[CANCER RESEARCH 51. 2239-2241, April 15, 1991]
Advances in Brief
Heterogeneity in Human Melanoma Cell Adhesion to Cytokine Activated
Endothelial Cells Correlates with VLA-4 Expression1
InèsMartìn-Padura, Roberta Mortarini, Davide Lauri, Sergio Bernasconi, Francisco Sanchez-Madrid,
Giorgio Parmiani, Alberto Mantovani, Andrea Anichini, and Elisabetta Dejana2
Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan [I. M-P., D. L., S. B., A. M., E. D.J and Division of Experimental Oncology, istituto
Nazionale Tumori, Via Venezian I, 20133 Milan [R. M., G. P., A. A.] Italy and Servicio InmunologÃ-a,Hospital de la Princesa, C/Diego de Leon 62, 28006 Madrid,
Spain ¡F.S-M.J
Abstract
Tumor cell attachment to endothelial cells (EC) is one of the critical
steps of the metastatic process. It was previously reported that interleukin
1 treatment of EC induces expression of membrane molecules that
promote tumor cell adhesion. In this paper we report that a panel of six
clones isolated from a human metastatic melanoma presented a marked
heterogeneity in their ability to adhere to interleukin 1 activated EC.
This was correlated with integrin VLA-4 expression by the clones.
Antibodies directed to VLA-4 and to its endothelial ligand INCAM110/
VCAM-1 abolished interleukin 1 induced increase in melanoma cell
adhesion to EC. These data demonstrate intratumor heterogeneity in the
expression of VLA-4 and that this can represent a crucial determinant of
tumor cell interaction with EC during secondary spread.
Introduction
An important step in the metastatic process is the arrest of
TC3 in the venous or capillary bed of the target organ (1,2). It
is therefore important to define the adhesive receptors on
endothelial and TC membrane that mediate their specific ad
hesive interactions. We and others have found that TC of
different origin adhere more effectively in vitro to EC activated
with inflammatory cytokines such as IL-1 and TNF (3, 4). This
phenomenon was correlated with an increase in the number of
human melanoma lung colonies in IL-1 and TNF treated nuce
mice (5).
Further studies4 showed that the increment of TC adhesion
to cytokine treated EC was mediated by newly synthesized
endothelial adhesive antigens (6). Some of these molecules have
been defined and could be equally recognized by leukocytes and
leukemic cell lines (7-9). TC, however, present origin depend
ent heterogeneity in the recognition of EC adhesive molecules.
Lines of colon carcinoma cells selectively bind to ELAM-1
while melanoma cells bind to INCAM 110/VCAM-l (6). The
receptor for INCAM 110/VCAM-l was recently identified on
leukemic cell lines as the integrin VLA-4 (10).
Tumors are composed of populations of cells heterogeneous
in a variety of biological and pharmacological properties in
cluding metastatic capacity ( 11). Anichini et al. ( 12) have shown
that human melanoma clones isolated from the same tumor
Received 1/21/91 ; accepted 2/22/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Italian Association for Cancer Research and
by the National Research Council special projection "Biotecnologie e Biostru
mentazione" and "Oncologia."
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: TC, tumor cells; EC. endothelial cells; IL-1,
interleukin 1; TNF, tumor necrosis factor; mAb, monoclonal antibody.
4 D. Lauri, L. Needham, I. Martìn-Padura. and E. Dejana, submitted for
publication.
differed in the pattern of integrins and in the amount of VLA4 expressed. Since it is important to define phenotypic and
functional parameters potentially relevant to predict TC met
astatic behavior, we investigated whether the amount of VLA4 expressed correlated with the capacity of the melanoma cells
to adhere to EC. The data reported indicate that VLA-4 only
partly mediates melanoma cell adhesion to unstimulated EC
but is required for the increase in adhesion to IL-1 treated EC.
This last parameter correlated with VLA-4 expression by the
cells.
Materials
and Methods
Cells. EC were isolated and cultured from human umbilical vein as
described previously (13). Melanoma cell clones from a human s.c.
metastasis (Mel 665/2) were obtained and cultured as reported (14).
Antibodies. mAb HP2/1 directed to VLA-4 «subunit has been
characterized previously (15); mAb B1E5 (16) directed to VLA-5 a
subunit was kindly donated by Dr. C. Damsky (University of California,
San Francisco, CA); both of them were incubated with TC suspension
ata 1:5 dilution.
mAb E1/6 (6) and mAb 4B5 (17) to INCAM 110/VCAM-l were
obtained through the courtesy of Dr. M. P. Bevilacqua (Harvard Med
ical School, Boston, MA) and Dr. J. M. Marian (University of Wash
ington, Seattle, WA), respectively; mAb 6.5B5 (18) directed to ICAM1 was generously donated by Dr. D. Haskard (Guy's Hospital, London,
United Kingdom) and mAb H18/7 (19) to ELAM-1 was from Dr. M.
