ICANCER RESEARCH57. 3040-3045. July 15. 19971

Suppression of Experimental

Lung Colonization of Mouse Colon Adenocarcinoma

26 in Vivo by an Anti-Idiotype

Monoclonal Antibody Recognizing a Platelet

Surface Molecule'Yukinari Kato, Naoya Fujita, Hideaki Yano, and Takashi Tsuruo2
Laborator? of Biomedical

Research, Institute of Molecular and Cellular Biosciences,

University of Tokyo, 1-1-I. Yayoi, Bunkyo-ku.

Tokyo 113 IY. K., N. F., H. Y., T. Ti, andCancer Chemotherapy Center. Japanese Foundation for Cancer Research, 1-37-1. Kami-ikebukuro, Toshima-ku, Tokyo 170 IT. TI, Japan

ABSTRACTThe interaction between platelets and tumor cells is important in theformationof pulmonary

metastasis.

We previously

establishedthe SF11

monoclonal antibody (mAb) by immunizing rats with the NL-17 cell line,
a highly metastatic variant of mouse colon adenocarcinoma 26. SF11 could
inhibit the platelet aggregationin vitro and suppress the pulmonary
metastasis in s'ivo by NL-17 cells. SF11 recognized the Mr 44'000 sialogly

coprotein (gp44) on NL-17 cells, and the affinity-purified gp44 alone could
induce platelet aggregation. Therefore, SF11 might inhibit gp44-induced
platelet aggregation by masking the epitope of gp44 that interacted withunknown molecule(s) on the platelet surface. To identify the platelet
antigen that interacted with gp44, we generated anti-idiotype mAbs by
Immunizing

rats with SF11. Two ofthe establishedmAbs, AWl and AIP4,

recognized not only SF11 but also the Mr 160,000 platelet surface protein.
AIP4

mAb

could

also inhibit

the NL-17

cell-induced

plateletaggregation

in a dose-dependent manner. Furthermore, pretreatment of mice with
AIP4 mAb suppressed the pulmonary metastasis of NL-17 cells in vivo.
These results suggest that the Mr 160@000platelet antigen participates in
the NL-17 cell-induced platelet aggregation and colonization of NL-17
cells in the lungby interacting

withthe gp44 of NL-17

cells.

INTRODUCTION
Recent studies on cancer metastasis have shown that some humanand animal tumor cells possess platelet-aggregating abilities that
correlate with their metastatic potential (1, 2). Actually, several inhibitors of platelet aggregation have been reported to retard tumor
metastasis in certain animal models (3â€”7).Interactions between tumor
cells and platelets have been considered to facilitate the arrest oftumor cell cluster in the microcirculation, with the subsequent forma
tion of experimental metastasis.
We previously established several clones possessing different met
astatic abilities from a murine colon adenocarcinoma 26 cell line (8).
Among these clones, a more highly metastatic clone, NL- 17, wasfound to exhibit a high platelet-aggregating activity, though a lessmetastatic clone, NL-l4, had a marginal platelet-aggregating activity(6, 9). Therefore, the ability to induce platelet aggregation was related
to the metastatic potential. Because one of these clones, NL-44,
possessing a high platelet-aggregating activity, did not form pulmonary metastasis even after platelet clusters formed, the platelet-aggre
gating activity alone is not enough for the formation of metastasis(10).
NL-44cells
werethereafter
foundtoproliferate
invivomore
slowly than NL-17 cells. Thus, the ability to proliferate in vivo may
also be important in the formation of pulmonary metastasis (10).
Received I/I 3/97: accepted 5/I 5/97.
The costs of publication of this article were defrayed in part by the payment of page@

charges. This article must therefore be hereby marked ads'ertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
This study was supported in part by a special grant for Advanced Research on Cancer

and a Grant-in-Aid for Cancer Research from the Ministry of Education, Science andCulture, Japan. and a Special Coordination Fund of the Science and Technology Agency,
Japan.
2 To

