From www.bloodjournal.org at DUKE MEDICAL LIBRARY on November 26, 2008. For personal use only.

2008 112: 1730-1739
Prepublished online Jun 9, 2008;
doi:10.1182/blood-2007-11-124693

Tetraspanin family member CD9 inhibits Aggrus/podoplanin-induced
platelet aggregation and suppresses pulmonary metastasis
Youya Nakazawa, Shigeo Sato, Mikihiko Naito, Yukinari Kato, Kazuhiko Mishima, Hiroyuki Arai,
Takashi Tsuruo and Naoya Fujita

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

Tetraspanin family member CD9 inhibits Aggrus/podoplanin-induced platelet
aggregation and suppresses pulmonary metastasis
Youya Nakazawa,1,2 Shigeo Sato,1 Mikihiko Naito,3 Yukinari Kato,4 Kazuhiko Mishima,5 Hiroyuki Arai,2 Takashi Tsuruo,1 andNaoya Fujita1
1Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo; 2Graduate School of Pharmaceutical Sciences and 3Institute of Molecular
and Cellular Biosciences, The University of Tokyo, Tokyo; 4Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science andTechnology, Tsukuba; and 5Saitama International Medical Center, Saitama Medical University, Saitama, Japan

CD9 has been reported to play a role intumor metastasis suppression. However,
it is not fully understood how CD9 affects
the hematogenous spread of tumor cells.
To clarify a new mechanism (or mechanisms), we generated HT1080 cells that
had been transfected with a CD9expressing plasmid. Ectopic expressionof CD9 in HT1080 cells actually reduced
their metastatic ability. CD9 expression
reduced lung retention and platelet aggregation activity of the transfectants.

Because HT1080 cells express themetastasis-promoting, platelet aggregation-inducing factor Aggrus/podoplaninon their surface, we examined the relationship between CD9 and Aggrus. We discovered that CD9 formed a complex with
Aggrus via transmembrane domains 1
and 2 (TM1 and TM2) of CD9. Investigation of the interaction revealed that eachCD9 and Aggrus interacted homophilically, and that they colocalized in lowdensity membrane fractions. Deleting TM1

and TM2 attenuated the ability of CD9 tointeract homophilically or to localize in
low-density membrane fractions. The expression of CD9–wild-type (WT), but not
CD9 lacking TM1 and TM2, attenuated theplatelet aggregation and metastasis induced by forced expression of Aggrus in
CHO cells. Therefore, CD9 may act as ametastasis suppressor, at least in part, by
neutralizing Aggrus-mediated platelet aggregation. (Blood. 2008;112:1730-1739)

Introduction
Four-pass transmembrane proteins of the tetraspanin family regulate cell migration, fusion, and signaling events by functioning asorganizers of a multimolecular membrane complex called thetetraspanin web or tetraspanin-enriched microdomains.1,2 The CD9protein is a member of the tetraspanin family and has been
identified as a suppressor of cancer spread.3 Numerous previous
studies have shown reduced CD9 expression in various cancers,which correlated with the presence of distant metastasis and apoorer prognosis.4-11 The clinical importance of CD9 in the
diagnosis, staging, and prognosis of tumors has been growing.12
Recently, it was reported that adenoviral transduction of CD9
inhibited lymph node metastasis in an orthotopic lung cancer
model.13 The investigators evaluated metastatic growth in the
mediastinal lymph nodes using gene delivery of CD9. Thesefindings strongly suggest that CD9 plays indispensable roles inmalignant tumor progression and shows the feasibility of genetherapy to prevent metastasis. However, we cannot say that thetherapeutic application of CD9 is now available. Functions oftetraspanins, to a great extent, may depend on expression patternsof their counterparts or other molecules on the tetraspanin web.
Actually, there are reports that overexpression of CD9 does notaffect metastatic properties,14 or that CD9 expression is downregulated at the primary site but reexpressed at the metastatic site.15
So, the precise mechanisms of the metastasis suppression should be
elucidated more clearly.
Aggrus/podoplanin, a type-I transmembrane glycoprotein, hasbeen shown to be up-regulated in a number of different cancers,16-23

suggesting that increased expression of Aggrus is associated withtumor malignancy and poor prognosis.24,25 We previously showedthat Aggrus expression induced platelet aggregation with norequirement for plasma components,26 and we have discoveredthat Aggrus promoted pulmonary metastasis in mice.27 Platelet
aggregation–inducing activity of Aggrus is directly associated withmetastasis formation because introducing a point mutation thatsuppresses platelet aggregation or administering aspirin diminishedthe formation of pulmonary metastasis.27 It was recently reported
that C-type lectin-like receptor 2 (CLEC-2) expressed on platelets
was identified as one of the counterparts of Aggrus.28,29 When
CLEC-2 binds to Aggrus expressed on tumor cells, it generates
activation signals, intra-platelet phosphorylation, and eventuallyinduces platelets to degranulate.30 Tumor cell–induced platelet
aggregation is believed to facilitate large tumor-platelet aggregate
formation, to embolize the microvasculature, and to promotetumor cell metastasis.31 Another survival advantage is tumor cell
protection from immunologic assault and shear forces in thecirculation.32,33 Indeed, mice without platelets, due to genetic
elimination of Nf-E2, showed marked resistance to hematogenous
metastasis formation.34 Therefore, Aggrus-mediated platelet aggregation may be a potential target for developing a hematogenous
metastasis inhibitor.
In this study, we investigated how CD9 affected the hematogenous spread of tumor cells as a metastasis suppressor. Ectopic
expression of CD9 in HT1080 cells reduced their metastatic ability
without affecting their growth in vitro and in vivo. CD9 expression

Submitted November 16, 2007; accepted April 22, 2008. Prepublished online
as Blood First Edition paper, June 9, 2008; DOI 10.1182/blood-200711-124693.

payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.

