The American Journal of Pathology, Vol. 170, No. 4, April 2007
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2007.060790

Tumorigenesis and Neoplastic Progression

The Platelet Aggregation-Inducing Factor Aggrus/
Podoplanin Promotes Pulmonary Metastasis

Akiko Kunita,*† Takeshi G. Kashima,†
Yasuyuki Morishita,† Masashi Fukayama,†
Yukinari Kato,*‡ Takashi Tsuruo,§ andNaoya Fujita*§
From the Cancer Chemotherapy Center,§ Japanese Foundation
for Cancer Research, Tokyo; the Department of Cell Growth andRegulation,* Institute of Molecular and Cellular Biosciences, andDepartment of Pathology,† Graduate School of Medicine, The
University of Tokyo, Tokyo; and the Glycogene Function Team of
Research Center for Medical Glycoscience,‡ National Institute ofAdvanced Industrial Science and Technology, Tsukuba, Japan

Tumor cell-induced platelet aggregation has beenreported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing
factor that is up-regulated in a number of humancancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor
growth , metastasis , and survival in vivo. Aggrus
expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differencesin the size of metastatic foci or in primary tumor
growth were found in either set of mice. Aggrusexpressing cells , which were covered with platelets , arrested in the lung microvasculature 30 minutes after injection. In addition , lung metastasis
resulting from Aggrus expression decreased thesurvival of the mice. By generating several Aggrus
point mutants , we revealed that point mutation at
the platelet aggregation-stimulating domain of Aggrus (Thr34 and Thr52) obliterated both platelet
aggregation and metastasis. Furthermore , administration of aspirin to mice reduced the number ofmetastatic foci. These results indicate that Aggrus
contributes to the establishment of metastasis bypromoting platelet aggregation without affecting
subsequent growth. Thus , Aggrus could serve as an
ideal therapeutic target for drug development toblock metastasis. (Am J Pathol 2007, 170:1337–1347; DOI:
10.2353/ajpath.2007.060790)

Metastasis is the major cause of death from cancer, yet
the optimal strategy against it remains uncertain. The
pathogenesis of metastasis is dynamic and consists ofthe following steps: 1) cellular transformation and tumor
growth, 2) angiogenesis, 3) detachment and local invasion of the host stroma, 4) entry into the bloodstream, 5)transport along circulation, 6) arrest in the capillary, 7)
extravasation, and 8) proliferation within the foreign tissue.1 However, even if tumor cells successfully proceed
through steps 1 to 3 and get into the circulation, almost all
circulating tumor cells are rapidly destroyed by the shear
forces or are attacked by the immune system, and lessthan 0.01% of these cells survive to produce metastasis.2
Thus, a key to success in metastasis relies on tumor
survival in the bloodstream. One of the strategies bywhich tumor cells achieve this is platelet aggregation, ie,tumor cell-induced platelet aggregation (TCIPA).
Platelet aggregation is believed to protect tumor cells
from immunological assault in the circulation. Indeed, it
has been shown that platelets protect tumors from tumor
necrosis factor ␣-mediated cytotoxicity.3 Another survival
advantage is the tendency for the large tumor-platelet
aggregate to embolize the microvasculature at a new
extravasation site.4 Platelets also facilitate the adhesionof tumor cells to the vascular endothelium5 (step 6) andrelease a number of growth factors that promote tumor
cell growth. Mice without platelets, because of genetic
elimination of Nf-E2, showed marked protection against
metastasis.6 Recently, it has been reported that platelets
contribute to tumor-induced angiogenesis by releasing
angiogenic growth factors, such as vascular endothelial
growth factor.7–9 There are several mechanisms involved
in TCIPA, and these can vary among different tumor cells.
For example, tumor cells can activate platelets by tumor
Supported in part by the Ministry of Education, Culture, Sports, Science,
and Technology, Japan (special grants 17016012 and 18390020 to T.T.
and N.F.); the Japanese Society for the Promotion of Science for Young
Scientists, Japan (grant for research fellowships to A.K.); the Kato Memorial Bioscience Foundation, Japan (to N.F.); and the Takeda Science
Foundation, Japan (to N.F.).
Accepted for publication December 15, 2006.
Address reprint requests to Naoya Fujita, Ph.D., Cancer Chemotherapy
Center, Japanese Foundation for Cancer Research, 3-10-6, Ariake, Kotoku, Tokyo 135-8550, Japan. E-mail: naoya.fujita@jfcr.or.jp.