whom

requestsforreprintsshouldbe

addressed,atLaboratoryof

These results indicate that platelet-aggregating ability and in vivo
growth potential are two major determinants for successful experi
mental lung metastasis of the colon adenocarcinama 26.
We previously generated mAbs3 by immunizing rats with NL-l7
cells to identify the molecule involved in the resulting platelet aggre
gation. One of the established mAbs, 8F1 1, exhibited an inhibitory
activity of the platelet aggregation in vitro and the lung colonizationof NL-l7 cells in vivo (1 1, 12). The 8F1 1 recognized a Mr 44'000
membrane sialoglycoprotein (gp44) on NL-17 cells (1 1). Using 8Fllaffinity column chromatography, we recently purified the gp44 from
NL-17 cells (13). The purified gp44 alone could also induce aggre
gation of washed platelets in a dose-dependent manner, with norequirement for plasma components. These results indicate that 8Fl 1
may recognize the specific epitope of gp44 that interacts with platelets.
In this study, we immunized rats with 8Fl 1 to identify the platelet
surface receptor of gp44. Some anti-idiotype mAbs of IgG2@and 1gM
subclasses were generated, and two of these mAbs, termed AWl and
AIP4, of IgG25 subclass, recognized not only 8F1 1 but also a Mr
160,000 platelet surface protein. AIP4 inhibited platelet aggregation
by NL-l7 cells in a dose-dependent manner. Furthermore, AIP4
inhibited the lung colonization of NL-17 cells in vivo as well as 8F1 1
did. These results suggest that the Mr 160,000 platelet antigen is acounterreceptor of gp44 and plays an important role in colonization of
NL-17 cells in the lung.

MATERIALS
Animals

AND METHODS

and Tumor

Cells. Female BALB/cmice and SD rats wereobtained from Charles River Japan, Inc. (Kanagawa, Japan). Female BALB/cmice, ages 8â€”12 weeks,
metastatic

were used throughout

clone NL-l 7 was establishedthese experiments.

The highlyfrom mouse colon adenocarcinoma

26,

as described previously (8). NL-l7 cells were cultured at 37Â°Cin a humidified
atmosphere of 5% C02-95% air in RPMI 1640 (Nissui Pharmaceutical Co.,Tokyo, Japan), supplemented with 10% heat-inactivated fetal bovine serum(Biocell, Carson, CA), 2 mM glutamine, and 100 @g/mlkanamycin (RPMI
growth medium).
Hybridoma Production. Six SD rats were immunized by neck s.c. injec
tions of 8FII

with Freund's

completeadjuvant(Difco,Detroit,

MI). One week

later, the secondary i.p. immunizations were performed. The booster injection
was given iv. 3 days before spleen cells were harvested. The spleen cells werefused with mouse myeloma P3U1 cells using polyethylene glycol (Mr 4000)'
and the hybridomas were grown in RPMI growth medium with hypoxanthine,aminopterin, and thymidine selection medium supplement (Sigma ChemicalCo., St. Louis, MO; Ref. 14). The culture supematantswere screened bycellular ELISA for the binding activity to platelets (see below).
Preparationof Mouse

PRP

and

WashedPlatelets.

PRP

was preparedfrom fresh heparinized blood drawn from BALB/Cmice by cardiac puncture,
followed by centrifugation at 230 X g for 7 mm at 23Â°C.Platelets in PRP wereseparated from plasma by centrifugation at 400 X g for 5 mm, washed, andresuspended in PBS (13).

Biomedical

Research, Institute of Molecular and Cellular Biosciences, University of Tokyo, I-I-I,
Yayoi,Bunkyo-ku,Tokyo 113,Japan.Phone:81-3-3812-2111,
ext.7861;Fax:81-33816-3592.
3040

3 The

abbreviations

used

NR IgG, normal rat IgG.

are:

mAb,

monoclonal

antibody;

PRP,

platelet-richplasma;SUPPRESSION OF LUNG METASTASIS BY AN ANTI-PLATELET mAb

Cellular ELISA. Platelets were seeded in each well of flat-bottomed
96-well plates and fixed with 0.5% glutaraldehyde. Hybridoma culture

