CXCL10PromotesOsteolyticBoneMetastasisbyEnhancing …Corresponding Authors: Hyunil Ha, Traditional Korean Medicine-Based Herbal Drug Research Group, Korea Institute of Oriental Medicine,

Microenvironment and Immunology

CXCL10 Promotes Osteolytic BoneMetastasis by EnhancingCancer Outgrowth and Osteoclastogenesis

Jong-Ho Lee1, Ha-Neui Kim1, Kyung-Ok Kim2, Won Jong Jin1, Seungbok Lee1, Hong-Hee Kim1,Hyunil Ha3, and Zang Hee Lee1

AbstractAmplification of the chemokines CXCL10 andRANKLhas been suggested to promote osteoclast differentiation

and osteolytic bone metastasis, but a function for endogenous CXCL10 in these processes is not well established.In this study, we show that endogenous CXCL10 is critical to recruit cancer cells to bone, support osteoclastdifferentiation and promote for the formation of osteolytic bone metastases. Neutralizing CXCL10 antibodyreduced migration of cancer cells expressing the CXCL10 receptor CXCR3, and loss of CXCR3 or CXCL10decreased bone tumor burden in vivo. Bone colonization augmented host production of CXCL10, which wasrequired for cancer growth and subsequent osteolysis. Direct interactions between cancer cells andmacrophagesfurther stimulated CXCL10 production from macrophages. Growth of bone metastases required CXCL10-stimulated adhesion of cancer cells to type I collagen as well as RANKL-mediated osteoclast formation. Together,our findings show that CXCL10 facilitates trafficking of CXCR3-expressing cancer cells to bone, which augmentsits own production and promotes osteoclastic differentiation. CXCL10 therefore may represent a therapeutictarget for osteolytic bone metastasis. Cancer Res; 72(13); 3175–86. �2012 AACR.

IntroductionThe importance of the interaction between cancer cells and

the surrounding environment in organ-specific metastasis hasbeen well characterized in bone metastasis. Bone is one of themost preferential metastatic sites for common human cancers,and bone metastases are a significant cause of morbidity andmortality in patients with cancer, causing severe bone pain,pathologic fractures, and spinal cord compression (1, 2). Thedevelopment and progression of osteolytic bonemetastasis arethought to depend on a complex cycle of bone destruction andcancer outgrowth (3). Colonized cancer cells in bone producevarious osteoclastogenic factors that stimulate osteoclast dif-ferentiation and activation either directly or indirectly bypromoting the ratio of receptor activator of nuclear factor-kBligand (RANKL) or its decoy receptor osteoprotegerin (OPG) in

osteoblasts (4, 5). Increased bone resorption by activatedosteoclasts releases growth factors such as transforminggrowth factor-b and insulin-like growth factors from thedegraded bone matrix. These growth factors in turn enhancethe growth of cancer cells and further stimulate them toproduce the osteoclastogenic factors, resulting in a viciouscycle of bone destruction and cancer outgrowth (4, 5).

The interaction between chemokine receptors expressed oncancer cells and the corresponding chemokines produced bythe target organs has emerged as a key mediator of organ-specific metastasis (6). For example, the chemokine receptorCXCR4 has been found to be expressed in many tumor types,and CXCR4-expressing cancer cells can metastasize to severalorgans where CXCL12 is abundantly expressed (7–9). Similarly,the chemokine receptor CXCR3 has also been recently iden-tified in various cancer cells including melanoma, breastcancer, colon cancer, and glioma (10–13). In addition, CXCR3expression correlates with poor prognosis in breast cancer,colon cancer, and melanoma (11, 12, 14). Furthermore, it hasbeen showed that CXCR3 expression on cancer cells promoteslung metastases of breast cancer and colon cancer as well aslymph nodemetastases ofmurinemelanoma and colon cancer(10–12, 15, 16). Despite the importance of CXCR3 expression incancer metastasis to lung and lymph nodes, nothing is knownabout the contribution of endogenous CXCR3 ligands in thetarget organs.