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AJP April 2007, Vol. 170, No. 4cell-induced thrombin generation through a coagulation
pathway,10,11 releasing adenosine 5⬘-diphosphate(ADP),12 thromboxane A2,13 matrix metalloproteinase
2,14 and the membrane protein Aggrus.15
Aggrus/podoplanin is a type I transmembrane sialomucin-like glycoprotein that consists of an extracellulardomain with abundant serine and threonine residues aspotential O-glycosylation sites, a single transmembrane
portion, and a short cytoplasmic tail with putative sites forprotein kinase C and cAMP phosphorylation.16 Because
Aggrus/podoplanin is expressed on the lymphatic endothelium but not on blood vessel endothelium, it is also
widely used in histopathology as a specific marker for
lymphatic endothelium and lymphangiogenesis.17 Aggrus expression has been shown to be up-regulated in a
number of different cancers, including squamous cell
carcinomas (oral cavity,16 lung,18 skin,19 and head andneck20), granulosa cell tumors,19 mesotheliomas,21 testicular seminomas,22 central nervous system tumors,23–25
and lobular breast cancers,26 suggesting that increased
expression of Aggrus is associated with tumor malignancy and poor clinical outcome.20 We previously
showed expression of Aggrus-induced platelet aggregation with no requirement for plasma components.15 We
also identified the segment of EDxxVTPG in the extracellular domain as the platelet aggregation-stimulating(PLAG) domain, which is critical for the platelet-aggregating activity of Aggrus.15 Aggrus contains three tandem repeats of the PLAG domain.27 In addition, these
PLAG domains were highly conserved among Aggrus
homologues from human, mouse, rat, dog, and hamster.27 However, it is yet to be elucidated whether its
platelet-aggregating activity is directly involved in the invivo metastasis-forming activity.
In this study, we investigated the role of human Aggrus
in tumor growth, metastasis, and survival. We established
Chinese hamster ovary (CHO) cells, which had beenstably transfected with wild-type (WT) Aggrus or its PLAG
domain mutants. We discovered that Aggrus expression
promoted pulmonary metastasis in both experimental
and spontaneous metastasis models and decreased survival of the mice. Platelet aggregation-inducing activity ofAggrus is directly associated with metastasis formation
because introducing a point mutation into the PLAG domains or administration of aspirin to mice decreased theformation of pulmonary metastasis.

Animals
Female BALB/c mice (5 weeks old) and BALB/c-nu/nu
mice (5 weeks old) were purchased from Charles River
Japan, Inc. (Kanagawa, Japan). Animals were housed
under pathogen-free conditions. The Animal Care and
Use Committee of the Institute of Molecular and Cellular
Biosciences approved the animal experiments describedherein.

Establishment of CHO Cells Stably Expressing
WT or Mutant Aggrus Proteins
The pcDNA3 vector containing WT human Aggrus cDNA
was established in our laboratory, as described previously.15 Substitution of the appropriate threonine codons toalanine codons in human Aggrus cDNA was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). CHO cells were thentransfected with the plasmids using LipofectAMINE 2000
reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Stable transfectants were selected by cultivating the cells in the medium containing 1
mg/ml Geneticin (G418; Sigma). We used two independent clones of each Aggrus-expressing clones. The expression level of human Aggrus was confirmed by Western blot analysis.

Western Blot Analysis
Cultured cell pellets or mice tumors were solubilized withlysis buffer (25 mmol/L Tris, pH 7.4, 50 mmol/L NaCl,
0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 1 mmol/L phenylmethyl sulfonyl
fluoride, and 50 mg/ml aprotinin), electrophoresed, andblotted onto a nitrocellulose membrane. The membranes
were incubated with an anti-human Aggrus YM-1 monoclonal antibody (hybridoma culture supernatant)28 or ananti-␤-actin antibody (Sigma). Membranes were subsequently washed and incubated with horseradish peroxidase-conjugated secondary antibody. After washing, themembranes were developed with an enhanced chemiluminescence system, according to the manufacturer’s instructions (GE Health Care UK Ltd., Buckinghamshire,
UK).

Flow Cytometry
Materials and Methods
Cell Culture Conditions
CHO cells were cultured in RPMI 1640 medium (Nissui,Tokyo, Japan) supplemented with 10% heat-inactivatedfetal bovine serum (Sigma, St. Louis, MO), 2 mmol/L
L-glutamine (Life Technologies, Inc., Grand Island, NY),and 100 ␮g/ml kanamycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

The cell surface expression of WT-Aggrus and Aggrus
point mutants was confirmed by flow cytometric analysis.
Cells were harvested by brief exposure to trypsin. Afterwashing with phosphate-buffered saline, cells weretreated with YM-1 monoclonal antibody for 1 hour at 4°C,
and then cells were incubated with Oregon Green 488conjugated antibody (Invitrogen Molecular Probes, Eugene, OR) for 30 minutes at 4°C. Flow cytometric analysis
was performed using a Cytomics FC500 flow cytometry
system (Beckman-Coulter, Miami, FL).

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Immunohistochemistry
Specimens were deparaffinized, rehydrated, and incubated with YM-1 monoclonal antibody at 23°C for 2 hours.
Then, the specimens were incubated with biotin-conjugated secondary anti-rat IgG antibody (DakoCytomation,
Glostrup, Denmark) for 30 minutes followed by incubation
with peroxidase-conjugated biotin-streptavidin complex (Vectastain ABC kit; Vector Laboratories, Peterborough, UK) for 30 minutes. Color was developed with
3,3-diaminobenzidine tetrahydrochloride tablet sets
(DakoCytomation).

Cell Proliferation Assays
The in vitro growth of stable transfectants was assessedusing Cell Counting Kit-8 (Dojin Laboratories, Kumamoto,Japan). Briefly, 1 ⫻ 103 cells were seeded into a 96-wellplate. The cells were allowed to grow for 1 to 4 days.
Then, the cells were incubated with 10 ␮l of the watersoluble tetrazolium salt-8 reagent for 2 hours. The opticaldensity was measured at 450 nm, with a 655-nm reference, using a microplate reader (model 550; Bio-Rad,
Hercules, CA).
To examine three-dimensional proliferation of stableclones, 1 ⫻ 103 cells were seeded into a 96-well plate(Sumilon Celltight Spheroid; Sumilon, Tokyo, Japan). The
spheroids were fed every other day by carefully replacing
half of the spent medium with fresh medium. To calculatemean size of the spheroids, diameters were measured
every other day. The cells were allowed to grow for 6
days.