RESULTS

Establishmentof Anti-Idiotype mAbs Recognizing a Platelet
Antigen. A highly metastatic variant of mouse colon adenocarcinoma
26 cells, the NL-l7 cell line, was known to express a platelet aggre
gation-inducing molecule (gp44) on the cell surface. Because ourpreviously generated rat mAb 8Fl I recognized gp44 and suppressed
gp44-mediated platelet aggregation (1 1), it might also recognize thespecific epitope of gp44 that would interact with platelets.
To identify the platelet receptor of gp44, we first tried to purify
gp44 for making affinity column immobilized of gp44. However, we
did not succeed in obtaining a large enough amount of gp44 because
gp44 was not stable protein. Then we tried to make anti-idiotype
mAbs of 8Fl 1 by immunizing six SD rats with 8F1 1 and selecting theability to recognize mouse platelets. We finally obtained six clones of
the hybridomas that produced mouse platelet-recognizing
mAbs.
Among these mAbs, AIP1 and AIP4 were IgG25 subclass, and AIP2,
AIP3, AlPS, and AIP6 were 1gM subclass (data not shown). Because
mAbs of 1gM subclass were difficult to purify and characterize, we
antibody biotinylation
kit, according to the manufacturer's
instructions(Amer
used the AIPI and AIP4 of IgG25 subclass throughout this study.
ican Qualex).
To confirm that the raised AIP1 and AIP4 were anti-idiotype mAbs
ELISA. AlPl , AIP4, anti-mouse CD62P (P-selectin) mAb (PharMin
to
8F1 1, we examined whether biotin-labeled 8F1 I could recognize
gen, San Diego, CA), and chromatographically purified NR IgG (Cappel,
Durham, NC) were immobilized on 96-well plates by incubation at 23Â°C AIP1 and AIP4, which were immobilized on 96-well plates. 8Fl 1
for 1 h. After washing with PBS, biotinylated 8Fl 1 (10 @.tg/ml)
was added reacted with the immobilized AIP1 and AIP4 in a dose-dependent
manner (Fig. 1, filled circles and filled squares, respectively). How
to the individual wells and incubated for an additional 1 h. Then, alkaline
ever, 8F1 1 could not bind to anti-P-selectin mAb or to NR IgG (Fig.
phosphatase-avidin (Zymed) was added to each well, followed by devel
1, open circles and open triangles, respectively). Therefore, the raised
oping with an alkaline phosphatase substrate kit (Bio-Rad), as described
AIP1 and AIP4 were determined to be anti-idiotype mAbs to 8Fl I.
above.
Then, using flow cytometry, we examined whether AIP1 and AIP4
Flow Cytometry. Mouse platelets were treated with AIP1, AIP4, 8F1I, or
NR IgG for 1 h at 4Â°Cand then reacted with FITC-conjugated anti-rat IgG recognized the membrane surface protein on mouse platelets. As(Cappel).
Fluorescencedata were collected using a Becton Dickinson(Sanshown in Fig. 2, AIP1 and AIP4 could recognize platelets, but 8Fl 1
Jose, CA) FACScan.
could not. To identify the molecular sizes of the recognized protein on
Immunoprecipitation and Western Blot Analysis. Platelets were washed mouse platelets, platelet lysates were immunoprecipitated with AlP 1,
with PBS and solubilized at 4Â°Cwith lysis buffer [25 mMTris (pH 7.4), 50 msi AIP4, anti-P-selectin mAb, or control NR IgG, and then the immu
NaCl, 0.5% Na deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 mM phenyl
noprecipitated proteins were immunoblotted by these antibodies (Fig.
supernatants

were

added

to the individual

wells

and incubatedat 23Â°C for

I h. After washing with PBS, biotin-labeled goat anti-rat lgG (Fc; American Qualex, La Mirada, CA) was added to the individual wells andincubated for an additional 1 h. Then, alkaline phosphatase-avidin (Zymed,
San Francisco, CA) was added to each well. After washing with PBS, thewells were developed with an alkaline phosphatase substrate kit, according
to the manufacturer's instructions (Bio-Rad, Richmond, CA), and theabsorbance at 405 nm was measured for each well using the model 450
microplate reader (Bio-Rad; Ref. 14).
Purification and Biotinylation of mAbs. Hybridoma cells (AIP1, AIP4,
and 8Fll) were cultured in RPM! growth medium, harvested by a brief
centrifugation, and suspended in PBS. Four-week-old female BALB/c nude
mice (Charles River Japan) were given i.p. injections of the hybridoma cells.
The mice were sacrificed 7 days later, and ascites fluid was collected. The
mAbs were purified using anti-rat lgG column (Sigma) or protein G-Sepharose
4B gel beads(Zymed),dialyzedagainstPBS, andthenused for in vitroandinvivo assays as follows. The purified 8Fl I mAb was biotinylated using an

methylsulfonyl

fluoride,

AIP4, anti-P-selectin

The proteinsbound to AIP1,

mAb, or NR IgG were immunoprecipitatedand 50@g/mlaprotinini.