CXCL10 is a member of the subfamily of IFN-g–inducibleand the non-ELR CXC chemokines and it signals throughCXCR3, a common receptor for IFN-g–inducible chemokines(CXCL9, CXCL10, and CXCL11). The fundamental role ofCXCL10/CXCR3 interaction has been well characterized inchronic Th1-inflammatory diseases. In inflamed tissues,

Authors' Affiliations: 1Department of Cell and Developmental Biology,Dental Research Institute, School of Dentistry, 2Institute on Aging, SeoulNational University, Seoul; and 3Traditional KoreanMedicine-BasedHerbalDrug Research Group, Korea Institute of Oriental Medicine, Daejeon,Republic of Korea

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Hyunil Ha, Traditional Korean Medicine-BasedHerbal Drug Research Group, Korea Institute of Oriental Medicine, 1672Yuseongdae-ro, Yuseong-gu, Daejeon 305-811, Republic of Korea.Phone: 82-042-868-9367; Fax: 82-042-868-9573; E-mail:[email protected]; and Zang Hee Lee, Department of Cell and Devel-opmental Biology, School of Dentistry, Seoul National University, 28Yeongon-dong, Jongno-gu, Seoul 110-749, Republic of Korea. Phone:82-02-740-8672; Fax: 82-02-747-6589; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-0481

�2012 American Association for Cancer Research.

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CXCL10 recruits Th1 cells and upregulates the production ofIFN-g . In turn, IFN-g stimulates CXCL10 expression in variouscell types, resulting in a positive feedback to amplify CXCL10and Th1 responses (17). In line with this, we previously foundthat reciprocal amplification of CXCL10 and RANKL plays acrucial role in bone destruction in a mouse model of rheuma-toid arthritis in which CXCL10 recruits CD4þ T cells andpromotes them to produce RANKL, and then RANKL inducesCXCL10 expression and osteoclast differentiation from itsprecursors (18).

These previous reports showing the important role of oste-oclast differentiation in bone metastasis, RANKL-inducedupregulation of CXCL10, and the participation of CXCR3 incancer metastasis to target organs led us to explore whetherCXCL10 plays a critical role in bone metastasis. To addressthis possibility, we examined CXCL10 expression in boneand then determined the effects of CXCR3 gene silencing incancer cells, systemic CXCL10 blockade with a neutralizingantibody, and CXCL10 deficiency in the host on experimentalmodels of bonemetastasis. We also investigated the molecularmechanisms underlying CXCL10-mediated osteolytic bonemetastasis.

Materials and MethodsReagents and cells

Mouse CXCL10, human RANKL, human macrophage col-ony-stimulating factor (M-CSF), anti-mouse CXCL10 anti-body (aCXCL10), and control rabbit IgG were obtained fromPeproTech. PD98059 was purchased from Calbiochem. AKTInhibitor VIII was purchased from Santa Cruz Biotechnol-ogy. B16F10 murine melanoma cell line, human breastcancer cell lines (MDA-MB-231, MDA-MB-453, and MCF7),MC3T3-E1 murine preosteoblastic cell line, NIH3T3 murinefibroblast cell line, and Raw264.7 murine monocytic cell linewere purchased from the American Type Culture Collectionand maintained in DMEM (Invitrogen) supplemented with10% FBS (Hyclone Laboratories) and 1% penicillin–strepto-mycin (Invitrogen). ST2 murine stromal cell line was kindlyprovided by Dr. Nacksung Kim (University of Chonnam,Korea) and cultured in a-MEM containing 10% FBS and1% penicillin–streptomycin. B16-FL cells were generatedfrom B16F10 cells by stable transfection of the firefly lucif-erase gene.

AnimalsCxcl10�/� mice and Toll-like receptor (Tlr)4�/� mice

(C57BL/6 background) were obtained from The JacksonLaboratory. Female BALB/C nude mice were purchasedfrom Charles River Korea. All animal procedures werereviewed and approved by the animal care committee ofthe Institute of Laboratory Animal Resources of SeoulNational University.

Migration assayCancer cells were harvested by treatment with cell dissoci-

ation solution (Sigma-Aldrich) and then resuspended inserum-free DMEM. Cells were seeded in the upper well of the

Boyden chamber (8-mm pore membranes; Corning Costar).Membranes were precoated with 10 mg/mL of fibronectin(Sigma-Aldrich). The lower well was loaded with serum-freeDMEM in the presence or absence of used attractants andincubated for 6 hours, and cells attached on the lower surfaceof the membrane were counted.

Antibody and RANKL injectionMice (6-week-old C57BL/6male) were treated intravenously

with 6.6 mg/kg aCXCL10 or control IgG the day before tumorintracardiac injection. Soluble RANKL (sRANKL: glutathione S-transferase fusion RANKL protein) was kindly provided by Dr.Hisataka Yasuda (Nagahama Institute for Biochemical Science,Oriental Yeast Company, Shiga, Japan) and used for in vivoexperiments only. Mice were injected intraperitoneally withsRANKL (20 mg per mouse), and serum CXCL10 levels weremeasured after sRANKL injection.