In Vivo Detection of Platelets Associated with
Aggrus-Expressing Cells and Cell Survival
The mock-transfected or WT-Aggrus-transfected CHO
cells (CHO/control or CHO/WT-Aggrus, respectively)
were stained with PKH67 using a green fluorescent celllinker kit (Sigma), according to the manufacturer’s instruction, and resuspended in Hanks’ balanced salt solution (HBSS) without calcium or magnesium to a final
concentration of 2.5 ⫻ 106 cells/ml. Female BALB/cnu/nu mice were injected intravenously with 200 ␮l of thelabeled cells (5 ⫻ 105 cells/mouse), and lungs were
removed 30 minutes and 6 hours after injection. The
number of arrested cells was determined using fluorescence microscopy on 12 images at ⫻200 magnificationfrom at least two mice at each time point per group. To
detect association of CHO/WT-Aggrus cells with platelets, frozen mouse lung sections were stained 30 minutes
after injection with an anti-mouse CD41 antibody (Becton
Dickinson, San Jose, CA) followed by incubation with abiotinylated second antibody (DakoCytomation) and avidin R-phycoerythrin (Biomeda Corporation, Foster City,
CA). The extent of platelet association with CHO/WTAggrus cells was quantitated by evaluating sections using conventional epifluorescence. Images were captured
on a fluorescence microscopy at ⫻200 magnification. At
least eight independent images for each lung were ana-lyzed. In some experiments, mice were injected intraperitoneally with 75 mg/kg aspirin (Sigma) for 3 days until the
day of intravenous injection of the stable clones (5 ⫻ 105
cells/mouse) into lateral tail vein of female BALB/c-nu/nu
mice. Thirty minutes after cell injection, blood was drawn
for testing platelet aggregation, and lungs were removed
to count the arrested cells.

Experimental Lung Metastasis andAnimal Survival
CHO/control, CHO/WT-Aggrus (clones 3 and 5), andCHO cells that were stably transfected with plasmid containing T34A-human Aggrus (CHO/T34A-Aggrus clones
4 and 5) or T52A-human Aggrus (CHO/T52A-Aggrus
clones 10 and 48), and were harvested, washed, andresuspended in HBSS (2.5 ⫻ 106 cells/ml). Then, thestable clones (5 ⫻ 105 cells/mouse) were inoculated
intravenously into lateral tail vein of female BALB/c-nu/nu
mice. After 17 days, the mice were euthanized, and surface lung metastatic foci were counted and measured.
The lung tissues from CHO/control and CHO/WT-Aggrusbearing mice were processed for hematoxylin and eosin(H&E) and elastica-van Gieson (EVG) staining and immunohistochemical analysis to confirm the expression ofhuman Aggrus in the metastatic foci. To investigate theeffect of aspirin on metastasis formation, female BALB/cnu/nu mice were injected intraperitoneally with aspirin (75mg/kg) or phosphate-buffered saline (PBS) for 3 days
until the day of intravenous injection of the stable clones(2.5 ⫻ 105 cells/mouse). Additional injections of aspirin or
PBS were given daily until day 17. After 17 days of cell
injection, lung foci were counted and measured. PBS wasused as a control. For the survival study, the stableclones (5 ⫻ 105 cells/mouse) were inoculated intravenously into lateral tail vein of female BALB/c-nu/nu mice.
The survival of the mice was checked daily for 50 days.

Tumorigenicity and Spontaneous
Metastasis Assays
CHO/control and CHO/WT-Aggrus cells were harvested,washed, and resuspended in HBSS (5 ⫻ 107 cells/ml).
Cells (5 ⫻ 106) in 0.1 ml of HBSS were injected subcutaneously into the back, close to the neck, of female
BALB/c-nu/nu mice. Tumors were measured with calipers
at 13, 15, 18, 20, 24, and 27 days after injection. Tumor
volume was calculated by the following formula: volume ⫽ W2 ⫻ L/2, where W ⫽ short diameter and L ⫽ long
diameter. Mice were euthanized 30 days after injection.
Lungs and primary tumor tissues were harvested for H&E
and EVG staining. Small portions of each tumor tissues
were analyzed to confirm the human Aggrus expression
by Western blot analysis.