using protein

G-Sepharose 4B gel beads coated with these antibodies. After electrophoresing
in reducing

conditionsusing

a 4â€”20% gradientgel polyacrylamide,

the im3). Although AlP! and AIP4 could immunoprecipitate and immuno
blot the Mr 1@Â°,Â°Â°Â°
and Mr 140,000 proteins (Fig. 3, A and B, Lanes
1 and 2, respectively), the Mr 140,000 protein might be nonspecifi

munoprecipitated proteins were transblotted onto a nitrocellulose membrane.
The membrane was incubated with 10 @g/mlof these antibodies in blocking
buffer [4% skim milk in 25 mMTris-HCI (pH 8.0), 125 nmiNaCl, 0. 1%Tween
20, and 0.1% sodium azide] for 1 h. After washing with the blocking buffer,
the membrane was incubated with peroxidase-conjugated anti-rat IgG (Amersham, Buckinghamshire,

UnitedKingdom).

After washing,

the membrane

0.3

was

developed with an enhanced chemiluminescence detection system (Amersham) and KOdakX-Omat AR film.
PlateletAggregation

turbidometricallyandInhibition.

by an NKK HEMA

PlateletTraceraggregation

was measured

I (Niko BioscientifictoJapan). A 200-id aliquot of PRP was incubated with AIP1, AIP4, 8Fl 1, or NR
IgG at an appropriate concentration in a cuvette at 37Â°Cunder constant stirringin theaggregometer.

suspension

After(2 X l0@ cells/ml)

incubation
or 10

for

30 mm,

10 pi

of NL-17@lof 1 m@i ADP (Amresco,

Solon,

of 0.2 ml of the tumor suspension

0.1

OH)

via the lateral tail vein. AIP4,

8Fll, or NR IgG was given i.v. 10 ruin before the tumor inoculation. Lungmetastasis

was examinedon day 14 after the tumor

inoculation(12).

Statistics. The two-tailed Student's t test was used to compare the control
groupwith the experimentalgroup.

0cell

were added. The changes in light transmittance were monitored for 10 mm(13).
Assay of Experimental Metastasis. NL-l7 cells were harvestedby brief
exposure to HBSS containing 0.05% trypsin and 0.02% EDTA. The cells were
washed and resuspended in HBSS supplemented with 1% BALB/c serum. The
cells were kept on ice for 10 mm and then adjusted to 1.25 X l0@cells/mI.
Under these conditions, >95% of the cells were viable. The mice were given
i.v. injections

0.2

0Co., Tokyo,

0-

0

10

30

50

Antibody coating concentration (j.tg/mI)
Fig. 1. Identification of ALP) and AIP4 as anti-idiotype antibodies to 8Fl 1. After
immobilization of the indicated concentrations of AIPI (â€¢),AIP4 (U), anti-P-selectinmAb (0), or NR IgG (L@)on 96-well plates, biotinylated 8Fl I (10 p@g/ml)was added tothe individual wells and incubated for I h. Alkaline phosphatase-avidin was added to theindividual wells, followed by developing with an alkaline phosphatase substrate kit. The

absorbanceat 405 nm of eachwellwas measuredby the model450 microplatereader(Bio-Rad). Data points, mean of triplicate determinations:

3041

bars, SD.

SUPPRESSION OF LUNG METASTASIS BY AN ANTI-PLATELET mAb

AFig. 2. Recognition of platelet surface proteins by

AIPI and AIP4 but not by 8Fl I. Mouse platelets weretreated with AIPI, AIP4, 8Fl I, or NR IgCi for I h at

4Â°Cand then reactedwith FITC-conjugatedanti-rat
IgG. The fluorescence intensity was analyzed using a

@

u@

E
C

AA

FACScan. Open cun'es, cells treated with AIPI (A),
AIP4 (B), or 8F1 I (C). Filled curves, cells treated withol

0

NR IgG.