The publication costs of this article were defrayed in part by page charge© 2008 by The American Society of Hematology

1730

BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

From www.bloodjournal.org at DUKE MEDICAL LIBRARY on November 26, 2008. For personal use only.
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

reduced platelet aggregation–inducing activity of HT1080 cells.
Consistent with the results, ex vivo imaging of tumor cells revealed
that CD9 expression reduced lung retention of HT1080 cells.
Because HT1080 cells express the platelet aggregation–inducing
factor Aggrus on their surfaces, we further examined the relationship between CD9 and Aggrus. We discovered that CD9 formed acomplex with Aggrus, via transmembrane domains 1 and 2 (TM1and TM2). The CD9-⌬6, lacking TM1 and TM2, could not form acomplex with Aggrus. Moreover, CD9 and Aggrus interacted
homophilically and localized in low-density membrane fractions(LMFs), while CD9-⌬6 could not interact homophilically andhardly existed in LMFs under Brij 97 treatment. Finally, weinvestigated the effects of CD9–wild-type (WT) or CD9-⌬6 onplatelet aggregation and pulmonary metastasis in CHO cells
overexpressing Aggrus. CD9-WT, but not CD9-⌬6, suppressed theabilities of Aggrus. Therefore, CD9 may act as a metastasis
suppressor, at least in part, by neutralizing Aggrus-mediatedplatelet aggregation.

Methods
Cell culture conditions
HT1080 and CHO cells were cultured in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St
Louis, MO). LN319 cells were cultured in Dulbecco modified Eagle
medium (DMEM) supplemented with 10% heat-inactivated FBS.
Animals
Female BALB/c mice, BALB/c-nu/nu mice, and C.B-17/Icr-scid mice werepurchased from Charles River Japan (Kanagawa, Japan). All animal
procedures were performed using protocols approved by the JapaneseFoundation for Cancer Research Animal Care and Use Committee.
Plasmids and cell preparation
The human aggrus, CD9, and CD44 cDNAs were subcloned into a
pcDNA3 vector. Substitution of the appropriate cysteine codons to serine
codons in human CD9 cDNA was accomplished using the QuickChangesite-directed mutagenesis kit (Stratagene, La Jolla, CA). Deletion of theappropriate region in human CD9 cDNA was accomplished by polymerase
chain reaction (PCR). HT1080 cells were transfected with vectors encoding
nothing (mock), CD9-WT, or CD44 using the LipofectAMINE 2000
reagent (Invitrogen, Carlsbad, CA). Stable transfectants were selected bycultivating the cells in medium containing 0.4 mg/mL geneticin (SigmaAldrich). A pRetroQ-AcGFP-N1 retroviral vector was purchased from
Clontech (Mountain View, CA). For ex vivo imaging, HT1080 cells were
infected with the retroviral vector according to the manufacturer’s instruction. Then, the established HT1080 (HT1080/AcGFP) was further transfected with plasmids using the LipofectAMINE 2000 reagent. After
selection by cultivating the cells in the medium containing 0.5 mg/mL
geneticin, stable transfectants were established. CHO cells were transfectedwith vectors encoding nothing (mock) or Aggrus using the LipofectAMINE
2000 reagent. Stable transfectants were selected by cultivating the cells in
medium containing 1.0 mg/mL geneticin. Then the stable transfectants
were transiently transfected with plasmids. To confirm the dependence on
Aggrus expression in HT1080-induced platelet aggregation, cells werepreincubated with rat anti–human Aggrus antibody (NZ-1) or control rat
IgG for 1 hour at 4°C in Hanks balanced salt solution (HBSS). In someexperiments, cells were transfected with aggrus-specific siRNA using the
LipofectAMINE 2000 reagent. The coding strand of the siRNA-directed
residues 183 through 201 of human aggrus mRNA was GUCUGGCUUGACAACUCUG. Nonsilencing control siRNA was purchased from Qiagen(Chatsworth, CA).

CD9 SUPPRESSES PLATELET AGGREGATION

1731

Flow cytometry
Cells were harvested by brief exposure to trypsin. After washing withphosphate-buffered saline (PBS), cells were treated with an anti–humanCD9 monoclonal antibody (Sigma-Aldrich) or control mouse IgG, thenwere incubated with Oregon Green 488–conjugated antibody (Invitrogen).
Flow cytometric analysis was performed using a Cytomics FC500 flow
cytometry system (Beckman-Coulter, Miami, FL).
Experimental lung metastasis and ex vivo imaging of lungretention
Cells were harvested, washed, and resuspended in HBSS (HT1080,
106 cells/mL; CHO, 2.5 ⫻ 106 cells/mL). Then, 200 ␮L of the cell suspension were intravenously inoculated into lateral tail veins (HT1080, 8-weekold female C.B-17/Icr-scid mice; CHO, 8-week-old female BALB/c-nu/nu
mice). After 28 days the mice were killed and surface lung metastatic fociwere counted. For ex vivo imaging of lung retention, AcGFP-expressing
HT1080 cells were resuspended in HBSS (106 cells/mL). Then 200 ␮L of
the cell suspension were intravenously injected into lateral tail vein
(8-week-old female BALB/c-nu/nu mice). After 5 minutes or 4 hours themice were killed and the lungs were extirpated. The fluorescence wasvisualized with an IV100 imaging system (Olympus, Tokyo, Japan;
6⫻/0.14) and analyzed by FLUOVIEW software (Olympus); the fluorescence area was quantitated with NIH ImageJ software (National Institutesof Health, Bethesda, MD).
In vitro and in vivo proliferation assays
To assess in vitro cell proliferation, the MTT assay was used after 24, 48, or
72 hours of culture. For the in vivo proliferation assay, HT1080 cells were
harvested, washed, and resuspended in HBSS. A total of 5 ⫻ 106 cells in
0.1 mL HBSS were subcutaneously injected into the backs of 8-week-old
female C.B-17/Icr-scid mice. Tumor volume was calculated by the following formula: volume ⫽ W2 ⫻ L / 2, where W is the short diameter and L is
the long diameter.
Platelet aggregation assay
Cells were harvested, washed, and resuspended in PBS (HT1080,
5 ⫻ 106 cells/mL; CHO, 107 cells/mL). Mouse platelet-rich plasma (PRP)
was prepared from fresh, heparinized blood extracted from retired female
BALB/c mice. A 200-␮L aliquot of PRP was incubated at 37°C. A total of
10 ␮L cell suspension was added, and then light transmittance wasmonitored at 660 nm with an MCM HEMA TRACER 313M (SSR
Engineering, Kanagawa, Japan). Platelet aggregation-inducing activity wascalculated using the following formula: platelet aggregation–inducing
activity ⫽ 1 / A, where A is the time from reaction-starting point tomaximum value point. For the assay of forced platelet activation, 10 ␮LADP (40 ␮M in PBS; WAKO, Osaka, Japan) and thrombin (50 units/mL in
PBS; Invitrogen) were added to 200 ␮L PRP that had been preincubatedwith CHO/mock or CHO/CD9 cells for 10 minutes. For intraplatelet
phosphorylation assay, mouse platelets were obtained by centrifugation of
PRP with heparin and citrate-dextrose solution and resuspended in modified
Tyrode buffer containing 1 mM GRGDS peptide. A total of 5 ⫻ 105
HT1080 cells were added to platelets, and reactions were terminated by theaddition of 2⫻ lysis buffer (2% Nonidet P-40, 20 mM Tris, 300 mM NaCl,
2 mM EDTA) after indicated times. The lysates were then electrophoresedand immunoblotted.
Immunoprecipitation and immunoblot analysis
Cells were harvested and solubilized in lysis buffer containing 1% detergent(Triton X, Brij 97, or CHAPS), 25 mM HEPES, 150 mM NaCl, 5 mM
MgCl2, 1 mM phenylmethanesulfonyl fluoride, and 1 mM aprotinin. The
FLAG-tagged proteins were immunoprecipitated with an anti-FLAG agarose (M2; Sigma-Aldrich). Endogenous CD9 and Aggrus were immunoprecipitated with protein G–Sepharose (Zymed Laboratories, South San
Francisco, CA) that had been conjugated with antibodies to CD9 (M-L13;
BD Biosciences, San Jose, CA) or Aggrus. Then, the immunoprecipitatedFrom www.bloodjournal.org at DUKE MEDICAL LIBRARY on November 26, 2008. For personal use only.
1732