Reverse transcription PCR and real-time PCR analysisTotal RNA isolation, reverse transcription (RT), and real-

time PCRwere conducted as described previously (19). In someexperiments, CXCR3 and RANK transcripts were analyzed byRT-PCR. The primers used for real-time PCRandRT-PCR listedin Supplementary Table S1.

Retroviral gene transductionShort hairpin RNAs (shRNA) targeting luciferase (shLuci;

CAAATCACAGAATCGTCGT),mouseCXCR3 (mouse shCXCR3;CAGGCGCCTTGTTCAACAT), and human CXCR3 (humanshCXCR3; CACACCACCCAGCAGCCAG) were inserted into thepSUPER.retro.puro vector (OligoEngine). Retrovirus packaging,cell infection, and selection were conducted as describedpreviously (19).

In vitro osteoclast formation assayIn vitro osteoclast formation from mouse bone marrow

macrophages (BMM) and from coculture of mouse calvarialosteoblasts and bone marrow cells (BMC) was done asdescribed previously (19). Tartrate-resistant acid phosphatase(TRAP)-positive multinucleated cells containing 3 or morenuclei were counted as osteoclasts.

ELISAThe protein levels of mouse RANKL, mouse OPG, mouse

CXCL10, human CXCL10, and mouse CXCL12 in samplescollected as indicated were measured using the correspondingELISA kits (R&D Systems). The detailed procedures weredescribed in Supplementary Materials and Methods.

Bone metastasis modelsFor intracardiac injections, mice were anesthetized and

injected with 1 � 105 tumor cells in 100 mL PBS into the leftcardiac ventricle, a route of administration that allows for bonemetastasis rather than lung infiltration of injected cells. Forintrafemoral injections, 1 � 104 tumor cells in 5 mL PBS wereinjected into the left femur using a 28-gauge Hamilton syringe.After metastases, mice were sacrificed and underwent blindednecropsy.

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Bioluminescence imaging and analysisMice (C57BL/6) were intraperitoneally injected with 150

mg/kg D-luciferin (Xenogen) in PBS 12 minutes before biolu-minescence imaging (BLI). Imaging was conducted using acharge-coupled device camera (IVIS 100, Xenogen; exposuretime of 3minutes, binning of 8,field of view of 15 cm, f/stop of 1,and no filter). Mice were anesthetized with isoflurane (2%vaporized in O2) and shaved to minimize attenuation of signalby pigmented black hair. For analysis, total photon flux(photons per second) was measured from a fixed region ofinterest in the hind limbs and the mandible/maxilla usingLiving Image software (Xenogen). Bioluminescent signalswith-in the fixed region of interest were normalized to the back-ground luminescence taken over the same region of interestfrom animals not injected with D-luciferin.

Radiographs and measurement of osteolytic lesion areaX-ray images of the mouse hind limbs were taken using a

cabinet-style soft X-ray unit (Softex, SOFTEXCo.; 30KVp, 2mA,exposure time: 90 seconds), scanned, and evaluated withoutknowledge of the experimental groups. The area of osteolyticlesions, defined as a radiolucent lesion in bone, was measuredusing image analysis system (Image J) and expressed as apercentage of the total tissue area.

Histology and histomorphometryParaffin-embedded sections of decalcified bones were pre-

pared as described previously (20). Sections were stained withhematoxylin and eosin (H&E) or separately for TRAP activity tovisualize osteoclasts. Histomorphometric analysis of tumorburden, osteoclast number, and total bone including bothtrabecular and cortical bones was conducted with the Osteo-measure analysis system (Osteometrics). Tumor burden wasexpressed as a percentage of tumor area per total tissue area.

ImmunohistochemistryImmunohistochemical analysis was conducted on decalci-

fied paraffin-embedded tissue sections. Antibodies againstKi67 and CD31 were purchased from Dako and Santa CruzBiotechnology, respectively. Detection of the primary antibodywas conducted using VECTASTAIN Elite ABC kit (VectorLaboratories), followed by 3,30-diaminobenzidine (Vector Lab-oratories) incubation and nuclear staining with hematoxylin.Ki67 and CD31 staining were quantified by the percentage ofpositively stained nuclei per 400� field and the number ofvessels per 200� field, respectively. Three or more randomlychosen fields per slide were analyzed and averaged.

ImmunoblottingImmunoblot analysis was conducted as described previous-

ly (19). Specific antibodies against phospho-ERK1/2 (Thr202/Tyr204), ERK, phospho-AKT (Thr308 and Ser473), and AKT(Cell Signaling Technology) were used.

