(Received for publication, August 3, 1995; and in revised form, January 2, 1996)
From the
Mechanisms mediating tumor cell attachment to the vessel wall
under flow conditions are largely unknown. Therefore we analyzed the
ability of human melanoma cells to adhere to an immobilized matrix
during blood flow and determined the role of platelets in this process.
In a parallel plate flow chamber, M21 melanoma cells were suspended in
human blood and perfused over a collagen I matrix at a wall shear rate
of 50 s (2 dynes/cm
) to simulate venous
flow over a thrombogenic surface. Melanoma cell interaction with the
matrix or blood cells and platelets was monitored and quantified by
fluorescence and confocal laser microscopy. Despite their ability to
adhere to collagen I under static conditions, M21 cells failed to
attach directly to this matrix during blood flow. However, they
associated with adherent thrombi, and this resulted in stable melanoma
cell arrest. Inhibition of platelet activation or platelet integrin
IIb
3 function abolished M21 cell attachment. Melanoma cell
interaction with thrombi was specific and required
3 integrin
expression. M21-L cells which lack integrin
v
3 failed to
associate with thrombi and to arrest during blood flow. Transfection of
these cells with the integrin subunits
v or
IIb resulted in
variants expressing
v
3, as in the wild type, or
IIb
3. Both variants were able to associate with thrombi and
to arrest during blood flow. Therefore,
3 integrin-mediated
binding to activated platelets represents an efficient mechanism for
melanoma cell arrest under flow, and this may contribute to the role of
platelets in hematogenous metastasis.
During metastasis, tumor cells disseminate to distant organs via the lymph or the blood stream(1) . The arrest of metastasizing tumor cells within the blood stream is a prerequisite for their extravasation. This step is rate-limiting during hematogenous metastasis. In the blood stream, tumor cells are exposed to flow-dependent shear forces, plasma proteins, blood cells, and platelets, all of which may affect tumor cell survival, arrest, and extravasation. Mechanisms which mediate tumor cell arrest involve adhesive interactions of tumor cells with vascular cells and their matrices. A major limitation of our current knowledge of such adhesive interactions originates from the fact that most experimental models used for their study are based on static conditions that are different from those present in the vasculature. Specifically, effects generated by blood flow and the resulting shear forces may critically affect adhesive cell interactions(2) . Therefore, the goal of the present study was to analyze tumor cell arrest under flow conditions. This was achieved by using an in vitro model that mimics flow in the vasculature. The system is based on a parallel plate flow chamber combined with confocal laser microscopy and real time recording to document and quantify tumor cell adhesive properties under flow.
Tumor cells originating from solid tumors express a variety of
adhesion receptors which support their attachment to extracellular
matrices present in tissues and the vessel wall, such as collagen,
fibrin, fibronectin, laminin, vitronectin, and von Willebrand
factor(3) . Adhesion receptors also participate in tumor cell
interactions with the intact endothelium of the vasculature. This has
been demonstrated mostly under static conditions, and many of these
adhesion receptors were identified as members of the integrin family
including 1 integrins containing the
subunits
1,
2,
3,
4,
5,
6,
7, and the
v
integrins
v
3 and
v
5(4) . Static conditions
are, however, unlikely to occur in the blood stream. It has been
suggested that passive entrapment of tumor cells in narrow capillary
vessels may favor their attachment to the endothelium or exposed sites
of the subendothelial matrix via adhesion receptors genuine to the
tumor cells. Yet shear conditions can reach maximal levels in capillary
passages(5) . Therefore, adhesion mechanisms mediating tumor
cell attachment to the vessel wall would have to be designed such that
adhesive interactions can be established under flow conditions and
continuously withstand shear stress. While mechanisms for
flow-resistant adhesion have been identified for platelets as well as
for leukocytes and enable them to fulfill their specific functions
during hemostasis and inflammation(6, 7) , it is yet
unknown whether metastasizing tumor cells possess similar mechanisms.
It has long been thought that platelets may assist hematogenous
dissemination of metastasizing
cells(8, 9, 10) . Perhaps the most convincing
evidence is the inhibition of metastasis by experimental
thrombocytopenia shown for a variety of
tumors(11, 12, 13, 14) . We
therefore hypothesized that tumor cells may interact with platelets and
thereby acquire specific mechanisms which mediate platelet anchorage
under flow. To test this hypothesis we selected a human melanoma cell
model, because melanoma is the most malignant form of skin cancer
characterized by a high frequency of metastasis during early stages of
the disease. M21 melanoma cells suspended in human blood failed to
attach directly to a collagen I matrix under flow, even at a relatively
low wall shear rate of 50 s
(2 dynes/cm
)
which corresponds to venous blood flow(5) . However, the
melanoma cells were able to associate with attached platelets that had
become activated and engaged in thrombus formation due to their initial
matrix contact. The interaction between melanoma cells and platelets
resulted in stable melanoma cell arrest during continued blood flow.