NAKAZAWA et al

BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5proteins or the cell lysates were electrophoresed and blotted onto a
nitrocellulose membrane. The membranes were incubated with antibodies
to CD9, Aggrus, FLAG tag (Sigma-Aldrich), V5 tag (V5-10; Invitrogen),caveolin (BD Biosciences), Erk1/2 (Cell Signaling, Beverly, MA), phosphoErk1/2 (Cell Signaling), or ␤-actin (Sigma-Aldrich). Subsequently, themembranes were incubated with a horseradish peroxidase–conjugatedsecondary antibody. After washing, the membranes were developed with anenhanced chemiluminescence (ECL) system.
Ultracentrifuge fractionation in sucrose gradients
CHO transfectants (107) were solubilized in 500 ␮L of lysis buffer
containing 1% detergent (CHAPS or Brij 97), 25 mM MES, 150 mM NaCl,and 5 mM MgCl2. Lysates were then mixed with an equal volume of 90%
sucrose. This 45% solution was overlaid onto 1.5 mL of 35% sucrose and
1 mL of 5% sucrose. Samples were centrifuged at 20 000g for 18 hours at
4°C, and 300 ␮L of each fraction were collected from the top of thegradient.35 The fractions were then electrophoresed and immunoblotted.
Immunostaining
LN319 cells were plated onto glass-bottomed dishes. After incubation for
20 hours, the cells were fixed and permeabilized with 4% paraformaldehyde and 0.2% Triton X in PBS for 10 minutes. Labeling was carried out by
incubation for 1 hour with antibodies to Aggrus (NZ-1) or CD9 (DU-ALL1), followed by incubation with Alexa Fluor 488–conjugated goat anti–rat
IgG (for NZ-1) or Alexa Fluor 633–conjugated goat anti–mouse IgG (for
DU-ALL-1) (Invitrogen). Subsequently, the cells were incubated with
Hoechst 33342 (Invitrogen). After washing, the specimens were visualizedusing a fluorescence confocal microscope (FV1000; Olympus. 60⫻/1.42 oilobjective) and analyzed by FLUOVIEW software (Olympus).
Invasion assay
Invasion of HT1080 cells was assessed by measuring their capacity to
migrate across transwell inserts using BD Bio-Coat Matrigel invasionchamber in 6-well plates, 8-␮m pore (BD Biosciences). A total of 2 ⫻ 105
HT1080 cells in serum-free RPMI 1640 medium were added to the uppercompartment, and RPMI 1640 medium supplemented with 20% heatinactivated FBS was added to the lower compartment. After incubation for
20 hours, invading HT1080 cells in the lower compartment were counted.
Gelatin zymography
Gelatin zymography was performed using a Gelatinzymo Electrophoresis
Kit (Yagai, Yamagata, Japan). Supernatant was collected from HT1080
cells cultured in serum-free RPMI 1640 medium (with or without 50 ng/mLof phorbol 12-myristate 13-acetate; TPA) for 20 hours. The culture
supernatants were concentrated with Centricon (Millipore, Bedford, MA)
and underwent SDS-PAGE using a 7.5% gel containing 0.1% gelatin. Afterelectrophoresis, the gel was soaked in 2.5% Triton X solution at 23°C withgentle shaking for 1 hour. After incubation for 40 hours in reaction buffer(50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM CaCl2, and 0.01% Brij
35) at 37°C, the gels were stained with Coomassie Brilliant Blue (WAKO).
Statistical analysis
All data are shown as means plus or minus SD. The Student t test was
performed. P values less than .05 were considered statistically significant.
All statistical tests were 2-sided.

Results
CD9 suppressed pulmonary metastasis and lung retention of
HT1080 cells

Forced expression of CD9 in tumor cells has been reported toreduce their metastatic ability.13,36-38 To confirm these reports, weprepared polyclonal HT1080 cells that had been stably transfected

Figure 1. Ectopic expression of CD9 suppressed pulmonary metastasis andlung retention of HT1080 cells. (A) Stably transfected polyclonal HT1080/mock (leftpanel) and HT1080/CD9 (right panel) cells were treated with a control mouse IgG (redline) or an anti-CD9 antibody (blue line). After incubation with Oregon Green
488–conjugated second antibody, CD9 expression was estimated by flow cytometer.
(B) HT1080/mock, HT1080/CD9, and HT1080/CD44 cells were intravenously injected into C.B-17/Icr-scid mice (n ⫽ 10). At 28 days after tumor inoculation, lungsurface nodules were counted. Representative pictures of the lungs (left panels) anddetailed numbers of the lung metastasis nodules in each mouse (right panel) are
shown. Data are means plus or minus SD. **P ⬍ .005 by Student t test. (C) Stablytransfected polyclonal HT1080/AcGFP⫹mock, HT1080/AcGFP⫹CD9, and HT1080/
AcGFP⫹CD44 cells were intravenously injected into BALB/c-nu/nu mice (n ⫽ 6).
After 5 minutes or 4 hours, the lungs were extirpated. The fluorescence wasvisualized using an IV100 imaging system, and the fluorescence area was quantitated. Representative pictures of the lungs (left panels) and detailed fluorescencearea (right panels) are shown. Data are means plus or minus SD. **P ⬍ .005;***P ⬍ .001 using the Student t test.

