with much higher affinity than the 530 and IF6 melanoma cell whether tumor cells undergo enhanced transendothelial
lines which have low metastatic potential. This finding is likely migration across activated VCAM-1 expressing endothelium.
due to constant activation of signaling pathways that lead to Transendothelial migration of the highly metastatic melanoma
affinity upregulation of the a4b1 integrin in the two melanoma cell lines MV3 and BLM occurred already through unstimulated,
cell lines of high metastatic potential. A role for PKC and VCAM-1 negative endothelial cells. As the melanoma cells used
intracellular calcium was described to be involved in a4b1 in this study are devoid of b2-integrins and do therefore not
dependent adhesion and cell spreading of human melanoma bind to ICAM-1, transmigration through unstimulated
cells (Saini et al., 1997; Seller and Hart, 2000). However, the endothelial cells is likely mediated via the interaction of other
exact signaling pathways that regulate a4b1 affinity in human adhesion molecules than VCAM-1 or ICAM-1. Possible

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP


TRANSMIGRATION OF MELANOMA CELLS 373

candidates are PECAM-1 or MCAM, which are expressed by might favor the transendothelial migration not only of effector
unstimulated endothelial cells (Newman et al., 1990; Albelda T-cells but also of a4b1 expressing tumor cells thereby
et al., 1990b; Bardin et al., 1996) as well as by melanoma cells promoting tumor metastasis.
(Luca et al., 1993; Lutzky et al., 2006), and which are known to The data presented in this study have also implications for gene
be involved in the process of leukocyte transmigration (Petri expression profiling of tumor cells using cDNA microarrays.
and Bixel, 2006). VCAM-1 is expressed by endothelial cells only Often cDNA microarrays are used to compare the expression
upon activation by inflammatory stimuli like TNF-a or levels of mRNAs between non-tumor and tumor cells
interferon-g (Xu et al., 1994; Haraldsen et al., 1996; Lee et al., (Gottschlich et al., 2006), or between non-metastatic and
2001; Park et al., 2001). Accordingly, the transendothelial metastatic tumor cells (Otsuka et al., 2001), in order to identify
migration of highly metastatic human melanoma cells was genes which are selectively up- or down-regulated in only one
enhanced upon treatment of endothelial cells with TNF-a. type of cell. In this study, we used two melanoma cell lines of
Treatment of the melanoma cells with an integrin a4b1 high (MV3 and BLM) and two melanoma cell lines of low
(VCAM-1 receptor) function blocking antibody completely (530 and IF6) metastatic potential. All of these melanoma cell
inhibited the enhanced transmigration seen after TNF-a lines expressed comparable levels of a4b1. By cDNA
treatment of the endothelial cells, while addition of an microarray analysis, expression of this integrin would,
unrelated antibody had no effect. Similar results were obtained therefore, not be associated with a specific phenotype.
by siRNA-mediated down-regulation of the integrin a4 subunit However, using a novel assay (ligand-complex-based adhesion
on the melanoma cells. Interestingly, the VCAM-1-dependent assay; Konstandin et al., 2006) which measures affinity plus
increase in transmigration of melanoma cells was completely avidity of the a4b1 integrin to VCAM-1 on the single cell level,
blocked by siRNA mediated knock-down of the integrin subunit we were able to show that, despite similar expression levels, the
a4, although expression of the a4 integrin subunit was not binding capacity of a4b1 to VCAM-1 is much lower in
completely down-regulated in these cells. Therefore it is likely melanoma cells with low metastatic potential as compared to
that a critical level of a4b1 integrin expression is required for melanoma cells with high metastatic potential.
efficient transendothelial migration of melanoma cells. This In conclusion, a4b1 integrin expression on melanoma cells
question could be addressed by the generation of subclones leads to enhanced transendothelial migration across VCAM-1
derived from a metastatic melanoma cell line which express expressing activated endothelial cells only if this integrin is
different levels of the integrin a4b1 in the high affinity present in the high affinity/avidity state. These data
conformation. demonstrate that rather than mere determination of a4b1
As endothelial cells express VCAM-1 on their surface only after integrin expression (Hart et al., 1991; Moretti et al., 1993;
inflammatory stimuli, as for example TNF-a, it is tempting to Schadendorf et al., 1993; Schadendorf et al., 1995), the
speculate, that integrin a4b1 expressing tumor cells might measurement of the affinity plus avidity of a4b1 to VCAM-1
preferentially leave the blood vessels at sites of inflammation, as might be used as a predictive marker for metastasis.
it is known for leukocytes (Ebnet et al., 1996; Worthylake and Consequently, blocking the a4b1 integrin on tumor cells and/
Burridge, 2001; Alon and Feigelson, 2002; Luscinskas et al., or blocking of VCAM-1 on endothelial cells may be a valuable
2002). Recent data have indeed shown that inflammation might approach to interfere with tumor metastasis.
be a critical component of tumor progression, and that many
cancers arise from sites of infection, chronic irritation and
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