Platelet Aggregation Assay
Platelet aggregation was monitored by measuring electric impedance29 using a whole-blood aggregometer

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Figure 1. Survival of Aggrus-expressing cells and its interactions with platelets in vivo. Nude mice were injected intravenously with the fluorescent dye
PKH67-labeled CHO/control (control) or CHO/WT-Aggrus clone 5 (WT). Mice were euthanized 30 minutes and 6 hours after injection. Frozen sections of thelungs were resected, and the green-colored cells were counted. A: Representative images of the PKH67-labeled cells that were arrested in lung 6 hours after cell
injection. B: The number of green-colored cells in lung sections. At least 12 independent images for each lung were analyzed. The number of foci in
CHO/WT-Aggrus-injected mice was statistically significant both 30 minutes and 6 hours. *P ⬍ 0.001 by analysis of variance with the Tukey-Kramer test. C: The in vivointeraction between platelets and intravenously injected PKH67-labeled cells after 30 minutes of inoculation. Frozen sections of the lungs were stained with an anti-CD41
antibody for platelets. The PKH67-labeled cells were observed in green, and platelets were observed in red. D: The numbers of PKH67-labeled cells covered with platelets
were quantitated by evaluating the sections. **P ⬍ 0.05 by Mann-Whitney U-test. Scale bars: 100 ␮m (A); 10 ␮m (C). Original magnifications, ⫻200.

(model 560; Chronolog, Havertown, PA). Heparinizedblood was drawn from BALB/c mice or aspirin-pretreated
BALB/c-nu/nu mice by cardiac puncture. Whole blood
was then diluted with an equal amount of normal saline.
The sample was placed in a plastic cuvette containing amagnetic stir bar and was kept at 37°C for 10 minutes
before analysis. The platelet aggregation was then initiated by adding CHO/WT-Aggrus cells (5 ⫻ 106 cells/
sample) or 10 ␮mol/L ADP (final concentration) and monitored for up to 20 minutes. In some experiments, whole
blood was pretreated with appropriate concentrations ofaspirin for 5 minutes at 37°C before stimulation.

Statistical Analysis
All data are shown as means ⫾ SEM, except for cellproliferation assay data that is shown by means ⫾ SD.
Student’s t-test, Mann-Whitney U-test, and one-way analysis of variance followed by Tukey-Kramer multiple comparisons were performed, where appropriate. The mousesurvival assay was evaluated by Kaplan-Meier analysis
and the log rank test. P values less than 0.05 wereconsidered statistically significant. All statistical tests
were two-sided.

Results
Aggrus Induced Platelet Aggregation andEnhanced Cell Survival in Vivo
Our previous in vitro studies suggested that Aggrus was
associated with TCIPA.15 To confirm further the role ofAggrus in in vivo TCIPA and metastasis formation, we
injected intravenously green fluorescent (PKH67)-labeled
CHO/control or CHO/WT-Aggrus cells into mice and analyzed their pulmonary retention. Several reports suggested that TCIPA was observed 30 minutes after tumor
cell injection.30,31 Thus, we examined the pulmonary retention of the Aggrus-expressing cells after 30 minutes
and 6 hours of cell injection. The number of PKH67labeled CHO/WT-Aggrus cells retained in the lung microvasculature was significantly higher than that of CHO/
control cells (Figure 1B, P ⬍ 0.001). Pulmonary retention

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of CHO/WT-Aggrus cells, but not CHO/control cells, wasalso observed 6 hours after injection (Figure 1, A and B).
Thus, Aggrus could be associated with the initial arrest
and the survival of CHO/WT-Aggrus cells in vivo. To detect Aggrus-induced platelet aggregation in vivo, frozen
lung sections were stained with an antibody to CD41, adefined marker for platelets in mouse.30,31 At 30 minutes
after cell injection, extensive platelet decoration (red)around CHO/WT-Aggrus cells (green) was observed in
38.8% of the cells, whereas only 7.6% of CHO/control
cells were covered with platelets (Figure 1, C and D; P ⬍0.05). These findings suggest that Aggrus facilitates initial arrest in the lung microvasculature by inducing platelet aggregation and contributes to initial survival of thetumor cells to promote metastasis formation.

Threonine-34 (T34) and Threonine-52 (T52) in
Human Aggrus Are Critical Sites for Platelet
Aggregation-Inducing Ability
We previously showed that human Aggrus had one PLAG
domain containing T52 (in the PLAG3 domain), thatmouse Aggrus had one domain containing T34 (in the
PLAG1 domain), and that these threonine residues werecritical for their platelet aggregation-inducing activities.15
Detailed alignment of the Aggrus homologues revealed
that Aggrus contained three tandem repeats of the PLAG
domains (PLAG1 to 3) and that the threonine residues in
the PLAG1 and the PLAG3 seem to be critical for theactivity.27 However, the significance of the T34 in the
PLAG1 domain of human Aggrus has not been determined yet. Therefore, we confirmed the importance ofT34 in platelet-aggregating activity of human Aggrus byestablishing stable transfectants. We established stable
CHO clones expressing WT human Aggrus (CHO/WTAggrus clones 3 and 5; WT-3 and WT-5), human Aggrus
containing a T34A mutation (CHO/T34A-Aggrus clones 4
and 5; T34A-4 and -5), human Aggrus containing a T52A
mutation (CHO/T52A-Aggrus clones 10 and 48; T52A-10
and -48), and vector alone (CHO/control; control). Western blot analysis showed all clones, with the exception of
the CHO/control, expressed Aggrus protein (Figure 2A).
Flow cytometric analysis suggested that WT and pointmutated Aggrus proteins were trafficking to the cell surface properly (Figure 2B). The stable clones grew atsimilar rate throughout 72 hours (Figure 2C), suggestingthat Aggrus expression did not affect CHO cell growth invitro. To examine anchorage-independent growth of theclones, three-dimensional spheroid growth was compared. Spheroids were measured for 6 days. We could
not see any difference in anchorage-independent growth(Figure 2D). These cells were further tested for their ability to induce platelet aggregation using whole blood aggregometer. Both CHO/WT-Aggrus clones (WT-3 andWT-5) induced platelet aggregation (Figure 2E). In contrast, no platelet aggregation could be observed when
CHO/control, CHO/T34A-Aggrus (T34A-4 and T34A-5),
or CHO/T52A-Aggrus (T52A-10 and T52A-48) was incubated with whole blood (Figure 2E). These results indicate that the T34 residue in the PLAG1 domain, in addi-

tion to the T52 in the PLAG3 domain,15 are essential sites
for exhibiting platelet aggregation-inducing ability of human Aggrus.