The association between melanoma cells and platelets was not due to
passive entrapment but was identified as a specific, receptor-mediated
interaction involving
3 integrin function on both the melanoma
cells and the platelets. Thus, our results demonstrate that melanoma
cell arrest to a matrix during blood flow can be mediated by their
specific interaction with platelets which establish the initial matrix
contact.
To test our hypothesis that platelets may assist melanoma
cell arrest during blood flow, we initially tested whether the melanoma
cells can stimulate and induce platelet aggregation by using the
classical stir aggregometer approach. This is important because the
activation state of platelets may be critical for platelet-melanoma
cell interaction(10) . M21 cells (or their variants, as below)
were added to human platelet-rich plasma from citrate-treated blood at
final concentrations of 5 10
, 1
10
, or 5
10
cells/ml and in the absence
of other stimulants. The melanoma cells failed to induce platelet
aggregation under these conditions, whereas 2 µM ADP, as a
control, did (data not shown). For this reason, and because we wanted
to examine the role of platelets on melanoma cell arrest during blood
flow at a low shear rate, we chose collagen type I as an experimental
matrix. At a low wall shear rate of 50 s
,
corresponding to a venous shear rate, collagen I promotes platelet
adhesion mediated by integrin
2
1 (23) and this
results in platelet stimulation (24) and thrombus
formation(25) .
Figure 1:
Time course of platelet adhesion and
M21 melanoma cell arrest on collagen I during blood flow. M21 cells
(2.5 10
/ml final) were suspended in whole human
blood containing PPACK as anticoagulant. This suspension was perfused
over a glass coverslip coated with collagen I at a constant wall shear
rate of 50 s
. Collagen I was chosen as matrix to
ensure platelet adhesion and activation at this low shear rate. The
melanoma cells were prestained with hydroethidine (red) and washed
before their addition to blood which contained mepacrine (green). All
cells and platelets in the suspension acquired a green fluorescence
(filter setting 488/515 nm) (upper panels), while the melanoma
cells could be identified by their unique red fluorescence (filter
setting 543/590 nm) (lower panels). At each time point, images
were captured with both filter settings at identical x and y coordinates. The bar in the lower right panel represents 10 µm. Locations of tumor cells within thrombi are
indicated by (+) in the upper right
panel.
Figure 2: Confocal z sections of an M21 melanoma cell arrested inside of an attached thrombus. Images of the z sections were captured using the confocal mode of the microscope under continued flow (experimental conditions as detailed in Fig. 1). Bottom sections are shown left; top sections, right. Numbers represent distances from the bottom surface of the flow chamber in micrometers. All cells and platelets were stained with mepacrine (top row) visualizing leukocytes and an M21 cell (top left), the M21 cell amid platelets (top middle), and the platelets on the surface of the thrombus (top right). The M21 cell was identified by its red fluorescence (bottom row). All images were acquired at identical x and y positions. The bar in the lower right panel represents 10 µm.
Figure 3:
Requirement of platelet function for M21
melanoma cell arrest during blood flow. A, M21 cells (1
10
in 1 ml DMEM) were suspended in 3 ml of human
blood and perfused over collagen I at a shear rate of 50 s
in the absence or presence of 50 µg/ml of mAbs LJ-CP8
(function blocking anti-
IIb
3) or AV-10 (not blocking
anti-
3), both IgG1, or 20 nM prostaglandin E
(PGE1). Without interrupting the flow, the chamber was
then perfused with 3% paraformaldehyde and attached melanoma cells or
thrombi were quantified by using a computer program which directed the
mobile stage of the microscope to 50 predefined positions and
controlled capturing of images with filter settings for green (all
cells and platelets) or red fluorescence (melanoma cells) at identical x and y positions. Attached melanoma cells were
identified as thrombus-associated by analyzing their position
coordinates on the ``green'' and ``red'' images of
identical optical fields using MetaMorph® image processing
software. Platelet thrombi were distinguished from adherent leukocytes
or individually attached melanoma cells by their larger size. Frequent
confocal sectioning along the z axis of attached objects
during control runs revealed objects, typically counted as thrombi, as
aggregates of platelets or heteroaggregates containing platelets,
leukocytes and/or melanoma cells ( Fig. 2shows an example).
