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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From Servicio de Inmunología, Hospital General
Universitario Gregorio Marañón, and Servicio de
Inmunología, Hospital de la Princesa, Madrid, Spain.
Heterotypic interaction among tumor cells (TCs) and endothelial
cells (ECs) may play a critical role during the vascular dissemination of neoplastic cells and during pathologic angiogenesis in tumors. To
identify molecules involved in these processes, the distribution of
vascular junctional proteins was first studied by immunofluorescence at
sites of heterologous intercellular contact using TC-EC mosaic monolayers grown on 2-dimensional collagen. Several members of the
tetraspanin superfamily, including CD9, CD81, and CD151, were found to
localize at the TC-EC contact area. The localization of tetraspanins to
the TC-EC heterologous contact area was also observed during the active
transmigration of TCs across EC monolayers grown onto 3-dimensional
collagen matrices. Dynamic studies by time-lapse immunofluorescence
confocal microscopy showed an active redistribution of endothelial CD9
to points of melanoma insertion. Anti-CD9 monoclonal antibodies were
found to specifically inhibit the transendothelial migration of
melanoma cells; the inhibitory effect was likely caused by a
strengthening of CD9-mediated heterotypic interactions of TCs to the EC
monolayer. These data support a novel mechanism of tetraspanin-mediated
regulation of TC transcellular migration independent of TC motility and
growth during metastasis and a role for these molecules in the
formation of TC-EC mosaic monolayers during tumor angiogenesis.
(Blood. 2001;98:3717-3726) The dissemination of cells from a primary tumor by
invasion and metastasis represents the hallmark of
malignance.1 Invasion is the local dissemination of
neoplastic tissue, whereas metastasis is the distant dissemination to
secondary places of growth involving the transport of neoplastic cells
through the fluid spaces of the body (blood, lymph, cerebrospinal, or
peritoneal fluid).2 Although tumor cell (TC) migration
through the extracellular matrix (ECM) is required for invasion,
success in metastasis requires migration through the ECM and across
host cell layers (transcellular migration).3 Because most
cancer cells reach distant sites by dissemination through blood or
lymphatic circulation, transendothelial migration of TCs is a crucial
event in vascular metastasis formation. In addition, 2 recent works in
human melanoma4 and colon carcinoma5 suggesting that tumor vessels are "mosaic" lined by endothelial cells (ECs) and malignant TCs extend the role of TC-EC
interactions to the process of tumor angiogenesis. Thus, TC
transendothelial invasion and molecules involved in TC-EC interactions
could contribute to the formation of new blood vessels in tumors.
Tetraspanins comprise a numerous group of proteins that have 4 putative
transmembrane domains and that have been implicated in the regulation
of cell development, proliferation, activation, and
motility.6,7 It has been suggested that tetraspanins may
provide a bridge between A large array of adhesion molecules has been described as mediating the
adhesion and migration of TCs through ECM and in the initial attachment
to EC monolayers, including integrins, selectins, cadherins,
immunoglobulins, and proteoglycans.18,19 However, little
is known about the mechanisms that regulate the passage of TCs through
endothelial junctions. Earlier studies20-22 suggest that
TCs disseminate by breaching the vasculature wall, and they point to
several tumor-derived factors to induce EC retraction. In contrast,
recent work23 indicates that the establishment of
heterologous gap junctions between melanoma and ECs may contribute to
in vivo metastasis formation. Furthermore, heterologous cell-cell interactions between TCs and other host cell layers can take place during TC dissemination through visceral cavities, and the cellular and
molecular pathways implicated could be related to those acting during
TC-EC interactions. In this regard, an essential role for the
activation of the Rho-ROCK pathway has been described during transcellular invasion of a mesothelial cell monolayer by hepatoma TCs,
and its inhibition reduced the peritoneal dissemination of TCs in an in
vivo model.24 An attractive hypothesis is that TCs may
establish intimate contact with ECs, and this TC-EC interaction could
coordinate the opening of interendothelial junctions, facilitating their transmigration. In the current study, EC monolayers grown onto
3-dimensional collagen matrices were used to study the TC-EC interactions that take place during active transmigration of melanoma cells. We have identified several members of the tetraspanin
superfamily that localize to the TC-EC contact region, and we have
investigated the functional role of these molecules during TC transmigration.