100

1O@ 102

iO@ io@ 10Â°10'

Fluorescense

inoculation of AIP4 at a dose of 1 mg/mouse. After 14 days, mice
were sacrificed, and the nodules in the lung were counted.
Mice given AIP4 and 8Fl 1 showed remarkably suppressed lungcolonization of NL-17 cells, as compared to the untreated mice (Fig.

cally immunoprecipitated protein because NR IgG immunoprecipi
tated the same Mr 140,000 protein (Fig. 3, A and B, Lanes 4).
Therefore, AIPI and AIP4 could recognize the Mr 160,000 protein. Mr
160,000 protein could not be recognized by anti-P-selectin mAb (Fig.
3C), indicating that it is not P-selectin.
Suppression of NL-17 Cell-induced Platelet Aggregation by

6A). The number of pulmonary nodules in the AIP4-treated mice wasabout 40% of that in the untreated mice (Fig. 6B). The lung weightsof the AIP4-treated mice were smaller than those of the untreatedmice (Fig. 60. These results suggest that AIP4 inhibits the lungmetastasis of NL-l7 cells in vivo by masking the binding epitope of

AIP1 and AIP4. NL-l7 cells wereknownto possessplateletaggre
gation-inducing ability. As 8Fl I could suppress the platelet aggregation induced by NL-17 cells, we examined the effect of AIP1 and
AIP4 on the aggregation. When platelets were preincubated with AIP1
and AIP4 at 100 p.g/ml (Fig. 4A, curves b and d, respectively), NL-l7cell-induced platelet aggregation was inhibited as compared with NR
IgG (Fig. 4A, curve a). The platelet aggregation was also repressed by
8Fl 1 at 100 @.tg/ml(Fig. 4A, curve c), as reported previously (1 1).
Moreover, the platelet aggregation was blocked by AIP4 in a dose
dependent manner (Fig. 4B). At a concentration of 150 @.tg/ml,theplatelet aggregation was almost completely suppressed (Fig. 4B, curvec).
To exclude the possibility that AIPI and AIP4 could nonspecifi
cally inhibit the activation of platelets by binding to the Mr 160,000
platelet antigen, we investigated the effects of AlP! and AIP4 on the
ADP-mediated aggregation of platelets. As shown in Fig. 5, ADP
induced platelet aggregation was not suppressed by the preincubationof platelets with AIP1 or AIP4 (Fig. 5, curve a or c, respectively).
These results suggest that AIPI and AIP4 specifically inhibit NL-l7cell-induced platelet aggregation by directly interfering with the association between gp44 on the NL-17 cells and the Mr 160,000 proteinon the platelets. As 8Fl 1 recognized AIP1 and AIP4 (Fig. 1), AIPI
and AIP4 might recognize the idiotope in the antigen combining siteof 8Fl I that mimicked the gp44-binding epitope on the Mr 160,000
plateletreceptor.
Inhibition of Experimental Lung Colonization of NL-17 Cells
by AIP4. As platelet aggregation induced by NL-17 cells was one of
the important determinants for the formation of experimental lungmetastasis of NL-17 cells (10), we examined the effects of platelet
aggregation-inhibitingmAb AIP4 on the formation of pulmonary
metastasis in vivo. NL-l7 cells were given i.v. after 10 mm of i.v.

A1234

@

@

200 â€”=Â°.@

97.4 â€”69â€”Mr 160,000

â€”

?@

proteinthat interacts

with the NL-l7cell surface

protein,

gp44.
DISCUSSION
The proposed mechanisms of platelet activation by tumor cells are:interactions of tumor cells with platelets via a sialolipoprotein and/or
sialoglycoprotein (1 1, 15); generation of thrombin through a coagu
lation pathway; activation by ADP (16); activation of the arachidonate
metabolism (17); release of cathepsin-B-like protease (18); and a
combination of these. Among these mechanisms, several factors intumor cells and microvesicles shed by tumor cells have been found to
induce platelet aggregation through thrombin generation in the presence of plasma components (18â€”23). Several cancer procoagulants
were identified. The procoagulant enzyme, purified from rabbit V2
carcinoma extracts, could directly activate Factor X (18). Mr 35,000
and Mr 28,000, isolated from human ovarian carcinoma, possessedprocoagulant activity (20). However, plasma membrane factors thatinduce platelet aggregation through their direct interactions withplatelets had not been identified.
Although molecules responsible for the initial tumor cell-plateletinteraction are totally unknown, integrins expressed on both tumor
cells and platelets (24â€”27), platelet membrane glycoprotein lb/DC(28), and P-selectin (29â€”31) on platelets are candidates for irrevers
ible formation of platelet-tumor cell aggregates. Several reports havesuggested that ligands containing recognition sequences, such as
Arg-Gly-Asp (ROD), modulate the affinity of integrmns; therefore,
resting integrins may be the molecules responsible for the first contact
between the tumor cells and platelets (32). In contrast to these mol