Optical field dimensions were 255
255 µm
. Each bar represents the mean number of attached objects in 50
predefined fields of triplicate runs ± S.D. The data presented
were obtained on the same day using blood from one donor. Numbers of
attached thrombi and dependent numbers of thrombus-associated melanoma
cells varied slightly due to blood donor variability. Controls runs at
the beginning and end of each session ensured unchanged performance of
the blood samples throughout the experiment. B, images of
representative optical fields captured with filter settings for
mepacrine (green fluorescence, upper row) or hydroethidine
(red fluorescence, lower row) each pair at identical x and y coordinates. The (+) signs in thrombi in the upper left panel indicate the locations of the arrested M21
cells (as shown in the lower left panel). The bar in
the lower right panel represents 10
µm.
Figure 4:
Relationship between thrombus size and
platelet-dependent melanoma cell arrest. M21 cells were suspended in
human blood and perfused over collagen I as in Fig. 3in the
absence or presence of increasing concentrations of mAbs LJ-CP8
(function blocking anti-IIb
3) (
) or AV-10 (not
blocking anti-
3) (
). For each experimental condition, images
were captured with filter settings for green (all cells and platelets)
or red fluorescence (melanoma cells) at 50 predefined positions. For
quantitation of attached objects the images were binarized, and object
classifiers were defined based on surface area coverage of individual
platelets, micro thrombi containing two to five platelets, small
thrombi, or large thrombi (1-70, 71-250, 251-1000, and
>1000 pixels, respectively, the total measured area corresponded to
215,384 pixels) using MetaMorph® image processing software.
Identification and quantitation of thrombus-associated melanoma cells
were done as in Fig. 3. Each data point represents mean numbers
± S.D. of attached objects of each class in 50 images expressed
as a percent of control. Controls were done in the absence of antibody
at the same day at the beginning and at the end of the experiment using
blood from the same donor. Note differences in the scaling of the y axes.
Figure 5:
Flow cytometric analysis of 3
integrin or collagen receptor
2
1 expression by M21, M21-L,
M21-L4, or M21-LIIb melanoma cells. The cells were stained with mAbs LM
609 for
v
3, 12F1 for
2
1, AV-8 for
v, AV-10 for
3, or LJ-CP8 for
IIb
3, respectively, followed by
anti-mouse fluorescein isothiocyante. The samples were analyzed in a
Becton Dickinson FACScan.
Figure 6:
Requirement of 3 integrin expression
for melanoma cell association with platelets and arrest during blood
flow. M21 (
v
3
,
IIb
3
), M21-L (
3
),
M21-L4 (
v
3
,
IIb
3
), or M21-LIIb
(
v
3
,
IIb
3
) cells
were suspended in human blood and perfused over a collagen I matrix and
attachment of large thrombi, or melanoma cells with or without thrombus
association, was quantified as in Fig. 3. Each bar represents the number of attached objects in 50 predefined fields
of multiple runs ± S.D. Bars for M21 cells correspond to the
mean of six runs, two of which were each done at the same date as
M21-L, M21-L4, or M21-LIIb, respectively. Bars for M21-L, M21-L4, or
M21-LIIb represent triplicate runs.
During platelet aggregation and thrombus formation, interaction of
platelets is mediated by integrin IIb
3. To investigate
whether expression of this integrin by melanoma cells supports their
association with platelets, we transfected M21-L cells with
IIb,
resulting in
IIb
3 expressing M21-LIIb cells (Fig. 5).
M21-LIIb cells associated with platelet thrombi during blood flow, and
this mediated their arrest to the matrix to an extent comparable to
that found for the
v
3-expressing M21 or M21-L4 cells (Fig. 6). Platelet-dependent arrest of both M21-L4 and M21-LIIb
cells was blocked by platelet inhibition through treatment with
PGE
or anti-
IIb
3 mAb LJ-CP8, corresponding to the
data shown for M21 wild type cells in Fig. 3. This indicates
that
3 integrin expression by melanoma cells supports their
interaction with platelets under flow and may suggest that the
mechanism involved is related to that of platelet-platelet interaction.