with vectors encoding nothing (HT1080/mock) or CD9 (HT1080/
CD9). Although endogenous CD9 expression could not be detectedin HT1080/mock cells, CD9 expression was clearly seen in
HT1080/CD9 cells (Figure 1A). CD44 is a transmembrane glycoprotein involved in cell adhesion. We also established stably
CD44-transfected polyclonal HT1080 cells (HT1080/CD44) as
negative control cells. We then intravenously inoculated theHT1080/mock, HT1080/CD9, or HT1080/CD44 cells into C.B-17/
Icr-scid mice. At 28 days after tumor inoculation, the number oflung surface nodules was counted, and the number in the HT1080/
CD9-injected mice was significantly lower than in the HT1080/
mock- or HT1080/CD44-injected mice (Figure 1B; **P ⬍ .005).
To assess the difference, we examined lung retention of tumor
cells 5 minutes or 4 hours after inoculation. To visualize tumor cellretention, we first tried to generate AcGFP-expressing HT1080
cells. The established HT1080/AcGFP cells were further transfected with vectors encoding nothing (HT1080/AcGFP⫹mock),CD9 (HT1080/AcGFP⫹CD9), or CD44 (HT1080/AcGFP⫹CD44).
Then, the established polyclonal HT1080/AcGFP⫹mock, HT1080/

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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

CD9 SUPPRESSES PLATELET AGGREGATION

1733

Figure 2. In vitro and in vivo growth and invasion of HT1080
transfectants. (A) Stably transfected polyclonal HT1080/mock (⽧),
HT1080/CD9 (〫), and HT1080/CD44 cells (E) were plated onto
each well of 24-well plates. After incubation for 24, 48, or 72 hours,
MTT assay was performed. Data are means plus or minus SD of
triplicate determinations. (B) HT1080/mock (⽧), HT1080/CD9 (〫),and HT1080/CD44 (E) cells were subcutaneously injected into thebacks of C.B-17/Icr-scid mice (n ⫽ 5). Tumors were measured withcalipers at 3, 6, 9, 12, and 15 days after injection. Tumor volume wascalculated using the following formula: volume ⫽ W2 ⫻ L / 2, where
W is the short diameter and L is the long diameter. Data are meansplus or minus SD. (C) HT1080/mock, HT1080/CD9, and HT1080/
CD44 cells in serum-free RPMI medium were added to the uppercompartment of Matrigel-coated invasion chambers. Data are meansplus SD of triplicate determinations. *P ⬍ .05; **P ⬍ .005 using theStudent t test. (D) HT1080/mock, HT1080/CD9, and HT1080/CD44
cells were cultured in serum-free RPMI medium for 20 hours. In an
experiment, HT1080/mock cells were stimulated with 50 ng/mL TPA(HT1080/mock⫹TPA). The culture supernatant was concentrated
and electorophoresed. The gels were incubated for 40 hours inreaction buffer and stained with Coomassie Brilliant Blue.

AcGFP⫹CD9, and HT1080/AcGFP⫹CD44 cells were intravenously injected into BALB/c-nu/nu mice. After 5 minutes or
4 hours the mice were killed and the lungs were extirpated. The
fluorescence area was visualized and quantitated (Figure 1C). After
5 minutes, there was little difference among the 3 samples. After
4 hours, however, lung retention of HT1080/AcGFP⫹CD9 cells
had drastically declined compared with others (Figure 1C;**P ⬍ .005; ***P ⬍ .001).
CD9 expression attenuated platelet aggregation induced by
Aggrus without affecting cell growth

To identify the determinants associated with CD9-mediated metastasis suppression in vivo (Figure 1), we first estimated the growthof HT1080/mock, HT1080/CD9, and HT1080/CD44 cells by MTT
assay. We also examined the in vivo growth of HT1080/mock,
HT1080/CD9, and HT1080/CD44 cells that had been injectedsubcutaneously into the back of C.B-17/Icr-scid mice. We foundalmost no difference between in vitro and in vivo cell growthamong HT1080/mock, HT1080/CD9, and HT1080/CD44 cells(Figure 2A,B). According to these results, CD9 expression might
prevent ectopic dissemination without blocking tumor formation.
We next examined the effects of CD9 on the invading ability oftumor cells. There are some reports that forced expression of CD9in tumor cells reduced their motility, resulting in reduction ofmetastatic ability.36,39,40 We thus estimated the invading ability ofHT1080/mock, HT1080/CD9, and HT1080/CD44 cells using a
Matrigel invasion assay (Figure 2C) and gelatin zymography(Figure 2D). Consistent with the previous reports, CD9 expression
slightly, but significantly, reduced HT1080 cell invasion (Figure2C). However, ectopic expression of CD9 did not affect the activityof matrix metalloproteinase (MMP)–2 and MMP-9 (Figure 2D).
CD9 probably reduced motility rather than invasion of HT1080
cells. CD44 expression also reduced HT1080 cell invasion to theMatrigel without affecting MMP-2 and MMP-9 activities. Interestingly, CD44 expression exhibited little effect on metastasis in vivo
(Figure 1B). These results suggest that attenuation of motility
might not be the reason for in vivo metastasis suppression by CD9in HT1080 cells.