Cells and cell cultures
Antibodies
Coculture experiments Collagen gels were used for 3-dimensional assays. Type I collagen (Cellagen solution AC-3; ICN Biomedicals) was mixed with 10 × medium 199 according to the manufacturer's directions. Appropriate volumes of this solution were allowed to gel at 37°C (1 hour) in each well of a 24-well tissue culture plate or on glass coverslips (12-mm diameter). Dehydrated collagen gels were made by allowing the gels on glass coverslips to dry in a laminar flow hood. Collagen gels were used as culture substrata for HUVEC cells. For 2-dimensional assays, glass coverslips were incubated with phosphate-buffered saline (PBS) containing 2% gelatin (Difco, Detroit, MI) for 30 minutes at 37°C, fixed with PBS containing 0.5% glutaraldehyde (Sigma) for 25 minutes at room temperature, washed 3 times in PBS, incubated with aqueous 0.1 M glycine (to block free aldehydes), washed 3 times in PBS, and stored for up to 1 week in HUVEC medium. BCECF-labeled A375 cells were added to the EC monolayer as indicated. This 2-dimensional model system did not allow the complete migration of the TCs underneath the endothelium.Silver nitrate staining Cell monolayers were fixed in 0.05% glutaraldehyde in PBS for 15 minutes at room temperature, washed twice with 5% glucose (Braun, Barcelona, Spain), and incubated for 30 seconds with 5% glucose containing 0.25% AgNO3 (Sigma). Cells were washed again with 5% glucose and covered with glycerol (87%) (Merck, Darmstadt, Germany). Silver lines were developed after the exposure of cells to UV light.Immunofluorescence analysis and confocal microscopy Cells were fixed for 15 minutes in 4% formaldehyde in PBS at room temperature. When necessary, cells were permeabilized with 0.2% Triton X-100 in Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, 0.1% NaN3, pH 7.6) for 3 minutes. After blocking nonspecific binding sites by incubation with TNB (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% blocking reagent; Boehringer Mannheim GmbH), cells were sequentially incubated with specific mAb or polyclonal antibodies and appropriate Cy3-labeled secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Cells were visualized using a Nikon Eclipse E800 photomicroscope (Nikon, Tokyo, Japan) with 100 × oil immersion objective, 1.4 NA. Preparations were photographed on either Ektachrome or TMAX 400 ASA film (Eastman Kodak, Rochester, NY). Fluorescence quantification was performed using Optimas 5.2 (Bioscan, Edmonds, WA). Series of optical sections distanced 0.5 µm on the z-axis were obtained with a confocal scanning laser microscope (MRC 1024; Bio-Rad, Hercules, CA) mounted on a Zeiss Axiovert 135 inverted microscope (Carl Zeiss, Oberkochen, Germany).Migration assays Transendothelial migration assays for A375 melanoma cells were performed in polycarbonate transwell inserts (8-µm pore; Costar, Corning, NY) coated with HUVECs and grown as a monolayer for 24 hours. BCECF-labeled A375 cells were resuspended in 10% FCS-RPMI-1640 and were preincubated with mAbs for 20 minutes before plating. HUVEC cells were also incubated with the same mAbs at this time. Typically, 1.5 × 105 BCECF-labeled A375 were seeded in the upper compartment (100 µL), and 10% FCS-RPMI-1640 was placed in the lower compartment (600 µL). After 16 hours at 37°C, the number of fluorescence-labeled A375 cells that had migrated through the monolayer was determined by direct counting on a FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA) using CellQuest software. Alternatively, migration