Statistical analysisAll quantitative data is presented as the mean � SD. Two-

group comparison was conducted by using 2-sided, 2-sampleStudent t test. Simultaneous comparison ofmore than 2 groups

was conducted by 1-way ANOVA (SPSS statistical package,version 12; SPSS Inc.). Values of P < 0.05 were consideredsignificant.

ResultsCXCL10 is produced in bone and its neutralizationreduces bone metastasis

To determine whether RANKL induces CXCL10 productionin vivo, mice were injected intraperitoneally with sRANKL.Serum CXCL10 levels were elevated on day 1 by sRANKL andsustained until day 3 (Fig. 1A). Because CXCL10 has beenshown to promote directional migration and invasion ofseveral cancer cells (10, 21, 22), we postulated that CXCL10,induced by RANKL during bone remodeling process, may beimplicated in bone metastasis via recruiting cancer cells to thebone. CXCL10 induced the chemotactic migration of severalcancer cells capable of developing bone metastasis, includinghuman MDA-MB-231 breast cancer cells and mouse B16F10melanoma cells (data not shown). In addition, blockingCXCL10 activity with aCXCL10 reduced mouse bone marrowfluid–induced migration of B16F10 cells, suggesting the bio-logic relevance of CXCL10 in recruitment of B16F10 cells to thebone (Fig. 1B).

When B16F10 cells are injected into the left cardiacventricle of syngeneic C57BL/6 mice, they result in metas-tases into several organs, such as bones, lungs, and adrenalglands (23). Bone metastasis of B16F10 cells causes blackpigmentation in bone lesions as a result of the melanin-pigment–producing B16F10 cells (23). To test whetherCXCL10 is involved in cancer bone metastasis, mice weretreated with 6.6 mg/kg aCXCL10 or control IgG the daybefore injection of B16F10 cells. Black pigmentation andhistomorphometric analysis revealed that although all micein both groups developed bone metastasis in their hindlimbs at 14 days after injection of B16F10 cells, aCXCL10reduced the number of bone lesions and tumor size in thehind limbs compared with control IgG (Fig. 1C and D).Similar to these results, the number of lesions and tumorsize in lungs and adrenal glands were also decreased byaCXCL10 (Supplementary Fig. S1A and S1B).

Reduction of CXCR3 expression in cancer cells decreasesbone metastasis

To investigate whether CXCL10-induced signaling in cancercells plays a crucial role in bone metastasis, we stably reducedCXCR3 expression in B16F10 cells by retrovirus-mediated genetransfer of shRNA. Compared with control (shLuci), shCXCR3reduced CXCR3 mRNA levels and impaired chemotaxis inresponse to CXCL10 but not RANKL in B16F10 cells (Fig. 2Aand B). Under the normal cell culture condition, shCXCR3 hadno effect on the proliferation of B16F10 cells (data not shown).The bone metastasis by intracardiac injection of B16F10 cellscarrying shLuci or shCXCR3 was analyzed by black pigmen-tation and histomorphometric analysis. Consistent with theresults of the aCXCL10 experiment, shCXCR3 decreased thenumber of lesions and tumor size in the hind limbs comparedwith shLuci (Fig. 2C and D).

Role of CXCL10 in Osteolytic Bone Metastasis

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The effect of CXCR3 knockdown in cancer cells on bonemetastasis was not restricted to the melanoma model but wasalso observed in a human breast cancer cell model. MDA-MB-231 cells stably expressing shLuci (MDA-shLuci) or shCXCR3targeting humanCXCR3 (MDA-shCXCR3) were generated, andthe functional knockdown of CXCR3 was confirmed byCXCL10-induced chemotaxis (data not shown). When MDA-shLuci cells were injected into the left ventricles of nudemice, 6of 8 mice developed severe osteolytic bone lesions in the hindlimbs 7 weeks after injection, as determined by X-ray imaging.In contrast, 4 of 8 mice injected with MDA-shCXCR3 cellsshowed relatively smaller bone lesions in the hind limbs (Fig.2E). Histomorphometric analysis further revealed that bone

tumor burden is dramatically reduced in mice-bearing MDA-shCXCR3 cells compared with mice bearing MDA-shLuci cells(Fig. 2F).

Host deficiency of CXCL10 reduces bone metastasisTo further validate the effects of CXCL10 on bone metas-

tasis, the bone metastasis in wild-type (WT) and Cxcl10�/�

mice after intracardiac injection of B16-FL cells stably expres-sing firefly luciferase was monitored by BLI. This analysisrevealed that the tumor burden in the hind limbs and themandible/maxilla was significantly less inCxcl10�/�mice thanin WT mice (Fig. 3A and B), which was further confirmed byhistologic and histomorphometric analyses (Fig. 3C and F).