We hypothesized that CD9 affects the state of tumor cells
circulating in the bloodstream (eg, protection and embolization oftumor cells in microvasculature). One possible target of CD9 wastumor cell-induced platelet aggregation. The aggregation was
associated with large tumor-platelet aggregate formation, protection of tumor cells from immunologic assault, and embolization in
the microvasculature.32,33 We previously identified Aggrus as aplatelet aggregation-inducing factor,26 and discovered that Aggrus
promoted pulmonary metastasis in vivo.27 Immunoblot analysis
showed that HT1080 cells endogenously expressed Aggrus. Ectopic expression of CD9 or CD44 did not affect the expression levelof endogenous Aggrus in HT1080 cells (Figure 3A). To estimate
the activity and dependency of Aggrus on HT1080-induced platelet
aggregation, a platelet aggregation assay was used with HT1080
cells that had been transfected with aggrus-specific siRNA (Figure3B) or cells that had been preincubated with a neutralizing antibody
to Aggrus (Figure 3C). Platelet aggregation induced by HT1080
cells was attenuated by both aggrus gene silencing and theneutralizing-antibody treatment. We also examined lung retention
and experimental metastasis of HT1080 cells in vivo, in thepresence or absence of the neutralizing antibody. We found that theantibody treatment reduced lung retention and pulmonary metastasis of HT1080 cells (data not shown). These results indicate that
Aggrus is associated with platelet aggregation, lung retention, andin vivo metastatic potency of HT1080 cells.
Next, we checked the platelet aggregation-inducing activity ofHT1080/mock, HT1080/CD9, and HT1080/CD44 cells. The activity was calculated using the following formula: platelet aggregationinducing activity ⫽ 1/A, where A is the time from reaction-starting
point to maximum value point. As shown in Figure 3D, ectopic
expression of CD9, but not CD44, down-regulated the platelet
aggregation-inducing activity in HT1080 cells (Figure 3D;**P ⬍ .005; *P ⬍ .05). Characteristics of this inhibition wereprolonged lag phase and minimal effect on either the slope of theaggregation curve or maximal aggregation. Therefore, we analyzed
intraplatelet phosphorylation of Erk1/2, which was used an indicator of platelet activation.41,42 The expression level of Erk1/2 in
HT1080 cells was very low compared with that of platelets (Figure

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NAKAZAWA et al

BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

Figure 3. Effect of CD9 expression on Aggrusinduced platelet aggregation. (A) HT1080 cells that
had been stably transfected with expression vector encoding nothing, V5-tagged CD9, or V5-tagged CD44 werelysed and immunoblotted with antibodies to Aggrus, V5tag, or ␤-actin. (B) HT1080 cells that had been transfected with nothing (black line), control siRNA (red line),or aggrus-specific siRNA (green line) were incubatedwith mouse PRP. Then, light transmittance was monitored for 15 minutes. The blue arrow indicates when cells
are added. Aggrus expression level in each sample wasestimated by Western blot analysis with an antibody toAggrus or ␤-actin (right panels). (C) HT1080 cells werepreincubated with control rat IgG (black line) or rat
anti-Aggrus antibody (red line). Then, the reactions wereincubated with mouse PRP. Light transmittance wasmonitored for 15 minutes. (D) Stably transfected polyclonal HT1080/mock (black line), HT1080/CD9 (red line),and HT1080/CD44 (green line) were incubated withmouse platelet-rich plasma. Then, light transmittance
was monitored for 15 minutes (left panel). The blue arrow
indicates when cells are added; red arrow, when platelet
aggregation begins. Platelet aggregation–inducing activity was calculated using the following formula: platelet
aggregation-inducing activity ⫽ 1 / A, where A is the timefrom reaction-starting point to maximum value point.
Each point represents a mean plus or minus SD of
triplicate experiments. *P ⬍ .05; **P ⬍ .005 using theStudent t test. (E) Stably transfected polyclonal
HT1080/mock and HT1080/CD9 cells were added toplatelets, and reactions were terminated by the additionof 2 ⫻ lysis buffer after indicated times. The lysates were
electrophoresed and immunoblotted with an antibody to
Erk1/2 and phospho-Erk1/2. (F) CHO cells were transiently transfected with a CD9-expressing vector (CHO/
CD9; red line) or an empty vector (CHO/mock; blackline). Mouse PRP was preincubated with CHO/mock or
CHO/CD9 for 10 minutes. Then, 10 ␮L of ADP (40 ␮M in
PBS; left panel) or thrombin (50 U/mL in PBS; rightpanel) was added to the reactions.

3E; tumor cell only). As shown in Figure 3E, addition of
HT1080/mock cells rapidly increased the phospho-Erk1/2 level.
However, addition of HT1080/CD9 cells slowly increased thephospho-Erk1/2 level. The delay of intraplatelet phosphorylationof Erk1/2 might be associated with the prolonged lag phase after
HT1080/CD9 addition (Figure 3D). Importantly, CD9 expression
seemed not to affect the maximal signal intensity of phosphoErk1/2 level, which was correlated with no alteration of maximum
platelet aggregation (Figure 3D). To exclude the possibility that
CD9 acts directly on platelets to inhibit their activation, we
prepared CHO cells that had been transiently transfected with avector encoding nothing (CHO/mock) or CD9 (CHO/CD9). Afterincubating cells with mouse platelet-rich plasma for 10 minutes,
ADP and thrombin were added. ADP and thrombin are the mostcommon platelet aggregation inducer of tumor cells. The ADP- or
thrombin-induced platelet aggregation was not affected by pretreatment of the platelets with CD9 (Figure 3F). These results suggestthat CD9 suppresses platelet aggregation not by directly affecting
platelet activity but by interfering with Aggrus function.

CD9 formed a complex via transmembrane domains 1 and 2
and colocalized with Aggrus

Next, we examined the direct interaction between CD9 and Aggrus.
When endogenous Aggrus was immunoprecipitated with an antiAggrus antibody, CD9 was coimmunoprecipitated (Figure 4A). To
determine the strength of interaction between CD9 and Aggrus, wetransfected CHO cells with FLAG-tagged Aggrus and V5-tagged
CD9. Then, cells were lysed with buffer containing 1% CHAPS,Brij 97, or Triton X. Interaction between CD9 and Aggrus wasobserved in the buffer containing 1% CHAPS or Brij 97 (Figure
4B). The results indicate that the interaction was a level 2interaction of tetraspanins and was likely to be significant for
tetraspanin function.35 We screened and found that a humanglioblastoma cell line, LN319, enodogenously expressed both CD9and Aggrus on its surface. When the endogenous CD9 protein
expressed on LN319 cells was immunoprecipitated with an antibody to CD9 in the buffer containing 1% CHAPS, endogenous
Aggrus was coimmunoprecipitated and vice versa (Figure 4C). An