assays were performed either with uncoated polycarbonate filters or after coating of the top side of the filter with fibronectin or collagen (10 µg/mL).Adhesion and aggregation assays For cellular adhesion assays, HUVECs were cultured on 96-microwell plates (Corning Glass Works) as a monolayer for 24 hours. BCECF-labeled A375 cells were resuspended in RPMI containing 0.4% bovine serum albumin (BSA) and the distinct mAbs for 15 minutes. HUVECs also were incubated with the same mAbs at this time. A375 cells (6 × 104) were allowed to adhere to each well for 5 minutes at 37°C. Unbound cells were removed by 3 washes with warm RPMI. The number of cells adhered to the wells was obtained by fluorescence intensity measurement in a microplate fluorescence reader (Victor Wallak, Turku, Finland). All assays were run in triplicate.Cell aggregation assays were performed as previously described33 with slight modifications. BCECF-A375 cells or SNARF-ECs were resuspended at 8 × 105 cells/mL in RPMI-1% BSA containing 10 µg/mL of either CD9 or control W6/32 mAb, mixed, and allowed to aggregate on BSA-coated plates for 30 minutes at 37°C and rotation (100 rpm). Numbers of particles before and after aggregation were quantified in a FACScan flow cytometer. After fixation, cell aggregates were photographed for either red or green fluorescence. Transient transfection and in vivo time-lapse confocal microscopy Cells were trypsinized and resuspended in RPMI 10% FCS medium supplemented with 5 µL 1.5 M NaCl, 20 µg Bluescript, and 5 µg CD9-green fluorescent protein (GFP)-DNA construct. Cells were electroporated at 975 µF/200 V in a Gene Pulser II (Bio-Rad) and were used for the time-lapse videomicroscopy experiments 24 hours after transfection. CD9-GFP fusion protein construct was obtained by polymerase chain reaction amplification of the CD9 cDNA (a kind gift of Dr E. Rubinstein, INSERM, Villejuif Cedex, France) and cloned in pEGFP-N1 Vector (Clontech Laboratories, Palo Alto, CA) in EcoRI sites of the cloning site.A375 cells were layered on a confluent HUVEC monolayer initially seeded on a collagen gel. Preparations were maintained at 37°C and 5% CO2 on an incubator coupled to a Leica TCS-SP confocal microscope (Leica Microsystems, Heidelberg, Germany). Series of images distanced 2 µm on the z-axis were acquired every 3 or 5 minutes. Fluorescence and DIC acquisition was performed simultaneously using a 63 ×, 1.4 NA oil-immersion objective. Statistical analysis Data were presented as the mean ± SD and were compared using Student t test or one-way analysis of variance.
Tumor cells transmigrate at the lateral borders of endothelial cells To investigate the transendothelial migration of TCs, EC monolayers were established on transwell inserts, and transendothelial migration (TEM) assays were performed using A375 melanoma cells labeled with the fluorescence dye BCECF (Figure 1A). In the absence of an endothelial monolayer, a significant number of A375 cells migrated after 16 hours, through the 8-µm pore polycarbonate filter, to the lower transwell compartment (28.9% ± 1.6% migrated cells). A slight reduction in the percentage of migrated A375 cells was observed using membrane inserts coated with ECM proteins, such as collagen or fibronectin (22.9% ± 0.9% and 24.6% ± 2.3% migrated cells, respectively). The presence of an endothelial cell monolayer coating the membrane filter resulted in an approximately 67% reduction in melanoma cell migration to the lower compartment (9.6% ± 1.2% migrated cells; P < .001).