Figure 1. Neutralization of CXCL10 inhibits bonemetastasis of B16F10melanoma. A, serumCXCL10 levels at the indicated days after sRANKL injection (n¼ 5per group; �,P<0.001). B,aCXCL10 (2mg/mL) significantly inhibited bonemarrow fluid–inducedmigration ofB16F10cells (�,P<0.05). C,mice pretreatedwithcontrol IgG or aCXCL10 (n ¼ 8 per group) were injected with B16F10 cells into the left cardiac ventricle, and their hind limbs were collected on day14 after injection. Representativemacroscopic images of femurs and the number of black pigmented lesions in the hind limbs (�,P < 0.01) are shown. Arrows,black pigmented lesions. D, representative H&E-stained images and histomorphometric quantification of tumor burden in femurs (�, P < 0.001). M,marrow; T, tumor; original magnifications, �40.

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Because it has been suggested that osteoclastic bone resorp-tion is implicated in cancer bone metastasis (3), we analyzedthe bone phenotype of Cxcl10�/� mice. There were no signif-icant differences in bone volume and osteoclast numberbetween WT and Cxcl10�/� femoral bones. However, metas-tasis of B16-FL cells to femoral bones resulted in a 3.5-foldincrease in osteoclast number with a decrease in bone volumein WT mice, whereas only a 1.3-fold increase in osteoclastnumber was observed in Cxcl10�/�mice (Fig. 3D and E). Thesedifferences were more clearly observed in the mandibularalveolar bone (Fig. 3F).

CXCL10 expression increases during bone colonizationof B16F10 cellsSerum CXCL10 levels were markedly elevated after intra-

cardiac injection of B16F10 cells, whereas CXCL12 levels werenot affected (Fig. 4A). Thus, we next investigated whether bonemetastasis affects CXCL10 production. We directly inoculatedB16F10 cells into the femur of syngeneic mice and monitored

the mRNA expression levels of the 3 CXCR3 ligands (CXCL9,CXCL10, and CXCL11). Among the CXCR3 ligands, CXCL10showed the highest basal gene expression. Although themRNAexpression levels of all 3 CXCR3 ligands were augmented afterinoculation of B16F10 cells, the increase of CXCL10 mRNAlevels was the most rapid and highest (Fig. 4B). The intrafe-moral inoculation of B16F10 cells also increased CXCL10protein levels in serum and bone marrow fluid in a time-dependent manner (Fig. 4C).

Cancer cells interact with macrophages to promoteCXCL10 production

We next investigated the mechanism of CXCL10 upregula-tion by bone colonization of B16F10 cells. Although B16F10cells secreted CXCL10 into the culture medium, synergisticaugmentation of CXCL10 releasewas observed in the cocultureof B16F10 cells with BMCs (Fig. 5A). Such synergistic CXCL10release was also observed when B16F10 cells were coculturedwith BMMs or RAW264.7, a monocyte/macrophage cell line

Figure2. GenesilencingofCXCR3 in cancer cells reducesbonemetastasis. A, specificknockdownofCXCR3 inB16F10cells carrying shCXCR3wasassessedby RT-PCR. B, the knockdown of CXCR3 abolished migration of B16F10 cells in response to CXCL10 (200 ng/mL) but not RANKL (2 mg/mL). n.s., notsignificant. C, representativemacroscopic images of femurs and the number of black pigmented lesions in the hind limbs on day 14 after intracardiac injectionof B16F10 cells carrying either shLuci or shCXCR3 (n ¼ 8 per group; �, P < 0.01). Arrows, black pigmented lesions. D, representative H&E-stainedimages and histomorphometric quantification of tumor burden in femurs from experiment (C). �, P < 0.001. M, marrow; T, tumor; original magnifications,�40.E, MDA-MB-231 cells carrying either shLuci or shCXCR3were injected into the left cardiac ventricle of female nudemice (n¼ 8 per group), and the hind limbswere collected 7 weeks after injection. Representative X-ray images and quantification of osteolytic lesions in femurs from each group (�, P < 0.01) areshown. F, representative H&E-stained images and histomorphometric quantification of tumor burden in femurs (�, P < 0.001). Original magnifications, �40.






(Fig. 5B). Prevention of direct contact between B16F10 cellsandBMMsbymembranefilters abrogated the augmentation ofCXCL10 release (Fig. 5C).