Figure 7:
Requirement of plasma proteins for
platelet mediated melanoma cell arrest during blood flow. M21 melanoma
cells were suspended in either plasma depleted, washed blood (open
bars), washed blood containing 2 mg/ml human fibrinogen (light
gray bars), or untreated whole blood (gray bars). The
suspensions were perfused over a collagen I matrix at a wall shear rate
of 50 s. Melanoma cell attachment and their
association with thrombi was measured as in Fig. 3. Surface area
coverage by platelets or thrombi of various sizes was determined as in Fig. 4. Each bar represents the mean numbers of pixels
(± S.D., n = 3 runs using the same blood and
washed blood preparation) for the surface coverage of attached objects
as classified in Fig. 4or the numbers of arrested tumor cells
at 50 predefined positions. Washed blood was prepared as detailed under
``Materials and Methods.''
This study was designed to analyze the ability of human
melanoma cells to adhere to an immobilized matrix during blood flow and
to determine the role of platelets in this process. This is pertinent
to tumor metastasis because tumor cell arrest within the vasculature is
required for extravasation and thus limits the metastatic capacity of
tumor cells that disseminate via the blood stream. In this report we
provide evidence that efficient melanoma cell arrest to a collagen I
matrix during blood flow depends on the interaction of the melanoma
cells with platelets which have already established matrix contact.
Despite their ability to adhere to collagen I under static conditions
in an integrin 2
1-dependent manner(19) , M21 human
melanoma cells largely failed to attach directly to this matrix during
blood flow. They were, however, able to associate with platelets, and
this interaction resulted in M21 cell arrest. The flow-resistant
interaction of M21 cells and platelets depended on platelet activation
and thrombus formation. Moreover, the interaction was found to be
specific and required
3 integrin function on both the melanoma
cells and the platelets.
The ability of platelets to support tumor
cell arrest during blood flow may contribute to the role of platelets
in hematogenous metastasis. A number of reports indicate that
interference with platelet function in vivo reduced tumor
metastasis in animal models. Moreover, reduction of platelet counts
inhibited metastasis of a variety of tumor cells including melanomas,
carcinomas, and
sarcomas(11, 12, 13, 14) . In these
cases, metastasis was reduced regardless of the ability of the tumor
cells to induce platelet aggregation in
vitro(26, 27, 28) . Tumor cells can
induce platelet aggregation by releasing
ADP(29, 30, 31, 32, 33) ,
generating tissue factor(34) , or by other mechanisms leading
to thrombin formation(35, 36, 37) . It is
conceivable that two distinct mechanisms are involved in
platelet-assisted tumor cell arrest in the vasculature: first, an
induction of platelet activation and aggregation by tumor cells or
their released factors which is possibly accompanied by passive
entrapment of tumor cells in platelet aggregates; and second, a
specific adhesive interaction between platelets and tumor cells. Both
types of interaction may allow tumor cells to utilize indirectly the
platelet-specific mechanisms for successful adhesion during blood flow.
Since M21 melanoma cells failed to induce platelet activation in
vitro, we sought to define conditions for their local exposure to
stimulated platelets. Therefore, we chose collagen I as the matrix
because it represents a thrombogenic surface and promotes platelet
adhesion and thrombus formation at low shear rates, corresponding to
venous blood flow(23, 24, 25) . Although
collagen I is present in the vessel wall, it becomes exposed to blood
only in deep vessel injury or upon rupture of atherosclerotic
plugs(38, 39) . Metastasizing cells are unlikely to
encounter this matrix in the vasculature. Therefore, collagen I was
used in this study solely as a model for a thrombogenic surface. The
collagen I matrix was perfused with M21 melanoma cells suspended in
whole human blood. It has to be considered that plasma contains
collagen-binding proteins, such as fibronectin, von Willebrand factor,
or vitronectin(40) , which potentially modified the matrix
during the experiments. However, it has been reported that a
function-blocking antibody to the 2 subunit of the platelet
collagen receptor
2
1 fully inhibited platelet adhesion to
collagen I during perfusion with whole blood at wall shear rates up to
1600 s
(41) . Other investigators showed that
at wall shear rates of 1500 s
or higher, von
Willebrand factor immobilized to the collagen I matrix during blood
flow becomes the relevant adhesive ligand for platelets, since a
recombinant fragment of von Willebrand factor, von Willebrand factor
445-733 containing the binding site for platelet receptor GPIb,
significantly inhibited platelet attachment at
1500 s
but not at lower shear rates(42) . In agreement with
these findings, platelet adhesion, thrombus formation, and
platelet-mediated melanoma cell arrest during blood flow over collagen
I at 50 s
in our experiments were not significantly
affected by the presence of mAb LJ Ib-1 which blocks GPIb function
(data not shown). Together, this suggests that collagen remained the
major matrix component for platelet adhesion at the low shear rate used
in our experiments. A possible modification of the collagen matrix did
not improve platelet-independent melanoma cell attachment even after
prolonged periods of blood flow.