To assess the interactions between TCs and ECs, endothelial monolayers were grown at confluence on collagen gels and were used to model the tightly apposed ECs that lined the vascular intima in vivo. Under these conditions, the EC junctions stained with silver nitrate and had well-developed adherens and tight junctions (not shown). BCECF-labeled A375 melanoma cells were added to confluent endothelial monolayer cultures and were observed for 16 hours. To monitor the integrity of the EC monolayer and the location of the TC-EC attachment in relation to the EC borders, TC-EC cocultures were fixed every 30 minutes and were stained with silver nitrate (Figure 1B) and with anti-VE-cadherin (Figure 1C). Initially, most TCs were observed above the EC monolayer and appeared round, with no silver deposition (data not shown). After 2 hours of coculture, most tumor cells were observed in direct contact with the silver-stained EC borders, showing a preferential localization where multiple ECs joined together (tricellular corners) (Figure 1B, panel ii, arrows). At this time, some TCs were observed in the same focal plane as the silver-stained vascular junctions, displaying pseudopods into the subendothelial matrix (Figure 1B, panels i-ii), and the silver reaction product stained the TC-EC contact area (Figure 1B, panel ii, arrows). VE-cadherin staining was observed to be continuous in the vicinity and under the TCs (Figure 1C). Only a focal loss of VE-cadherin staining was seen at the bottom of the EC-junction depression induced by the attached TCs, without a significant disruption of the neighboring EC monolayer (Figure 1C, right panel). At 16 hours, some TCs displayed a spread morphology and were observed under the plane of the silver-stained vascular junctions (Figure 1B, panel iii). Continuous lines of silver stain were observed overlying the TCs that appeared to have completed their passage through the monolayer to the abluminal surface (Figure 1B, panels iii-iv), indicating that EC cell-cell junctions were re-established after TC passage. Tetraspanins CD9, CD81, and CD151 localize at the heterotypic TC-EC intercellular junctions To identify EC molecules involved in the interaction with TCs, mAbs against several vascular junction proteins were studied for their ability to stain the TC-EC heterotypic contact areas by immunofluorescence. To study these interactions, a mosaic monolayer of TCs and ECs was prepared by seeding fluorescently labeled TCs over a confluent EC monolayer grown onto cross-linked gelatin. After 16 hours of coculture, the A375 cells were observed inserted between the ECs, forming a mosaic TC-EC monolayer. VE-cadherin staining completely disappeared from the edges of the ECs in contact with the TCs (Figure 2Ai), as previously described.34 Similarly, a lack of staining at sites of TC-EC contact was observed for -catenin (Figure 2Aii), a cytoplasmic
protein that links cadherins with the actin cytoskeleton, and for
PECAM-1 (Figure 2Aiii), a receptor involved in the transmigration of
leukocytes. The tight junction protein ZO-1 was localized at EC-EC and
TC-TC junctions, but it was absent from heterologous TC-EC contacts
(Figure 2Aiv). Interestingly, the tetraspanin superfamily members
(TM4SF) CD9, CD81, and CD151, which have been previously described to
localize at the lateral junctions of endothelial and epithelial
cells,6,30 were concentrated at TC-EC contact areas and at
EC-EC junctions (Figure 2B, panels i-iii). The  3
integrin that associates with members of the TM4SF30 was
also observed to be partially redistributed to the TC-EC contact area
(Figure 2Biv). In addition, the intensity of the fluorescence signal in
the images was quantified along lines traced over TC-EC contact regions
and over homotypic (EC-EC or TC-TC) junctions (Figure 2, histograms).
Although cadherin/-catenin, ZO-1, and PECAM-1 showed a sharp
increment in the intensity of the fluorescence signal only at homotypic
junctions, the quantification of the stainings of CD9, CD81, CD151, and
3 integrin peaked at sites of TC-EC contact and at EC-EC
contacts, indicating that these molecules are indeed concentrated at
heterotypic intercellular contact regions. The concentration of CD9,
CD81, and CD151 was only observed at sites of intercellular contact
(heterologous or homologous), and no accumulation was observed in the
periphery of TC or EC when cell-cell contact was lost (data not
shown).
The distribution of the tetraspanins CD9, CD81, and CD151 was also
studied during the active transmigration of TCs across ECs grown on
3-dimensional collagen matrix and analyzed by laser scanning confocal
microscopy. TCs above the endothelium (yet to transmigrate) were
observed in proximity with EC junctions, where CD9, CD81, and CD151
staining was highly concentrated at TC-EC contact areas (Figure
3A and data not shown). Transmigrating
TCs were observed inserted between ECs along several focal planes. Staining for CD9 (Figure 3B), CD81 (Figure 3D), and CD151 (data not
shown) revealed a strong concentration along the TC-EC contact regions.