P. Bevilacqua. mAbs were used to a final concentration of 1:10, except
mAb 4B5 and mAb 6.5B5 at concentrations of 1:100 and 1:20,
respectively.
A rabbit polyclonal antiserum to purified fibronectin (20) was added
during the adhesion assay at 1:50 dilution.
Antibody concentration was selected to obtain maximal biological
activity as described in the pertinent references.
Fluorescence flow cytometric analysis was performed by an EPICS
C cytofluorimeter as described previously (12).
Adhesion Assay. EC were activated with 20 units/ml of human
recombinant IL-1/3 (specific activity, IO3units/Mg; Sciavo, Siena, Italy)
for 6 h (3). mAbs directed to EC adhesive receptors were preincubated
with EC for 30 min/37°Cbefore addition of TC and then allowed to
stay during the adhesion assay. [125I]Iododeoxyuridine (1 jiCi/ml for 18
h; Amersham International, Amersham, United Kingdom) labeled mel
anoma cells were detached, washed twice with phosphate buffered
saline, and finally resuspended in M199-20% newborn calf serum at 6
x 10s cells/ml as described (3). mAbs directed to integrins were added
to TC 30 min before the adhesion test. Then an aliquot (50 u\) of
radiolabeled TC suspension containing the mAbs was added to each
EC well and incubated for 30 min at 37°C.Nonadherent cells were
then removed by washing the plates three times with phosphate buffered
saline plus 2% newborn calf serum. The content of each well was
solubili/ed with 100 ¿ilof NaOH-1% sodium dodecyl sulfate and the
lysate was counted in a 125Igamma counter (Beckman, Fullerton, CA)
as described (3).
2239
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MELANOMA CELL ADHESION TO THE ENDOTHELIUM
400 -,
Panel A
D
2/60
2/17
18
.1
tn
300 H
13
<B
ra
E
o
IL-1
C+VCAM-I Ab
2/61
10
2/62
13
Õ
& 200
IL-UVCAM-1 Ab
-
cr
2/4
31
2/14
26
8
10020
40
60
% VLA-4
80
100
Panel B
I
2/14
jI
2/4
2/62
1
2/61
2/17
2/60
Melanoma clones
2'60
Fig. 3. Effect of the INCAM110/VCAM-1 mAb H l /6 on adhesion of different
melanoma clones to EC. Unstimulated (C) and IL-1 stimulated (20 units/ml) EC
monolayers were incubated in medium containing either INCAM110/VCAM-1
mAb (Ab) or an irrelevant mAb (final concentration, 1:10) for 30 min at 37°C.
2<17
2/61
Radiolabeled melanoma cells were added without removal of mAbs and incubated
at 37°Cfor an additional 30 min to allow adhesion [mean ±SD (bars) of 3
experiments, 6 replicates/experiment). *, P < 0.01 versai control adhesion; O, P
< 0.01 versus adhesion to IL-1 activated EC. Statistical analysis by Dunnet's test.
2'62
2/4
2'14
Results
300
100
200
% increment
Fig. 1. (A) Fluorescence flow cytometric analysis of VLA-4 integrin expression
on melanoma clones from Me665/2. Columns, percentage of positive cells for
VLA-4 after subtracting the nonspecific background staining with an irrelevant
antibody. Numbers inside columns, mean intensity of fluorescence (arbitrary units
on a linear scale). The analysis was performed at least three times for each clone
with comparable results. (B) Adhesion of human melanoma clones to IL-1 (20
units/ml, 6 h) stimulated EC for 30 min at 37°C.Results are expressed as
percentage of increment of cell adhesion. Percent increment was calculated as
(TC bound to stimulated EC - TC bound to untreated EC)/(TC bound to
untreated EC x 100). Mean ±SD (bars) of 3 experiments. 6 replicates/experi
ment. Control adhesion to unstimulated EC was 1059 ±169 (cells/well) for clone
2/60. 2287 ±595 for clone 2/17, 2281 ±456 for clone 2/62. 1931 ±154 for
clone 2/61, 2159 ±238 for clone 2/4, and 3744 ±526 for clone 2/14.
400
I
•'
n |L1
-,
300
f2
C»VLA-4 Ab
Cn
IL-1 »VLA-4 Ab
Fig. 1 compares different clones of melanoma cells for the
percentage and intensity of fluorescence of VLA-4 expression
(Fig. IA) and for IL-1 induced increment in adhesion to EC
(Fig. \B). The melanoma clones were quite heterogeneous for
these parameters. However, a correlation was observed between
VLA-4 expression and cell adhesion to IL-1 treated EC.