Aggrus Promotes Pulmonary Metastasis in a
Platelet Aggregation-Dependent Manner
To address the function of Aggrus in vivo, we inoculated
intravenously the stable Aggrus transfectants into female
BALB/c-nu/nu mice. Injection of CHO/WT-Aggrus led todevelopment of multiple lung metastatic foci (Figure 3A).
Pulmonary metastasis was rarely observed in mice injected with CHO/control, platelet aggregation-deficient
CHO/T34A-Aggrus, or CHO/T52A-Aggrus (Figure 3A).
The median number of pulmonary metastatic foci (range)
was 4.9 (0 to 21) in control, 151 (34 to 235) in WT-3, 209(150 to 266) in WT-5, 3 (0 to 18) in T34A-4, 19 (2 to 49) in
T34A-5, 2.7 (0 to 12) in T52A-10, and 3.2 (0 to 16) in
T52A-48 (Figure 3B). The number of metastatic lung nodules in mice injected with CHO/control, CHO/T34AAggrus, or CHO/T52A-Aggrus clones was significantly
lower than that in mice injected with CHO/WT-Aggrus
clones (Figure 3B, P ⬍ 0.001). Consistent with the increase in the number of metastatic foci, lung weight was
higher in CHO/WT-Aggrus-injected mice than in mice
injected with CHO/control, CHO/T34A-Aggrus, or CHO/
T52A-Aggrus (Figure 3C). We did not observe, macroscopically, metastatic foci in the liver, kidney, spleen,colon, or ovary in any of the mice (data not shown).
Aspirin is known to attenuate metastasis formation by
inhibiting platelet aggregation.32,33 To confirm thatthe platelet aggregation was directly associated with
Aggrus-mediated metastasis, we examined the effects
of aspirin on Aggrus-mediated pulmonary metastasis.
When platelets were pretreated with aspirin in vitro (0 to100 ␮mol/L) for 5 minutes before analysis, Aggrus-induced platelet aggregation was inhibited in a concentration-dependent manner (Figure 4A). We then examinedaggregating ability of platelets that were prepared fromaspirin-treated mice. The mice were administered withaspirin for 3 days until the day of platelet preparation. As
shown in Figure 4B, in vivo aspirin-treated platelets could
not aggregate even when platelet aggregation was induced by 10 ␮mol/L ADP. In contrast, PBS administration
did not affect the platelet-aggregating ability.
Detailed analysis of lung sections revealed that pulmonary retention of CHO/WT-Aggrus cells was inhibited byaspirin administration (Figure 4C, P ⬍ 0.0001). Consistent with the results, treatment of the mice with aspirin
suppressed metastasis formation of CHO/WT-Aggrus
cells (Figure 4D). Lung weight of aspirin-treated mice
was significantly lighter than that of PBS-treated mice
(Figure 4E, P ⬍ 0.05). These results demonstrate thatplatelet aggregation was directly associated with theAggrus-induced pulmonary metastasis.
Histological analysis of the lung tissues from CHO/WTAggrus-injected mice shows the typical features of themetastatic tumors. First, metastatic tumors were detectedin the lungs of the CHO/WT-Aggrus-injected mouse. Metastatic tumors and the tumor embolus, occupying and1342
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Figure 2. Characterization of WT-Aggrus-expressing CHO clone 3 and clone 5 (WT-3 and WT-5), platelet aggregation-deficient Aggrus mutant-expressing CHO
clones (T34A-4, T34A-5, T52A-10, and T52A-48), or mock-transfected CHO clone (control). A: Cell lysates were electrophoresed and immunoblotted with ananti-human Aggrus antibody (top) or an anti-␤-actin antibody (bottom). B: Flow cytometric analysis to confirm the cell surface expression of Aggrus. Cells weretreated with an anti-human Aggrus antibody, and then cells were incubated with Oregon Green 488-conjugated secondary antibody. C and D: Growth of CHO
clones in monolayer culture (C) or in three-dimensional spheroid culture (D). Data are means ⫾ SD of triplicate determinations. E: Platelet aggregation-inducing
ability of each clone was estimated by incubating with the diluted mouse whole blood, as described in Materials and Methods.

attaching to the pulmonary arteriole (indicated by arrowheads in Figure 5B), are sequential. The lungs from micethat were injected with CHO/control showed no metastatic lesions (Figure 5A). Second, EVG staining clarified

that the venule was totally occluded by the tumor embolus of the CHO/WT-Aggrus-expressing cells (Figure 5C).
Third, Aggrus staining was observed in the metastatic
lesions when the lung tissues were stained with our pre-