This was further demonstrated when vertical confocal sections were
analyzed (Figure 3B, D, lower panels, arrows). Transmigrated TCs were
observed in the subendothelial matrix in proximity with the overlying
endothelium (Figure 3C) and deep in the collagen matrix (Figure 3D,
lower panel). In both cases, the integrity of the EC monolayer
overlying the migrated TCs was restored (arrowheads). Thus, during the
active migration of TCs across the lateral borders of the ECs, the
TM4SF members CD9, CD81, and CD151 were redistributed around the
transmigrating TCs. These data support a possible role for tetraspanins
CD9, CD81, and CD151 in the molecular interactions required for TC
transendothelial invasion.
Anti-CD9 monoclonal antibodies impair the transendothelial migration of tumor cells To determine the possible functional involvement of members of the TM4SF in the passage of TCs across an EC monolayer, TEM assays of A375 cells were performed in the presence of different mAbs to these molecules. As shown in Figure 4A, the anti-CD9 VJ1/10 mAb markedly inhibited (approximately 69%; P < .005) the transendothelial migration of TCs (4.4% ± 1.4% vs 14.2% ± 1.3% control anti-HLA-class I W6/32). In contrast, mAbs to either the tetraspanins CD81 and CD151 or to the integrins1 and 3 did not affect
TEM findings of TCs. When the ability of several other anti-CD9 mAbs to
inhibit TC transmigration was assessed, we found that VJ1/10, VJ1/20, and GR2110 mAbs also blocked TC transmigration (Figure 4B). Moreover, the divalent F(ab')2 fragment of VJ1/10 was inhibitory,
thus ruling out any Fc-mediated effect (Figure 4C). The inhibition of
TC transmigration was dependent on the mAb dose, with a maximal
induction in the range of 1 to 20 µg/mL for VJ1/10 mAb (data not
shown).
Previous reports14,15,30 have addressed the roles of CD9, CD81, and CD151 in cell migration, describing the inhibition of cell motility by several mAbs against these molecules. Thus, a general impairment of TC motility induced by the anti-CD9 mAb could be the mechanism involved in the prevention of TC transendothelial migration. To ascertain the effect of the anti-CD9 mAb in TC migration versus TC transendothelial migration, parallel assays were carried out either in the absence or in the presence of an EC monolayer coating the transwell filter (Figure 4C). Consistent with previous data, a mild reduction of approximately 5% (36.4% ± 0.4% vs 41.1% ± 3.4%) in TC migration was induced by the anti-CD9 VJ1/10 mAb in the absence of endothelium. In contrast, when the transwell filter was coated with an EC monolayer, the inhibitory effect of the anti-CD9 mAb was markedly increased by more than 60% (9.4% ± 2% vs 23.6% ± 2.3%; P < .001). To explore the mechanism responsible for the inhibition of TC
transmigration, the ability of anti-CD9 mAbs to modify TC adhesion to
an EC monolayer was analyzed. BCECF-labeled A375 cells were allowed to
attach for 5 minutes to an EC monolayer in the presence of the
different mAbs. Anti-CD9 mAbs consistently enhanced the adhesion of
A375 cells to endothelium (Figure 5A).
Because CD9 is highly expressed by TCs and ECs, experiments were
performed to rule out the possibility that CD9 mAb influenced cell
adhesion by cross-linking TCs to ECs. As shown in Figure 5A, monovalent Fab fragments of anti-CD9 VJ1/10 and VJ1/20 mAbs induced TC adhesion to
EC monolayers, thus ruling out any antibody-mediated cross-linking effect. Furthermore, other mAbs against a different tetraspanin molecule, CD81/TAPA-1, also expressed by both cell types, did not
enhance TC adhesion to EC monolayers (Figure 5A). The CD9-mediated enhancement of TC attachment to EC occurred rapidly, at a maximum at 5 to 10 minutes, and was dependent on mAb dose, with induction of the
proadhesive effect in the range of 1 to 10 µg/mL VJ1/10 that
correlated well with inhibitory doses in the transmigration assays
(data not shown). These data suggest that the mechanism responsible for
the inhibition of transendothelial migration might be related to an
increase in heterotypic TC-EC adhesion mediated by CD9.