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CD9 SUPPRESSES PLATELET AGGREGATION

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Figure 4. Interaction between CD9 and Aggrus. (A) HT1080
cells were stably transfected with V5-tagged, CD9-encoding vector(HT1080/CD9). Then, endogenous Aggrus was immunoprecipitatedwith an anti-Aggrus antibody in buffer containing 1% CHAPS. Exogenous CD9 was coimmunoprecipitated with the anti-Aggrus antibody.
(B) CHO cells were transfected with expression plasmids encoding
FLAG-tagged Aggrus and V5-tagged CD9. Then, the cells were solubilized with buffer containing 1% CHAPS, Brij 97, or Triton X. The
FLAG-tagged Aggrus was immunoprecipitated with an anti-FLAG agarose. The immunoprecipitated proteins were electrophoresed and immunoblotted with an antibody to V5 tag or Aggrus. (C) LN319 cells weresolubilized with buffer containing 1% CHAPS. Then, endogenous
proteins were immunoprecipitated with control IgG or antibody to CD9 or
Aggrus. The immunoprecipitants were electrophoresed and immunoblotted with an antibody to Aggrus or CD9. (D) LN319 cells were immunostained with antibodies to CD9 and Aggrus. Aggrus was visualized byincubation with Alexa Fluor 488–conjugated goat anti–rat IgG (leftpanel). CD9 was visualized by incubating with Alexa Fluor 633–
conjugated goat anti–mouse IgG (middle panel). Merged image isshown in right panel.

immunostaining experiment showed that CD9 was localized in a
dotlike pattern on the LN319 cell membrane, suggesting its
localization in tetraspanin-enriched microdomains (tetraspanin
web; Figure 4D). Aggrus was colocalized on the overall cell
membrane, but in specific regions, strong colocalization of CD9and Aggrus could be detected (Figure 4D right panel, inset). These
results suggest that CD9 and Aggrus interacted and colocalized on
specific membrane regions.
To identify the binding site in CD9, we prepared several CD9point and deletion mutants. We generated a deletion mutant lacking
the shorter extracellular loop (EC1) (⌬1: 39-56 deletion) or lacking
the longer extracellular loop (EC2) (⌬2: 114-133 deletion; ⌬3:
134-153 deletion; ⌬4: 154-173 deletion; and ⌬5: 174-192 deletion;
Figure 5A). We also established a deletion mutant lacking TM1,
EC1, and TM2 (⌬6: 13-82 deletion). Because palmitoylation
supports assembly and function of tetraspanins, we generated amutant that was unable to be palmitoylated (MT: C9S, C78S,
C79S, C218S, and C219S; Figure 5A). Immunoprecipitation
analysis showed that all the generated mutants, except CD9-⌬6,
interacted with Aggrus under both CHAPS and Brij 97 treatment(data not shown), just as CD9-WT did (Figure 5B left panels).
CD9-⌬6 interacted with Aggrus in the presence of CHAPS but not
Brij 97 (Figure 5B right panels). CD9 TM1 and TM2 have beenreported to be important in intra- and intermolecular interactions,and the interactions are fundamental in forming primary units
within the tetraspanin web.43 In addition, the 3 other tetraspanins
we tested could form complexes with Aggrus (data not shown).
These results suggest that Aggrus constantly localizes and interacts
with CD9 in the tetraspanin web.
We investigated localization of Aggrus, CD9-WT, and CD9-⌬6
in the tetraspanin web using 2 different criteria. First, the tetraspanin web is consisted of many kinds of molecules, so
molecules within tetraspanin web are likely to have a homophilicinteraction. Second, molecules within the tetraspanin web are
expected to localize in LMFs in sucrose gradients because the ratioof lipid to protein in the tetraspanin web is higher than insurrounding parts. To prove a homophilic interaction, we prepared
CHO cells expressing FLAG-tagged Aggrus together with V5tagged Aggrus. We also generated CHO cells expressing FLAGtagged CD9-WT together with V5-tagged CD9-WT or V5-tagged
CD9-⌬6. Immunoprecipitation after immunoblot analysis revealed
the homophilic interaction of Aggrus and CD9-WT (Figure 5C). In
contrast, CD9-⌬6 could not bind to CD9-WT. Next, to examine
localization in LMF, we solubilized CHO/Aggrus, CHO/
Aggrus⫹CD9-WT, and CHO/Aggrus⫹CD9-⌬6 cells in lysis buffer
containing 1% CHAPS or Brij 97. Lysates were centrifuged insucrose gradients and were fractioned. Because caveolin is wellknown to appear in lipid-enriched microdomains, we used it as a

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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

NAKAZAWA et al

Figure 5. Identification of CD9 mutant forming a weakcomplex with Aggrus and the tetraspanin web.
(A) Schematic representation of the generated CD9mutants: shorter extracellular loop (EC1)–deletion mutant (⌬1: 39-56 deletion); longer extracellular loop (EC2)–
deletion mutants (⌬2: 114-133 deletion; ⌬3: 134-153
deletion; ⌬4: 154-173 deletion; and ⌬5: 174-192 deletion); TM1, EC1, and TM2 deletion mutant (⌬6: 13-82
deletion); and palmitoylation-deficient point mutant (MT:C9S, C78S, C79S, C218S, and C219S). (B) CHO cells
were transiently transfected with V5-tagged CD9-WT(left panels) or CD9-⌬6 (right panels) together with orwithout FLAG-tagged Aggrus. After solubilization with 1%
CHAPS or Brij 97, the FLAG-tagged Aggrus was immunoprecipitated with an anti-FLAG agarose. The immunoprecipitants were analyzed by Western blot with an antibody
to V5 tag. (C) CHO cells were transfected with plasmid
encoding nothing or FLAG-Aggrus together with V5Aggrus (left panels). In some experiments, CHO cells
were transfected with plasmids encoding V5-CD9-WT or
V5-CD9-⌬6 together with FLAG-CD9-WT (right panels).
These cells were solubilized with lysis buffer containing
1% CHAPS. After immunoprecipitation with an antiFLAG agarose, the immunoprecipitants were analyzed
by Western blot with antibodies to V5 or FLAG. (D) CHO
cells were transiently transfected with FLAG-Aggrus
expressing plasmid together with plasmids encoding
nothing (CHO/Aggrus), V5-CD9-WT (CHO/Aggrus⫹CD9WT), or V5-CD9-⌬6 (CHO/Aggrus⫹CD9-⌬6). Then, cells
were solubilized in lysis buffer containing 1% CHAPS or
Brij 97. Each cell lysate was centrifuged in sucrose
gradients and fractioned. Then, each fraction was electrophoresed and analyzed by Western blot with antibodies
to Aggrus, caveolin, FLAG tag, or V5 tag. LMFs wereconcentrated in fraction numbers 2 through 4 (F2-F4).