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Figure 4. Aspirin suppressed Aggrus-mediated platelet aggregation invitro and metastasis formation in vivo. A: Diluted whole blood was
incubated with the indicated concentrations of aspirin for 5 minutes. The
platelet aggregation was then initiated by adding CHO/WT-Aggrus clone
5. B and C: Nude mice were intraperitoneally administered aspirin or PBS
for 3 days, as described in Materials and Methods. B: The platelet aggregation was then initiated by adding 10 ␮mol/L ADP. C: Frozen sections of
the lungs were resected after 30 minutes of intravenous injection of
CHO/control (control) or CHO/WT-Aggrus clone 5 (WT), and the arrested cells were counted. At least eight images for each lung wereanalyzed. *P ⬍ 0.0001, by analysis of variance with Tukey-Kramer test. D
and E: Nude mice were injected intraperitoneally with aspirin or PBS for
3 days until the day of the intravenous injection of CHO/WT-Aggrus clone
5. After cell inoculation, mice were further administered aspirin or PBS
daily for 17 days. Number of lung nodules (D) and lung weight (E) ineach mouse were analyzed after 17 days of injection. Data are means ⫾SEM. **P ⬍ 0.05, by Mann-Whitney U-test.

Figure 3. The expression of WT-Aggrus, but not platelet aggregation-deficient Aggrus mutants (T34A and T52A), promotes pulmonary metastasis invivo. Nude mice were euthanized 17 days after intravenous injection of
CHO/control (control, n ⫽ 9), CHO/WT-Aggrus clone 3 (WT-3, n ⫽ 6),
CHO/WT-Aggrus clone 5 (WT-5, n ⫽ 9), CHO/T34A-Aggrus clone 4 (T34A-4,
n ⫽ 9), CHO/T34A-Aggrus clone 5 (T34A-5, n ⫽ 6), CHO/T52A-Aggrus
clone 10 (T52A-10, n ⫽ 7), and CHO/T52A-Aggrus clone 48 (T52A-48, n ⫽6). A: Representative pictures of the lungs of control, WT-5 (WT), T34A-4(T34A), and T52A-10 (T52A) are shown. B and C: Detailed numbers of thelung metastasis nodules (B) and weight of the lung (C) in each mouse areshown. B: Bars indicate mean value. C: Data are means ⫾ SEM. *P ⬍ 0.001,
compared with control; ⫹P ⬍ 0.001, compared with T34A-4; #P ⬍ 0.001,
compared with T34A-5; -P ⬍ 0.001, compared with T52A-10 or ‡P ⬍ 0.001,
compared with T52A-48; **P ⬍ 0.05, compared with control; and ##P ⬍ 0.05,
compared with T34A-4 by analysis of variance with Tukey-Kramer test.

viously generated YM-1 monoclonal antibody that could
specifically recognize human Aggrus but not mouse Aggrus.24,25,28 At high-power magnification, drifted tumor
cell clump in the venule showed strong membranous
staining (Figure 5D, inset). In addition, the Aggrus-ex-pressing cells made a mass with platelets, leukocytes,
and erythrocytes (Figure 5D, arrow, inset). Aggrus-expressing cells also attached to the endothelial cells (Figure 5D, asterisk, inset). These attachments of tumor cells
to both leukocytes and endothelial cells are suggested topromote extravasation in the metastatic cascade.34
There were no differences in the in vivo growth of WT orpoint-mutated Aggrus-expressing clones when theseclones were inoculated subcutaneously (Figure 6A). Because pulmonary metastasis was slightly observed inmice injected with CHO/control (Figure 3B), we compared the tumor size at the metastatic nodules of CHO/
control and CHO/WT-Aggrus clones. As shown in Figure
6B, the size of nodules was almost the same. These
results suggest that Aggrus expression does not contribute to the in vivo growth of CHO cells. Therefore, Aggrus
may promote the formation of pulmonary metastasis via
its platelet aggregation-inducing activity without affecting
the growth potential.
We also evaluated the survival of mice after intravenous injection of CHO/control or CHO/WT-Aggrus
clones. We observed significantly poorer survival ratesof mice injected with CHO/WT-Aggrus cells than thoseof mice injected with CHO/control (Figure 6C, P ⬍1344
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Figure 5. Formation of emboli in CHO/WT-Aggrus-formed metastatic tumors. Nude mice were euthanized 17 days after intravenous injection of CHO/control or
CHO/WT-Aggrus clone 5. Tumor specimens of the lungs were subjected to histochemical analysis. A and B: H&E staining shows CHO/WT-Aggrus cells, but not
CHO/control cells, formed metastatic tumors. Embolized tumor cells, shown by an asterisk, were seen in the pulmonary arteriole (B: arrowheads, pulmonary
arteriole; Br., bronchi), whereas CHO/control cells rarely formed metastasis (A). C: The lumen of the venule (stained by EVG, arrowheads) was occluded by
CHO/WT-Aggrus-formed tumor embolus. D: Expression of Aggrus in the lesions was confirmed by immunohistochemical studies. Aggrus expression wasobserved in the lungs of mice injected with CHO/WT-Aggrus cells. Aggregates of circulating Aggrus-expressing tumor cell clump with platelets, erythrocytes, andleukocytes were detected in the venule (D: inset, arrow). The inset shows magnification of areas of interest. Attachment of Aggrus-expressing cells to endothelial
cells are shown by an asterisk. T, tumor cells. Scale bars ⫽ 100 ␮m.