To assess the functional role of CD9 in heterotypic TC-EC adhesion, cell aggregation assays were performed with TCs and ECs labeled with BCECF (green) or SNARF (red), respectively. VJ1/10 anti-CD9 mAb, at doses of 1 to 10 µg/mL, was able to induce the formation of heterotypic cell aggregates (Figure 5B). CD9-mediated TC-EC aggregation was strong, involving most of the cells, and most aggregates contained both TC and EC cell types (98% of TC-EC heterotypic aggregates). A representative TC-EC aggregate is shown in Figure 5C. These data point to a role for CD9 in the heterotypic adhesion between TCs and ECs that may have profound implications for metastasis and development of mosaic blood vessels in tumors. Dynamic assessment of CD9-mediated heterotypic TC-EC interaction To assess directly the possible role of CD9 in heterotypic TC-EC cell-cell interaction, a CD9-GFP DNA construct was transfected in either A375 or EC cells. First, A375 cells were transfected with GFP-tagged CD9 and were seeded over an endothelial cell monolayer. At the focal plane of TC contact with the endothelium (Z = 0 µm), CD9 was present in filopodia extensions that seemed to survey the endothelial surface (Figure 6A, panels 0-9 minutes). A reconstruction of the signal in different confocal planes showed that there was no clear relocalization of melanoma CD9 to the TC-EC contact (Figure 6A, upper panels, 0 minutes). Once the melanoma cell found an appropriate insertion site, it spread in less than 15 minutes forming a mosaic monolayer (Figure 6A, panels 15-24 minutes).
In parallel studies, EC cells were transfected with the CD9-GFP construction (Figure 6B). CD9 appeared diffusely on the endothelial cell surface, and only in some fields did it concentrate at EC-EC contact sites (upper panel arrowheads). When melanoma cells were layered on top, endothelial CD9 clearly redistributed to TC-EC contacts at the time of insertion (Figure 6B, upper and lower panels 10-20 minutes, arrows). These data support a role for endothelial CD9 in EC heterotypic adhesion with melanoma cells during transmigration.
The spread of cancer cells from a primary tumor to a site of metastasis formation involves multiple steps, including migration of TCs through the surrounding stroma, entry into the circulatory system, and arrest, extravasation, and growth at a distant secondary site.2 Metastasizing TCs accomplish 2 rounds of vessel wall invasion. First, during intravasation, TCs invade the basal lamina and migrate across ECs lining the capillaries that service the tumor to the vascular space. Second, during extravasation, blood-borne TCs bind a specific ligand on the surfaces of ECs and transmigrate across ECs and the basal lamina into the different tissues.3 TC-EC interactions may participate in the metastatic process during TC intravasation into the vascular space and during TC extravasation into tissues. Either distinct or common molecular mechanisms could be involved in direct (luminal to abluminal) and the reverse (abluminal to luminal) TC transendothelial invasion. Because little is known about the molecular interactions between TCs
and ECs during transendothelial migration of cancer cells, we
established several model systems that allow the in vitro
characterization of this process. We used either a 3-dimensional system
that allows the complete migration of TCs under the EC monolayer into
the subendothelial collagen matrix or a 2-dimensional system that prevents the complete migration of the TCs and creates a TC-EC mosaic
monolayer. These cocultures were used to analyze, by immunofluorescence staining and confocal microscopy, the subcellular distribution of
vascular junctional molecules at sites of TC-EC contact. Using this
approach, several members of the tetraspanin family Our results show that TCs transmigrate through an EC monolayer by paracellular routes, spreading into the subendothelial matrix. During their passage, TC lateral borders were closely apposed to the adjacent edges of ECs, indicating tight association between TCs and ECs. TC-EC interactions appeared to be critical for vascular dissemination of TCs as assessed in the B16 melanoma mouse model.23 In this in vivo model, the ability of TCs to spontaneously metastasize was associated with the TC expression of connexin 26 and the establishment of heterologous gap junctions with ECs. Interestingly, the formation of TC-EC gap junctions was highly dependent on EC culture conditions, which was optimal on a vein segment23 or when ECs were cultured on collagen matrix (P.S.