positive control for LMFs. We found that Aggrus, CD9-WT and
CD9-⌬6 were localized in LMF (fraction numbers 2-4) under
CHAPS treatment (Figure 5D). Under Brij 97 treatment, Aggrus,
CD9-WT, and CD9-⌬6 all shifted to higher-density membrane
fractions; however, it was most complete for CD9-⌬6. CD9expression did not affect Aggrus distribution to LMFs under both
CHAPS and Brij 97 treatment. These results suggest that Aggrus
constantly localized and interacted with CD9 in the tetraspaninweb. Because CD9-⌬6 could not strongly interact with thetetraspanin web, CD9-⌬6 could not form a complex with Aggrus.
CD9 TM1 and TM2 involvement in Aggrus-induced platelet
aggregation and metastasis formation

To clearly show the direct link between CD9-Aggrus interaction
and Aggrus inactivation, we prepared CHO/mock or CHO/Aggrus
cells that had been transfected with plasmids encoding nothing(CHO/mock⫹mock or CHO/Aggrus⫹mock), CD9-WT (CHO/
Aggrus⫹CD9-WT), CD9-⌬6 (CHO/Aggrus⫹CD9-⌬6), or CD44
(CHO/Aggrus⫹CD44). The expression of CD9 and CD44 did notaffect the expression level of Aggrus in CHO/Aggrus cells (Figure
6A top right panel). Consistent with the data on HT1080 cells(Figure 3D), overexpression of CD9-WT attenuated the platelet

aggregation-inducing activity of Aggrus (CHO/Aggrus⫹CD9-WT;
Figure 6A). In contrast to CD9-WT, CD9-⌬6 exhibited minimum
inhibitory effect on Aggrus function (Figure 6A). We also examined pulmonary metastasis of the transfectants and confirmed thatthe number of lung surface tumor nodules in mice injected with
CHO/Aggrus⫹CD9-WT cells was significantly lower than that inmice injected with CHO/Aggrus⫹mock cells (Figure 6B; *P ⬍ .05).
Although the number of CHO/Aggrus ⫹ CD9-⌬6 was slightly
reduced compared with the number of CHO/Aggrus⫹mock, theextent of reduction was not significant (Figure 6B; NS indicates
not significant).
These results indicate that platelet aggregation and pulmonary
metastasis induced by Aggrus could be attenuated by interaction
with CD9 on the tetraspanin web.

Discussion
Some would argue that treatments directed against metastasis are
too late because cells have already escaped from the primary tumor.
However, there has been considerable improvement in survival
after adjuvant radiation and chemotherapy designed to eliminateFrom www.bloodjournal.org at DUKE MEDICAL LIBRARY on November 26, 2008. For personal use only.
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

CD9 SUPPRESSES PLATELET AGGREGATION

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Figure 6. Effect of CD9-⌬6 expression on platelet aggregation andmetastasis induced by forced expression of Aggrus. (A) CHO/mockand CHO/Aggrus cells were transiently transfected with vectors encoding nothing (mock), CD9-WT, CD9-⌬6, or CD44. The transfected polyclonal CHO/mock⫹mock (pink line), CHO/Aggrus⫹mock (black line),
CHO/Aggrus⫹CD9-WT (red line), CHO/Aggrus⫹CD9-⌬6 (green line),
and CHO/Aggrus⫹CD44 (blue line) cells were incubated with mouse
PRP. The light transmittance was monitored for 15 minutes. Blue arrow
indicates time when cells are added, and red arrow indicates time whenplatelet aggregation begins (top left panel). Platelet aggregationinducing activity was calculated using the following formula:volume ⫽ W2 ⫻ L / 2, where W is the short diameter and L is the long
diameter. Data are means plus or minus SD of triplicate experiments(bottom panel). *P ⬍ .05; NS indicates not significant by Student t test.
Expression level of each protein was confirmed by Western blot with
antibodies to Aggrus, V5 tag, or ␤-actin (top right panels). (B) CHO/
mock⫹mock, CHO/Aggrus⫹mock, CHO/Aggrus⫹CD9-WT, CHO/
Aggrus⫹CD9-⌬6, and CHO/Aggrus⫹CD44 cells were injected intravenously into BALB/c-nu/nu mice (n ⫽ 6). At 28 days after inoculation, thenumber of lung surface tumor nodules was counted. Representative
pictures of the lungs (left panels) and detailed numbers of the lungmetastasis nodules (right panel) are shown. Data are means plus orminus SD. *P ⬍ .05; NS indicates not significant by Student t test.