The association of M21 melanoma
cells with platelets under flow depended on platelet activation and
thrombus formation since PGE or anti-
IIb
3
abolished M21 cell interaction with platelets. In the presence of these
inhibitors, attachment of individual platelets and micro thrombi was
strongly enhanced; however, they failed to support M21 cell adhesion.
The increase in single platelet and micro thrombus attachment in the
presence of platelet inhibitors may be explained by the fact that
platelets were not depleted from the streaming blood due to
incorporation into large thrombi as observed in the absence of platelet
inhibitors. Association of M21 cells with thrombi coincided with the
occurrence of large size thrombi. Detailed analyses of mural
thrombogenesis revealed that the blood flow pattern in the vicinity of
growing thrombi is characterized by standing vortices that develop
upstream and downstream of the thrombi. This leads to considerably
reduced velocities in and near the recirculating regions which in turn
permit the local accumulation of platelet-activating agents that are
released or induced by the aggregating platelets within the
thrombi(43) . It is therefore plausible that tumor cells may be
passively trapped close to or between nearby growing thrombi. We have
ruled out that the M21 melanoma cell interaction with thrombi during
blood flow was due to nonspecific entrapment. We found melanoma cells
associated with thrombi at random sites, indicating that changes in
flow did not affect the interaction. Moreover, we demonstrated that the
M21 cell association with thrombi was a specific, receptor-mediated
process. Failure of M21 cells to bind to adhered platelets in which the
integrin
IIb
3 function had been blocked suggests that
IIb
3 participates in the interaction between platelets and
melanoma cells. This concept is supported by previous reports (44, 45) and is substantiated by the finding that
platelets from Glanzmann's thrombasthenic patients, which lack
IIb
3 expression, failed to interact with tumor cells in
vitro(46) . In order to identify a potential counter
receptor for platelet integrin
IIb
3 expressed by the melanoma
cells, we reasoned that
IIb
3 may bind to a related receptor
expressed by the melanoma cell using a mechanism similar to that which
governs platelet-platelet interaction in thrombus formation. Evidence
suggests that an
IIb
3-related integrin expressed by tumor
cells might be involved in their interaction with
platelets(46, 47) . This
IIb
3-related
molecule may represent tumor cell integrin
v
3, since these
receptors share characteristic homologies and a ligand recognition
repertoire. In order to examine the role of melanoma cell integrin
v
3, we utilized an M21 cell variant that lacks
v
3
expression (M21-L) (16) in comparison to a variant in which
v
3 expression was reconstituted upon transfection
(M21-L4)(19) . Using this system, we demonstrated that
v
3 expression was required for M21 cell association with
platelets, and that this resulted in efficient M21 cell arrest on a
collagen I matrix during blood flow.
In human melanoma, expression
of v
3 was found to be restricted to the metastatic phenotype (48) and to commence with the onset of vertical growth within
the primary lesion(49) . Furthermore, we have evidence that
v
3 contributes to the tumorigenicity of human melanoma cells (19) and to adhesive interactions with fibrin(ogen) and its
breakdown products which occur in the tumor stroma(50) . Thus,
melanoma cell
v
3 seems to be involved in early stages of
tumor development. We now provide evidence suggesting that this
adhesion receptor may also be involved in later stages of melanoma
progression by contributing to melanoma cell-platelet binding and
arrest during blood flow.
It is yet unknown whether platelet binding
to the melanoma cells is based on a direct interaction between the two
cell types or whether bridging ligands are involved. We hypothesize
that platelet integrin IIb
3 and melanoma cell integrin
v
3 interact via divalent or multivalent RGD-containing plasma
proteins, such as fibrinogen, von Willebrand factor, fibronectin, or
thrombospondin. This concept is supported by a number of reported
inhibition studies. Antibodies directed to either platelet
IIb
3 or to its ligands, as well as RGD-containing peptides
and snake venoms, significantly reduced the interaction between tumor
cells and platelets in vitro and metastasis in
vivo(14, 44, 51, 52, 53, 54, 55, 56, 57) .