To determine the origin of enhanced CXCL10 release,B16F10 cells were cocultured with BMMs from WT orCxcl10�/� mice. A marked reduction of CXCL10 release wasobserved in coculture with Cxcl10�/� BMMs (Fig. 5D). Toclarify the issue further, mouse BMMs were cocultured withhuman breast cancer cell lines (MDA-MB-231, MDA-MB-453,andMCF-7), and the production of mouse and human CXCL10was differentially measured by each specific ELISA (Fig. 5E).These breast cancer cells secreted undetectable levels (MDA-MB-231 and MDA-MB-453) or very low levels (MCF-7) ofhumanCXCL10. Cocultures ofmouse BMMswith these human

breast cancer cells led to augmentation of mouse CXCL10release without changes in human CXCL10 release in all thecocultures.

CXCL10 promotes tumor outgrowth in the bonemicroenvironment

The ability of cancer cells to bind type I collagen, the mostabundant extracellular matrix molecule (ECM) within thebone, is closely related to cancer outgrowth within the bone(24).We found that CXCL10 promotes adhesion of B16F10 cellsto fibronectin and type I collagen (Supplementary Fig. S2A).Furthermore, CXCL10 significantly enhanced survival ofB16F10 cells in the serum-free condition but did not affectcell proliferation under the normal (10%) and low (0.1%) serum

Figure 3. Host deficiency of CXCL10 reduces bone tumor burden and bone loss by intracardiac injection of B16-FL cells. B16-FL cells or PBSwas injected intothe left cardiac ventricle of WT and Cxcl10�/� mice (n ¼ 10 per group), and bone metastases were analyzed on day 14 after injection. A, representativebioluminescence images. B, BLI analysis of tumor burden in the hind limbs (�, P < 0.001) and the mandible/maxilla (�, P < 0.01). C, representativeH&E-stained images and histomorphometric quantification of tumor burden in femurs (�, P < 0.001). M, marrow; T, tumor; original magnifications, �40.D, representative images of TRAP-stained femur sections and quantification of osteoclast number per bone surface (N. Oc/BS; �, P < 0.001). T, tumor;original magnifications, �100. E, bone volume per total tissue volume (BV/TV) in femurs by histomorphometric analysis (�, P < 0.05). F, as in D, exceptfor mandibles from each group (�, P < 0.001). A, alveolar bone; T, tumor; original magnifications, �100.

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condition (Supplementary Fig. S2B). These results promptedus to investigate the possibility that the augmented CXCL10production during bone metastasis provides a growth advan-tage for B16F10 cells within the bone. B16-FL cells wereinoculated directly into the femoral cavity of WT andCxcl10�/� mice and the progression of bone metastasis wasmonitored by BLI analysis. There was no significant differencein bone tumor burden between WT and Cxcl10�/� mice onday 7. On day 14, however, tumor burden was significantlylower in Cxcl10�/� mice than in WT mice (Fig. 6A and B). Thereduction of tumor burden in Cxcl10�/� mice was confirmedby histomorphometric analysis (Fig. 6C) andwas accompaniedby a marked decrease in osteoclast number and bone destruc-tion (Fig. 6D and E). Notably, Ki67 staining revealed a signif-icant decrease in the number of proliferating tumor cells inCxcl10�/� mice (Fig. 6F). In contrast, there was no significantdifference in tumor angiogenesis between WT and Cxcl10�/�

mice as determined by CD31 staining (Fig. 6G).

CXCL10 stimulates osteoclast differentiationBecause CXCL10 deficiency dramatically reduced osteoclast

number induced by bone metastasis of B16F10 cells, we nextexamined whether CXCL10 stimulates osteoclast differentia-tion. CXCL10 did not impact RANKL-induced osteoclast dif-ferentiation from its precursors, BMMs. However, CXCL10induced osteoclast formation in coculture of osteoblasts andBMCs in a dose-dependent manner (Fig. 7A and B). We foundthat CXCL10 increases RANKL mRNA and protein levels inosteoblasts in dose-dependent manner with no significantchange in OPG levels (Fig. 7C and D). Interestingly, CXCR3knockdown had no effect on CXCL10-induced RANKL mRNAexpression in osteoblasts. In contrast, CXCR3 knockdown

impaired the slight increase in RANKL mRNA expression inresponse to 2 other CXCR3 agonists, CXCL9 and CXCL11 (Fig.7E). Next, we sought to identify the CXCL10 receptor thatmediates RANKL induction in osteoblasts. LPS, a TLR4 ligand,increases RANKL expression in osteoblasts (25), and CXCL10has been shown to activate TLR4 signaling by binding toTLR4 (26). Indeed, CXCL10-induced RANKLmRNA expressionwas impaired in Tlr4�/� osteoblasts (Fig. 7F). In addition,CXCL10-stimulated osteoclast formation was dramaticallyreduced in the coculture of WT BMCs and Tlr4�/� osteoblasts(Fig. 7G).