B 1234(kDa)

intensity

C1234

D 1234

(kDa)

(kDa)

(kDa)

200 â€”

200 â€”

200 â€”

97.4

97.4 â€”69 â€”

97.4â€”
69 â€”

69â€”Fig. 3. Recognition of the same Mr 160,000 platelet surface protein by AIP1 and AIP4. The proteins bound to AIPI (Lane 1), AIP4 (Lane 2), anti-P-selectinmAb (Lane 3), or NR

IgG(Lane4) wereimmunoprecipitatedandelectrophoresed
inreducingconditionsusinga 4â€”20%
gradientpolyacrylamide
gel,asdescribedin â€œMaterials
andMethods.â€Afterblotting•
onto a nitrocellulose membrane, the membrane was incubated with 10 @sg/mlAIPI (A), AIP4 (B), anti-P-selectin mAb (C), or NR IgG (D) for 1 h. After washing, the membrane wasincubated with peroxidase-conjugated anti-rat IgG and was developed with an enhanced chemiluminescence detection system (Amersham).
3042

SUPPRESSION OF LUNG METASTASIS BY AN ANTI-PLATELET mAb

8F1 1 affinity chromatography (13). Because the purified gp44 alone
could induce aggregation of washed platelets containing no plasmacomponents, it could directly interact with platelets and induce ag
gregation. These results from a sequential enzymatic hydrolysis ofcarbohydrates linked to gp44 indicate that gp44 contains a large
quantity of O-glycans (13). Because treatment of gp44 with O-gly
canase resulted in the loss of the reactivity of 8Fl I to gp44, thecarbohydrate chains linked to Ser/Thr residues were assumed to be
responsible for the gp44-mediated platelet aggregation.
Murine Bl6 melanoma cell line also induces aggregation of platelets (34). When 8Fl 1 was reacted on highly metastatic variants of B16
melanoma cells, it recognized the Mr 41,000 glycoprotein as an
antigen (34). Moreover, 8Fl 1 inhibited platelet aggregation inducedby the melanoma cells under conditions that prevent thrombin
activity. These data indicate that Mr 41,000 glycoprotein is a platelet
aggregation-inducing factor in metastatic variants of B 16 melanoma
cells.
Because the platelet receptor of gp44 has not yet been identified, we
immunized rats with 8Fll to establish anti-idiotype antibody to it. As
8Fl 1 inhibited the gp44-induced platelet aggregation, it might mask
the epitope involved in the binding of gp44 to the platelet receptor.
Making an anti-idiotype antibody is a useful method of identifying an
unknown receptor of the known ligand when the immunizing mAb
could suppress the binding between the ligand and the receptor or
could mimic the ligand (35, 36). We here report successful generationof anti-idiotype mAbs of IgG2@and 1gM subclass that recognize not
only 8F1 1 but also platelet surface molecules (Figs. I and 2 and data
not shown). Because mAbs of 1gM subclass were difficult to purify,
we used mAbs of IgG25 subclass (AIPI and AIP4) for additional
experiments. AIPI and AIP4 recognized the same Mr 160,000 platelet
surface protein (Fig. 3). We assumed that AIP1 and AIP4 mAbs might
induce platelet aggregation by mimicking gp44. However, they could
not induce platelet aggregation by themselves (data not shown). Then,we examined whether AIP1 and AIP4 could inhibit platelet aggregation by NL-17 cells. AIPI and AJP4 inhibited platelet aggregationinduced by NL-l7 cells, like 8Fl 1 did (Fig. 4A). As ADP-induced
platelet aggregation was not inhibited by AIP1 and AIP4 (Fig. 5), theirbinding to platelets did not lead to platelet inactivation, or blockade of

A

80
a
C
0

60 -

b

U)

C

E 40 U)
C

d

20 -I

0
@

I

0

I

I

I

I

246

I

I

8

10

I

I

I

12

Time(mm.)