We then directly investigated the role of VLA-4 and of its
EC ligand INCAM 110/VCAM-1 by antibody inhibition stud
ies. A VLA-4 mAb (Fig. 2) and an INCAM 110/VCAM-1 mAb
(Fig. 3) abolished IL-1 induced increment in adhesion of mel
anoma clones, except clones 2/14, 2/4, and 2/61 which were
only partially inhibited by an INC AM 110/VCAM-1 mAb. The
data reported in Fig. 3 have been obtained with mAb H1/6 but
comparable results were found with mAb 4B5.
Both mAbs slightly but consistently inhibited melanoma cell
adhesion to untreated EC. This inhibition reached statistical
significance for clones 2/4, 2/62, 2/61, and 2/60 with both
antibodies. ELAM-1, ICAM-1, VLA-5, and fibronectin anti
bodies did not affect adhesion of melanoma clones to either
untreated or IL-1 treated EC (data not shown).
O)
€
ra
g
Discussion
200 -
8
100 -
2/14
2/4
2/62
2/61
2/17
2/60
Melanoma clones
Fig. 2. Effect of the VLA-4 mAb on adhesion of different melanoma clones to
EC. Melanoma cells (C) were preincubated in medium containing either VLA-4
mAb or an irrelevant mAb (final concentration, 1:5) for 30 min before addition
to unstimulated and IL-1 stimulated EC and maintained for an additional 30 min
to allow adhesion at 37°C.Results are expressed as percentage of control adhesion
[mean ±SD (bars) of 3 experiments, 6 replicates/experiment. "P < 0.01 versus
control adhesion: O, P < 0.01 versus adhesion to IL-1 activated EC. Statistical
analysis by Dunnet's test.
The characterization of the molecules involved in TC adhe
sion to the endothelium might help one to understand how TC
localize and eventually metastasize in different organs.
The data reported indicate that VLA-4 expression by clones
of human melanoma cells correlates with the increase in adhe
sion induced by IL-1 treatment of EC and that a VLA-4 mAb
can block this increment.
The recently identified EC ligand for VLA-4 was named
INCAM 110/VCAM-1 (6) and belongs to the immunoglobulin
superfamily (7). INCAM 110/VCAM-l mAbs strongly inhib
ited cell adhesion to IL-1 activated EC. However, while the
VLA-4 antibody was fully inhibitory with all the clones used,
INCAM 110/VCAM-1 antibodies in three cases (clones 2/14,
2/4, and 2/61) were only partially inhibitory. We do not have,
at present, a direct explanation for this discrepancy but it is
2240
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MELANOMA CELL ADHESION TO THE ENDOTHEL1UM
conceivable that these clones recognize an additional VLA-4
ligand on activated EC. A possible candidate could be fibronectin since VLA-4 can also bind this protein and fibronectin is
present in EC extracellular matrix and possibly on EC surface.
However, anti-fibronectin antibodies did not affect adhesion of
melanoma clones to either untreated or IL-1 activated EC. The
possibility that the cells bind to other known IL-1 dependent
EC adhesive antigens is also unlikely since ELAM-1 and
ICAM-1 mAbs were uneffective. The existence of a second
ligand for VLA-4 therefore remains unclarified and requires
further and more direct studies.
VLA-4 and INCAM110/VCAM-1
antibodies slightly inhib
ited adhesion of melanoma clones to untreated EC. This obser
vation is in agreement with previous reports indicating the
presence of low but biologically significant amounts of
INCAM110/VCAM-l
on the surface of resting EC (21, 22).
The biological relevance of VLA-4/VCAM-1 type of recog
nition in melanoma cell dissemination and metastasis remains
to be elucidated.
IL-1 and TNF can be released in tissues and reach appreciable
circulating concentrations during inflammatory reactions (23).
Many cell types including vascular cells, macrophages, and TC
of hematopoietic and nonhematopoietic origin have the poten
tial to release these cytokines (23).
Circulating or locally released TNF and IL-1 can activate EC
to express adhesive structures for TC and this in turn facilitates
the metastatic process. We reported (5) that IL-1 treatment of
nude mice caused a strong increase in the number of melanoma
lung colonies. It will be important to investigate whether mel
anoma cell localization correlates with VLA-4 expression and
could be inhibited by specific antibodies directed to this integrin.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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2241
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1991 American Association for Cancer Research.
Heterogeneity in Human Melanoma Cell Adhesion to Cytokine
Activated Endothelial Cells Correlates with VLA-4 Expression
Inès Martìn-Padura, Roberta Mortarini, Davide Lauri, et al.
Cancer Res 1991;51:2239-2241.
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