The antimetastatic activity of RGD peptides was associated with an
accelerated disappearance of isotope-labeled tumor cells from the lungs
of treated mice. This indicates that specific adhesion processes were
involved in stable tumor cell arrest in the lung capillaries. To
address the question whether plasma proteins, envisaged as containing
adhesive ligands for the
3 integrins, were required for
platelet-mediated melanoma cell arrest during blood flow, we depleted
the plasma proteins from blood by repeated washes. As expected,
platelet adhesion to the collagen I matrix was not impaired; however,
the formation of large size thrombi was drastically reduced, and
consequently, platelet-mediated melanoma cell adhesion was also
inhibited. Thrombus formation and melanoma cell arrest were partially
restored in the presence of added fibrinogen. The lack of a full
recovery of thrombus formation and melanoma cell arrest could be due to
one or both of the following reasons. First, platelets as well as
leukocytes become refractory during the blood-washing procedure, and
second, other plasma proteins, such as fibronectin, von Willebrand
factor, vitronectin, or thrombospondin, may be involved in addition to
fibrinogen. Together, our results indicate that plasma protein(s) are
required for platelet-mediated melanoma cell arrest under our
experimental conditions. Logically, the mechanism of melanoma
cell-platelet heteroaggregate formation needs to be addressed in
greater detail, employing experimental procedures which involve minimal
disturbance of platelet responsiveness.
Integrin IIb
3
mediates platelet cohesion via fibrinogen and/or von Willebrand factor
as major bridging ligands in thrombus formation. Therefore, we sought
to compare
3 integrins
v
3 and
IIb
3 expressed
by the same type of melanoma cell for their potential to mediate
melanoma cell interaction with platelets. To accomplish this, we
transfected M21-L cells with
IIb by a method similar to that
described by Kieffer et al.(58) , resulting in the
M21-LIIb variant which expresses
IIb
3 but not
v
3.
Several reports suggest that integrin
IIb
3 may be expressed
by certain tumor cells(59) . M21-LIIb cells were able to
associate stably with platelet-containing thrombi and utilized this
mechanism for efficient arrest during blood flow. The extent of
M21-LIIb cell interaction with platelets was comparable to that found
using
v
3-expressing M21 or M21-L4 cells. This indicates that
3 integrins in general can function as receptors on melanoma cells
during their interaction with platelets. This finding may lend further
support to the concept that the mechanism involved in melanoma
cell-platelet interaction is related to that which governs platelet
cohesion in thrombus formation.
We propose that 3
integrin-mediated tumor cell-platelet interaction may represent one
possible mechanism to facilitate hematogenous dissemination of tumor
cells. It is yet unknown whether platelets promote tumor cell arrest on
intact endothelium, a process which may involve more than one receptor.
These may include other integrins and selectins, the latter of which
could contribute to the initial binding
events(60, 61, 62, 63, 64, 65) .
Among the integrins,
4
1 represents an example for supporting
leukocyte rolling on and attachment to endothelial cells under
flow(66, 67) .
4
1 is expressed by a variety
of tumor cells including certain melanoma cells, and it may be involved
in the melanoma cell interaction with the vascular endothelium.
5
1 and
v integrins were shown to support tumor cell
arrest under flow to fibronectin or vitronectin
substrates(68) . Stabilization of the initial binding events
seemed to depend on the activity of transglutaminase expressed by the
tumor cells(69) . Under our experimental conditions, one likely
candidate to contribute to melanoma cell interaction with thrombi is
P-selectin. P-selectin is expressed by activated platelets and was
shown to mediate tumor cell-platelet binding under stationary
conditions(70) . Under flow conditions, P-selectin has been
reported to support leukocyte rolling. In our flow experiments, we
recorded adhesive events in real time and found no evidence of melanoma
cell rolling. A monoclonal antibody directed to human P-selectin, known
to block P-selectin-mediated interactions, such as adherence of
activated platelets to neutrophils (71) or histamine-induced
rolling of leukocytes in postcapillary venules(72) , failed to
interfere with platelet-mediated melanoma cell arrest under our
experimental conditions (data not shown). A more detailed analysis of
the initial binding events between activated platelets and melanoma
cells during blood flow will reveal a potential contribution of
P-selectin. In vivo, association of platelets with tumor cells
attached to vascular endothelium has been reported (73) and
suggests that platelets may stabilize and protect attached tumor cells
during blood flow. In vivo studies will help to clarify
whether
3 integrin-mediated tumor cell-platelet interaction
contributes to the complex process of tumor cell arrest in the
vasculature.