B
a
C

0

U)
U)

E

U)
C

b

â€˜C
C

0
2

4

6

Time(mm.)Fig. 4. Suppression ofNL-17 cell-induced platelet aggregation by both AWl and AIP4.
A, mouse platelets were preincubated with 100 @.tg/mlAIPI (curve b), A!P4 (curve d),

80 -8Fl 1 (curve c), or NR IgG (curve a) in a cuvefle at 37Â°Cunder constant stirring in an

NKKHEMATracerI, as describedin â€œMaterials
andMethods.â€Afterincubation•for 30mm, 2 X lO@cells of the NL-I7 cell suspension were added (arrow). B, mouse platelets
were preincubated with 100 @g/ml(curve b) or 150 p.g/ml (curve c) AIP4. They were also60

preincubated with 100 @g/mlNR IgG(curve a). After incubation for 30 mm, 2 X l0@cells

Cof the NL-l7 cell suspension were added (arrow). The changes in light transmittance weremonitored for 10 mm.
E

40

U)

C

@

@

ecules, P-selectin needs activation for its expression, so P-selectin
might not be involved in the initial contact (30, 33).
We previously found that the highly metastatic clone NL-l7, derived from mouse colon adenocarcinoma 26, had an ability to induce
platelet aggregation (8). The NL-17 cell-induced platelet aggregation
was mediated by a trypsin-sensitive cell surface protein and not bythrombin generation in a procoagulant mechanism or by ADP release(11). By generating rat mAb 8Fll that could suppressthe aggregation,
we found that the platelet aggregation-inducing protein on NL-17
cells

was

a Mr 44'000.

membraneglycoprotein(gp44;

Ref.

a

20 -

b

C

o I

I 1). We

I

I

I

I

I

I

I

2
4
6
Time (mmFig. 5. The effects of Awl and AIP4 on ADP-mediated.

platelets

recently punfied gp44 from NL-l7 cells by two affinity chromatographic methods, wheat germ agglutinin affinity chromatography andwere

incubated

with

100

@g/ml AlP)

(curve

a),

I

8

10

platelet aggregation. Mouse
AIP4(curve

c)

or

NR

lgG

(cu,,@@eb). Afterincubationfor 30 mm, 10 @dof 1 msi ADPwas added(arrow).Thechangesin lighttransmittanceweremonitoredfor 10mm.

3043

SUPPRESSION OF LUNG METASTASIS BY AN ANTI-PLATELET mAb

A00
control

AIP4

8F11

B@

@:
200
m:

control

AIP4

C
a@ .
@

â€”

â€˜CA'L

REFERENCES.
*a)v.â€•a)I. Honn, K. V., Tang, D. G., and Crissman. J. D. Platelets and cancer metastasis: acausal relationship? Cancer Metastasis Rev., I I: 325â€”351, I992
2. Bastida. E., and Ordinas, A. Platelet contribution to the formation of metastatic foci:U)@suppressed the pulmonary metastasis of CT26 colon adenocarcinoma(37). Because we injected AIP4 10 mm before tumor inoculation inthis study, the suppression of pulmonary metastasis by AIP4 seemed
to be independent of immune-mediated destruction of platelets. Further studies are needed to determine the involvement of immune
mediated destruction by making F(ab')2 fragments of AIP4. As with
8Fl 1-mediated suppression of lung colonization by NL-l7 cells,
F(ab')2 fragments of 8F1 1 also inhibited the lung colonization (12).
Therefore, mechanisms unrelated to immune-mediated destruction
may be involved in the 8F1 1 suppression of pulmonary metastasis.
Therefore, we expected that the immune-mediated destruction ofplatelets might not be involved in the AIP4-mediated suppression oflung colonization of NL-17 cells.
In summary, we here identified the Mr 160,000 platelet surface
component as the receptor of the platelet aggregation-inducing factor,
gp44 of NL-l7 cells, by generating anti-idiotype mAbs to 8Fl 1. Oneof these mAbs, AIP4, possesses the ability to suppress the gp44mediated platelet aggregation and the pulmonary metastasis of NL-l7
cells. Because some human tumor cells possess the platelet-aggregat
ing abilities that correlate with their metastatic potential, further
studies are needed to identify the human homologue of the Mr 160,000
platelet antigen.

0.2

the role of cancer cell-induced platelet activation. Haemostasis, 18: 29â€”36,1988.

LT@@1

3. Mehta, P. Potential role of platelets in the pathogenesis of tumor metastasis. Blood,
63: 55â€”63,1984.
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C

_J

0@
control

AIP4

Fig. 6. Suppression of pulmonary metastasis of NL-l7 cells in vivo by AIP4. NL-l7
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