0.0001). All mice in the CHO/control group survived
until 40 days of cell injection, whereas 9 of the 10 mice(90%) in CHO/WT-Aggrus group died from cachexia
with a large number of lung metastatic nodules within
40 days. One of the ten CHO/control-injected micedied on day 49, accompanied by forming two large
lung nodules (data not shown). These results suggestthat Aggrus expression contributes to metastatic
tumor formation in the lung and decreases survival ofthe mice.

Aggrus Expression Promotes Spontaneous
Metastasis
As shown in Figure 3, Aggrus could promote the formation of pulmonary metastasis in the experimental metastasis model. To confirm this metastasis-promoting poten-

tial, we next examined whether CHO/WT-Aggrus could
form pulmonary metastasis when these cells were inoculated subcutaneously into nude mice. Mice were euthanized at day 30. The lungs were examined for metastases
by H&E staining, and the pulmonary blood vessels werevisualized by EVG staining. CHO/WT-Aggrus cells
formed metastatic nodules in the lung, whereas CHO/
control cells showed much fewer or no metastatic lesions(Figure 7A, left and middle). A significant increase in thenumber of metastatic foci in the lung and the lung weight
was seen in mice injected with CHO/WT-Aggrus, whencompared with those in mice injected with CHO/control(Figure 7, B and C). Examination of all specimens
revealed that six of seven mice (86%) injected with
CHO/WT-Aggrus formed pulmonary micrometastases,
whereas only one of seven (14%) control mice did. Consistent with the experimental metastasis model, tumor

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AJP April 2007, Vol. 170, No. 4

to be different between the two groups (data not shown).
Western blot analysis revealed that Aggrus expression in
the primary tumors of CHO/WT-Aggrus cells continued
30 days after injection (Figure 7D). These results indicatethat Aggrus acts as a metastasis-promoting factor without affecting tumor cell growth.

Discussion

Figure 6. Aggrus expression does not confer in vivo CHO cell growth but
decreases mouse survival. A: CHO/control (control), CHO/WT-Aggrus clone
5 (WT), CHO/T34A-Aggrus clone 4 (T34A), and CHO/T52A-Aggrus clone 10(T52A) were inoculated subcutaneously into nude mice. Tumor diameter
was measured with calipers at 13, 15, 18, 20, 24, and 27 days after injection,
as described in Materials and Methods. Data are means ⫾ SEM. B: Surface
lung tumor size in the nude mice injected with CHO/control (control) or
CHO/WT-Aggrus clone 5 (WT) was measured. Data are means ⫾ SEM. C:
Survival rate of mice bearing CHO/control (control) or CHO/WT-Aggrus
clone 5 (WT) throughout time. *P ⬍ 0.0001 versus control evaluated by
Kaplan-Meier analysis and the log rank test.

embolus within the pulmonary arteriole was observed
and was attached to the metastatic lesions (Figure 7A,
right). There was no difference in tumor volume at the
primary sites between these two groups (Figure 6A).
Moreover, morphology of the primary tumors seemed not

Platelets have long been suspected of having a role incancer progression and metastasis.35 We previously
identified human Aggrus/podoplanin as a platelet aggregation-inducing factor that was expressed on the surfaceof various types of tumor cells.15 Because CHO cells
expressed very low level of Aggrus homologue and several CHO mutants that display inability in distinct steps ofglycosylation (eg, Lec series) were available for the analysis of O-glycans attached to Aggrus, we used CHO for
the analysis of Aggrus function. Although ectopic expression of WT human Aggrus on CHO cell surface could
induce platelet aggregation in vitro,15 its role in metastasis formation in vivo was still unknown. Thus, we examined
whether stable Aggrus-transfected CHO cells could form
pulmonary metastasis in experimental and spontaneous
metastasis models. In the experimental metastasis
model, increase in the number of pulmonary metastatic
nodules was observed by WT-Aggrus expression (Figure3). In addition, in vivo interaction between WT-Aggrusexpressing cells and platelets could be detected within

Figure 7. Aggrus expression promotes spontaneous metastasis. A: Nude mice were injected subcutaneously with CHO/control (control, left) or CHO/WT-Aggrus
clone 5 (WT, middle and right) cells. Mice were euthanized at day 30, and metastases in the lungs were examined by H&E staining (left and middle). EVG staining
was used to detect pulmonary blood vessels (right). T, tumor; arrowheads, pulmonary arteriole. The arteriole was filled with tumors. B and C: The numbers of
micrometastases in the lungs (B) and the lung weight (C) were determined. Data are means ⫾ SEM. *P ⬍ 0.01, and **P ⬍ 0.05 by Mann-Whitney U-test. D: The
lysates of CHO/control (control, left) or CHO/WT-Aggrus clone 5 (WT, right) xenografts were electrophoresed and immunoblotted with an anti-Aggrus antibody(top) or an anti-␤-actin antibody (bottom). Scale bars ⫽ 100 ␮m. Original magnifications: ⫻100 (A, left and middle); ⫻400 (A, right).