-M., unpublished observations, 1999). Similarly, EC junctions are stained with silver only when cells are grown on 3-dimensional matrix or cross-linked gelatin.35,36 Under these conditions, resembling those of the vascular intima found in vivo, TCs migrate through the EC lateral junctions interacting with the ECs; thus, it is an appropriate model to study TC-EC molecular interactions. Most works on the transendothelial migration of cancer cells show the formation of gaps in the EC monolayer that allow direct access of TCs to the exposed subendothelial matrix.21,22,37-39 In this regard, it has been reported that TC-EC interactions induce a rapid EC-EC dissociation, which correlates with a dramatic loss of VE-cadherin staining around the TCs.34 In contrast, only local changes in VE-cadherin staining at sites of TC-EC contact were reported using a 3-dimensional EC culture system on matrigel,40 in accordance with our results with collagen gels. These differences may be explained either by the EC culture conditions or by differences between TCs in their ability to interact with ECs. Reversible focal changes in VE-cadherin complex have recently been described during the transendothelial migration of monocytes.41 It is conceivable that TC transmigration shares some properties with leukocyte transmigration; hence, an active TC interaction with the EC may trigger the focal loss of VE-cadherin. Notably, our data on the identification of several members of the
tetraspanin superfamily, such as CD9, CD81, and CD151, as molecules
that localize along the TC-EC contact area indicated that they can play
a crucial role in TC transendothelial migration. Thus, during
transmigration, TCs may establish dynamic heterologous junctional
structures with ECs containing TM4SF/ Our data on the inhibitory effect on TC transmigration exerted by
several anti-CD9 mAbs provide demonstrative evidence of the involvement
of CD9 in the TC transmigration process. A possible mechanism
accounting for the inhibition of TEM could reside in the
anti-CD9-mediated enhancement of TC adhesion to EC monolayers and the
promotion of a strong heterotypic TC-EC adhesion, without affecting
adhesion to extracellular matrix proteins (P.S.-M. et al, unpublished
observations, 1999). Interestingly, tetraspanins are
associated with different Members of the tetraspanin superfamily of proteins usually become down-regulated in metastatic tumors,9-13,46 and the transfection of CD9, CD63, or CD82 reduces metastasis in vivo.15-17 Inhibition in either bare cell migration or cell growth had been previously invoked to explain the reduction in metastatic potential of tetraspanin-overexpressing TCs, and a direct involvement in extravasation and colonization of secondary sites has only been suggested for CD151.14 Our data support a new mechanism for tetraspanin regulation of tumor cell metastasis independent of tumor cell growth or motility, and they highlight a role for endothelial CD9 in active recognition of TCs during insertion. At any rate, the contribution to the in vivo suppression of metastasis of the inhibitory effect of transcellular migration deserves further investigation.
We thank J. Villarejo and I. Treviño for their technical assistance, the members of Servicio de Obstetricia, Hospital General Universitario Gregorio Marañón for the material provided, and M. A. Olazcarizqueta for technical assistance with confocal microscopy. We also thank Dr José Luis Rodríguez for critical reading of the manuscript.
Submitted April 2, 2001; accepted August 15, 2001.
Supported by grants SAF96-0092 from Comisión Interministerial de Ciencia y Tecnología and PM98-0016 from Dirección General de Enseñanza Superior e Investigación Científica (P.S.-M.); and SAF99-0034-CO2-01 from the Ministerio de Educación y Cultura and QLRT-1999-01036 from the European Community (F.S.-M.).
G.R. is the recipient of a fellowship from the Fundación Científica de la Asociación Española Contra el Cáncer.
N.L. and M.Y.-M. contributed equally to this paper.
The online version of the article contains supplemental videos.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Paloma Sánchez-Mateos, Hospital Gregorio Marañón, Servicio de Inmunología. C/Dr Esquerdo 46, 28007 Madrid, Spain; e-mail: inmunoonc{at}hispacom.es.
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Abstract
Introduction
(i) VE-cadherin, (ii) 