disseminated cells after surgical removal of the primary tumor. To
establish treatments against metastasis, identifying and understanding the specific genes that control metastasis is necessary. Generally, a metastasis suppressor is distinguished from a tumor suppressor and is defined as a gene that prevents ectopic dissemination
without blocking tumor formation. More than 20 metastasis
suppressors have been identified, but their precise mechanisms ofaction are largely unknown. Recently, a tetraspanin family member,
CD82/KAI1, believed to be a metastasis suppressor, has beenshown to interact with a protein (DARC [Duffy antigen receptor forchemokines]) on endothelial cells and to induce senescence oftumor cells,44 indicating a role in preventing ectopic dissemination.
Another tetraspanin member, CD9, is believed to suppress migration signals by forming membrane complexes with integrin receptors,4,5,45 and has been identified as a metastasis suppressor.13,36-38
However, it has not been clearly or concretely elucidated how the
motility change acts, or which metastatic step is affected. We
confirmed herein that CD9 expression reduced motility of HT1080
cells (Figure 2C). Tumor cell motility might not be directly
associated with in vivo metastatic ability because CD44 expression
also reduced motility of HT1080 cells, without affecting in vivo
metastatic potential (Figure 1B). We also found that CHO cells did
not exhibit an invasive ability in vitro, even though the cells weretransfected with Aggrus or CD9 (data not shown), while
Aggrus-transfected CHO cells formed many lung metastatic fociin vivo (Figure 6B).
One key to success in metastasis formation is tumor survival in
the bloodstream. Tumor cell–induced platelet aggregation is be-lieved to form the large tumor-platelet aggregate, leading to tumor
cell protection from host immunity and cell embolization in the
microvasculature.32,33 The metastatic potential is not simply an
inherent trait of cancer cells, but it is substantially modified bytumor microenvironment. Platelets release a number of soluble
factors (eg, PDGF, TGF-␤, ADP, fibronectin) after aggregation,
which may make the microenvironment suitable for metastasis
formation.46-48 Considering these features, tumor cell–induced
platelet aggregation promotes ectopic dissemination without affecting primary tumor formation. Conversely, this suggests that
inhibition of platelet aggregation may slow down the rate of tumor
progression and metastasis.
In this study, we showed that CD9 expression reduced lungretention of tumor cells 4 hours after inoculation (Figure 1C). Thismeans that CD9 expression affected the extremely early steps ofthe metastatic process (ie, circulation and arresting in microvasculature). We confirmed that CD9 expression reduced plateletaggregating activity of HT1080 and CHO cells (Figures 3D and
6A). Because HT1080-mediated platelet aggregation occurred after
6 minutes of contact between HT1080 cells and platelets (Figure3), tumor cells in the lung after 5 minutes of HT1080 inoculation
might be temporary entrapment before platelet aggregation–induced stable lung retention. That might be the reason why no
difference in lung retention after 5 minutes of tumor inoculation
could be found among 3 samples (Figure 1C). It is well known thattumor cells induce platelet aggregation mainly by the synthesis ofthrombin and ADP. However, platelet activation induced bythrombin and ADP was not suppressed by CD9 (Figure 3F). TheseFrom www.bloodjournal.org at DUKE MEDICAL LIBRARY on November 26, 2008. For personal use only.
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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

NAKAZAWA et al

results suggest that CD9 suppressed platelet aggregation byinterfering with other factors involved in platelet function. Aggrusinducing platelet aggregation is observed in the presence of specificthrombin and ADP inhibitors,49 and Aggrus can activate platelets
washed free from other plasma components26 through activating
intraplatelet signaling cascade.28 We have shown here that reducing
platelet aggregation and metastasis by CD9 expression required
interaction between CD9 and Aggrus via TM1 and TM2 (Figure
6A,B). Because CD9 lacking TM1 and TM2 could not stronglyinteract with the tetraspanin web (Figure 5C,D), CD9-⌬6 could notinhibit Aggrus function on the tetraspanin web. We confirmed that
CD9 expression delayed intraplatelet phosphorylation without
affecting maximal signal intensity (Figure 3E). There is a correlation between the delayed ERK phosphorylation and the increased
lag time in the aggregation studies when platelets were incubatedwith CD9 expressing HT1080 cells, although we cannot determine
whether this is causal. In the formation of metastasis in vivo, thedelay of platelet aggregation would be sufficient in metastasis
suppression because tumor cells in circulation are destroyed very
rapidly by sheer force or the host immune system.32 In addition, we
examined whether CD9 expression affected Aggrus binding to
CLEC-2, which is one of the counterparts of Aggrus on platelets.28,29 CD9 expression did not affect the Aggrus binding to
CLEC-2 (data not shown). This result suggested that CD9 expression might inhibit some platelet-aggregating pathways after Aggrus
binding to CLEC-2 (eg, CLEC-2 multimerization). Cross-linking
of CLEC-2 by anti–CLEC-2 antibody can induce platelet aggregation in the absence of specific ligands.30 Thus, CLEC-2 multimerization was reported to be associated with CLEC-2–mediatedplatelet aggregation. CD9 might interfere with CLEC-2 aggregation and suppress platelet aggregation. Thus, we need to examine
its effect on CLEC-2 multimerization in the future.
Therapeutic use of platelet inhibitors against metastasis has
been challenged by many investigators, and an anticancer effecthas often been suggested.50-52 However, applying these results toeffective anticancer therapies has not been established because thestate of the platelets of a patient with cancer is extremely fluid andelusive. Patients with cancer can show thrombocytosis or thrombocytopenia, depending on their circumstances. High platelet counts,or thrombocytosis, are often seen in patients with cancer. It isthought that tumors secrete substances to stimulate platelet production.53 Furthermore, in patients with advanced cancer, the expression of adhesion molecules on platelets is increased, indicating theplatelet activation.54 In fact, concomitant venous thromboembolism (VTE) is well recognized in cancer. An antiplatelet approach iseffective not only for metastasis but also for hypercoagulability,
such as in VTE. On the other hand, patients with cancer undergoingtreatment with cytotoxic agents often suffer from bleeding due toplatelet toxicity, or thrombocytopenia. Because platelets play animportant role in maintaining normal hemostasis even in theabsence of acute injury, patients with cancer suffering fromthrombocytopenia regularly receive platelet transfusions. It is
possible that this life-saving therapy simultaneously facilitates
metastasis. Effective platelet-modulating therapies must exhibit
specificity for the pathologic tumor cell–platelet interaction without affecting normal platelet function. In this study, suppression ofplatelet aggregation was confirmed as an entirely new function of
CD9 with regard to metastasis suppression. This suppression
depended on Aggrus expression on the tumor cell surface(Figure 3). These tumor cell–platelet–specific mechanisms must be
more clearly elucidated in order to determine the feasibility of
antiplatelet therapy.

Acknowledgments
We thank Drs N. Sugimura and N. Onda (Olympus) for theirtechnical support.
This study was supported in part by special grants from
Ministry of Education, Culture, Sports, Science and Technology,
Japan 17016012 and 18390020 (to T.T. and N.F., respectively); a
grant from Takeda Science Foundation, Japan (to N.F.); and a grantfrom the Cell Science Research Foundation, Japan (to N.F.).

Authorship
Contribution: Y.N. and N.F. designed the research; Y.N. performed
the research; M.N., Y.K., K.M., H.A., and T.T. contributed new
analytical tools; and Y.N. and N.F. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Naoya Fujita, Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation forCancer Research, 3-10-6, Ariake, Koto-ku, Tokyo 135-8550,
Japan; e-mail: naoya.fujita@jfcr.or.jp.

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