We then studied the molecular mechanism underlyingTLR4-mediated RANKL expression in response to CXCL10.Treatment of WT osteoblasts with CXCL10 increased ERK andAKT phosphorylation within 5 minutes, which was sustainedfor at least 1 hour. In contrast, these increases were notobserved in Tlr4�/� osteoblasts (Fig. 7H). TLR4-mediatedactivation of ERK and AKT was linked to CXCL10-inducedRANKL expression. Pretreatment of WT osteoblasts witheither the ERK kinase inhibitor (PD98059) or the AKT inhibitor(AKT inhibitor VIII) blocked CXCL10-induced RANKL expres-sion (Fig. 7I).

DiscussionEvidence is accumulating for a positive role of CXCR3

expression on cancer cells in organ-specific metastasis (10–12, 15, 16). However, CXCR3 ligands including CXCL10 haveexhibited opposing effects on cancer cells and on stromal cellsin the tumor microenvironment. CXCL10 has been shown toincrease migration, invasion, proliferation, survival, and adhe-sion to ECMof diverse cancer cells (12, 13, 21, 22). In contrast to

Figure 4. Bone colonization ofB16F10 cells increases CXCL10production in bone. A, the proteinlevels of CXCL10 and CXCL12 inserum on day 14 after intracardiacinjection of B16F10 cells or PBS ascontrol (n ¼ 8 per group; �, P < 0.01).B, relativemRNAexpression levels ofCXCR3 ligands in the femurs on theindicated days after directinoculation of B16F10 cells into thefemoral cavity of syngeneic mice(n ¼ 4; �, P < 0.05 vs. day 0). C,quantification of CXCL10 proteinlevels in bone marrow fluid andserum from mice treated as in B.P < 0.05, day 7; P < 0.01, day 14.






the direct effects on cancer cells, it has also been reported thatCXCL10 exerts antitumor activities by inducing immune-stim-ulating and angiostatic effects (27–30). However, the role ofendogenous CXCL10 in the development of primary cancersand theirmetastatic foci has not been addressed. Herewe showthat host-derived CXCL10 plays an important role in osteolyticbone metastasis by promoting cancer outgrowth and osteo-clast differentiation in bone as well as by trafficking CXCR3-expressing cancer cells to bone.

In this study, CXCR3 gene silencing in cancer cells inhibitedbone metastasis by intracardiac injection of cancer cells.Among the CXCR3 ligands, CXCL10 is likely to be a majorcontributor to the effects of CXCR3 gene silencing on bone

metastasis, because CXCL10 expression is much higher thanthat of the 2 other CXCR3 ligands, CXCL9 and CXCL11, in bonetissues. Our finding of RANKL-induced CXCL10 productionmay provide a possible explanation for the high expression ofCXCL10 in bone tissues. Loss of CXCL10 by neutralizingantibody or genetic knockout also suppressed bonemetastasisby intracardiac injection of cancer cells. These observationscollectively indicate that CXCL10 plays an important role inbone metastasis, at least in part, by directing CXCR3-expres-sing cancer cells to the bone. In addition to bone metastasis,the CXCL10/CXCR3 axis may also be implicated in cancermetastasis to other organs such as lung. In our study, CXCL10neutralization reduced the number of lesions and tumor size

Figure 5. Cancer cells promoteCXCL10 production frommacrophages. A, CXCL10 levelsin culture supernatants from theindicated culture conditions byELISA. B, augmented production ofCXCL10 by coculture of B16F10cells with BMMs or Raw264.7 cells(�, P < 0.01). C, abrogation of theaugmented CXCL10 production bymembrane filters (�, P < 0.001). D,coculture of B16F10 cells withCxcl10�/� BMMs resulted in amarked reduction of CXCL10production compared withcoculture with WT BMMs(�, P < 0.001; n.d., not detected).E, mouse (m) and human (h)CXCL10 productions in thecultures were measured by eachspecific ELISA. All human breastcancer cell lines augmentedmCXCL10 production fromBMMs (�, P < 0.05).