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30 minutes of infusion (Figure 1). Furthermore, the numbers of WT-Aggrus-expressing cells initially arrested in
the lung were higher than those of control cells (Figure
1B). These findings indicate that Aggrus induced platelet
aggregation soon after the cell injections, and that might
help the survival of the cells and formed metastasis.
Aggrus expression also promoted spontaneous lung metastasis (Figure 7) without affecting the growth at the
primary site (Figure 6A). This finding was supported by
the fact that the size of metastatic foci in the experimental
model was not affected by Aggrus expression (Figure
6B). In agreement with the above results, the survival ratein the mice bearing WT-Aggrus-expressing cells was
significantly lower than that of the mice carrying control
cells (Figure 6C). These findings suggest that Aggrus
could be a potential molecular marker to estimate thepossibility to form metastasis and could portend a poor
prognosis.
We also clarified previously that the sialylated O-glycan of human Aggrus was critical for its platelet aggregation-inducing activity using the glycosylation-deficient
CHO cell lines.28 We identified three tandem repeats ofthe segment of EDxxVTPG in the extracellular domain as
the PLAG domain, which is critical for the platelet-aggregating activity of Aggrus.27 In human Aggrus, we here
confirmed the importance of the threonine-34 (T34) in the
PLAG1 domain, in addition to the threonine-52 (T52) inthe PLAG3 domain, by analyzing the platelet aggregation-inducing activity of their point mutants T34A or T52AAggrus (Figure 2). Because threonine followed by proline
is likely to be O-glycosylated36 and Edman degenerative
microsequencing of the equivalent residues in the dog
homologue of Aggrus revealed gaps at the threonine
residues,37 sialylated O-glycan attached to the T34 andthe T52 in human Aggrus might be associated with its
platelet aggregation-inducing capability. Using the mutants, we performed the experimental metastasis assay
and found that the platelet aggregation-deficient T34A or
T52A-Aggrus lacked the metastasis-forming activity (Figure 3). Therefore, the platelet-aggregating activity is directly involved in Aggrus-derived metastasis formation.
To confirm further that role of platelets in the Aggrusmediated pulmonary metastasis in vivo, we examined theeffect of aspirin as an inhibitor of platelet aggregation.
Gasic and colleagues32,33 and Kolenich and colleagues38 reported that the aspirin administration intomice decreased the frequency of lung metastases. Aspirin inhibited Aggrus-induced platelet aggregation in vitro(Figure 4A). Because the ADP-induced aggregation ofplatelets was suppressed in aspirin-administered mice
(Figure 4B), aspirin might inhibit Aggrus-mediated platelet aggregation in vivo. As expected, pulmonary arrest of
CHO/WT-Aggrus cells was significantly lower in mice
pretreated with aspirin than that in PBS-treated mice
(Figure 4C). Moreover, aspirin administration decreased
the formation of experimental metastasis (Figure 4, D and
E). These results strongly indicate that platelet aggregation is indispensable for the Aggrus-mediated pulmonary
metastasis.
With careful examination of the lung specimens derived from both experimental and spontaneous metasta-

sis models, we observed that the metastatic lesions wereattached to the tumor emboli within the pulmonary arterioles (Figure 5B and Figure 7A). Drifting tumor cellclumps, which expressed Aggrus, were found in thevenule (Figure 5D). These tumor cell clumps seem tohave higher metastatic efficiencies than single cells.34 In
addition, attachment of Aggrus-expressing cells to bothleukocytes and endothelial cells, which could be facilitated by platelets,39 was also observed. This step wasreported to promote the essential step of arrest in thecapillary5 and extravasation in the metastatic cascade.39
We speculate that Aggrus-induced platelet aggregation
might facilitate the survival of the tumor cells by protecting against immune systems, promoting proliferation
within the vasculature using platelet-releasing growth
factors, forming tumor emboli, and expanding continuously to the extravascular lung tissues that resulted in theformation of metastatic foci.
Aggrus expression could also be detected in highly
metastatic B16 melanoma variant, B16-F10,40 and in
highly metastatic mouse colon adenocarcinoma 26 variant, NL-17.41 Our previously established anti-mouse Aggrus antibody 8F11 inhibited platelet aggregation induced by both cell lines.40,41 Moreover, the antibody
suppressed pulmonary metastasis of NL-17 cells.42
These results suggest that the present data are notunique to CHO cell line.
In conclusion, the present study shows that Aggrus
promoted experimental and spontaneous metastasis,
without affecting tumor growth, and diminished survival of
mice. Pulmonary metastasis was suppressed by abrogation of its platelet-aggregating activity. Our studies provide clear evidence for a causative role of platelet aggregation in cancer metastasis. Based on these studies, wesuggest that small molecule inhibitors or antibodies
against human Aggrus could be a therapeutic strategy
for inhibiting tumor metastasis and for enhancing cancer
patient survival.

Acknowledgments
We thank Drs. Kazuo Harada, Yoshinao Kikuchi, andMika Kato Kaneko for the critical reading and invaluablecomments on the manuscript; Dr. Takao Shimizu for theaggregometer; Shigeo Sato and Yasuyuki Kihara for their
excellent technical advice on the animal experiments;
and Chizuko Hashimoto for technical support withimmunocytochemistry.

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