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only in bones but also in lungs and adrenal glands, and CXCR3gene silencing and a pharmacologic CXCR3 antagonist wereshown to inhibit lung metastases of breast cancer cells andcolon cancer cells (11, 15, 16).Notably, CXCL10 production in bone was augmented by

bone colonization of cancer cells. In accordance with theimportance of host-derived CXCL10 in bonemetastasis, cancercells stimulated CXCL10 production from macrophages in a

cell–cell contact manner. Tumor-associated macrophages arethe major leukocyte population present in tumors and providemany trophic factors that promote tumor progression andmetastasis (31). In addition, macrophages have been shown totransmit survival signals in breast cancer cells during lungmetastasis (32) and provide prostate cancer cells with resis-tance to selective androgen receptor antagonists/modulators(33) through direct interaction with cancer cells. Also,

Figure 6. Host deficiency ofCXCL10 decreases tumor outgrowthwithin the bone. B16-FL cells wereinjected directly into the left femur ofWT or Cxcl10�/� mice (n ¼ 10 pergroup). A, representative images ofbioluminescence on day 14 afterinjection of B16-FL cells. B, BLIanalysis of tumor burden on days 7and 14 after injection (�, P < 0.005).C, representative H&E-stainedimages and histomorphometricanalysis of tumor burden in femurs onday 14 (�, P < 0.005). M, marrow; T,tumor; original magnifications, �40.D, representative images of TRAP-stained femur sections andquantification of osteoclast numberper bone surface (N. Oc/BS;�, P < 0.001); original magnifications,�100. E, histomorphometric analysisof bone volume per total tissuevolume (BV/TV) in femurs from eachgroup (�,P < 0.05). F, quantification ofpercent Ki67þ tumor cells in femurson day 14 (�, P < 0.05). G,quantification of microvessel numberper �200 field on CD31-stainedtumor burden on day 14.






macrophages mainly produce TNF-a in tumor microenviron-ment (34). TNF-awas shown to increase CXCL10production inbone marrow-derived mesenchymal stem cells (21). We alsoobserved that TNF-a increases CXCL10 expression in osteo-blasts and in macrophages (data not shown). These observa-tions suggest that macrophages can contribute to the aug-mented production of CXCL10 by bone metastasis via directinteraction with cancer cells as well as via TNF-a production.Our findings may thus provide new insight into the role ofmacrophages in bone metastasis.

The CXCL10 augmentation by bone metastasis raises thepossibility that CXCL10 might do much more than simplyrecruit CXCR3-expressing cancer cells to bone. Indeed, it hasbeen reported that when colon cancer cells are inoculated inthe rectum of nude mice, forced expression of CXCR3 oncancer cells does not affect the initial dissemination of cancer

cells to lymph nodes but facilitates expansion of cancer cells inlymph nodes (12). In line with these findings, we found that theaugmented production of CXCL10 is required for canceroutgrowth within the bone. It has been suggested that oste-oclastic bone resorption facilitates successful outgrowth ofbreast cancer cells in bonemarrow by providing growth factorssuch as transforming growth factor-b stored in bonematrix (3).However, it is worth noting that osteoclast-defective Src�/�

mice are resistant to melanoma-induced bone destruction butnot melanoma metastasis to bone after intracardiac injectionof B16F10 melanoma (35). Those findings suggest that func-tional osteoclasts or osteoclastic bone resorption is notrequired for cancer entry into and growth in bone marrow incase of B16F10 melanoma. Therefore, the reduced canceroutgrowth observed in Cxcl10�/� mice is unlikely because ofthe decreased bone destruction. The success of solid tumor

Figure 7. CXCL10 inducesosteoclast differentiation. A,BMMswere cultured in the presence ofM-CSF (30 ng/mL) and RANKL (100ng/mL) with indicated doses ofCXCL10 for 4 days. TRAP-positivemultinucleated cells containing 3ormore nuclei were counted asosteoclasts. B, CXCL10 dosedependently increasedosteoclastsformation in coculture ofBMCsandosteoblasts for 8 days (�, P < 0.05).C, relative mRNA expression levelsof RANKL and OPG in osteoblaststreated with or without CXCL10 for1 day (�, P < 0.05). D, quantificationof the protein levels of RANKL andOPG by ELISA (�, P < 0.05). E,mRNA expression levels of RANKL(left; �, P < 0.05) and CXCR3 (right;�, P < 0.01) in osteoblasts stablyexpressing shLuci or shCXCR3 inresponse to CXCR3 ligands (200ng/mL). F, relative RANKL mRNAlevels in osteoblasts (OB) from WTor Tlr4�/� mice in response toCXCR3 ligands (�, P < 0.001). G,CXCL10-induced osteoclastformation in co-culture of WTBMCs with WT or Tlr4�/�

osteoblasts (�, P < 0.01). H,CXCL10-induced activation ofERK and AKT in osteoblasts fromWT or Tlr4�/�mice. I, prevention ofCXCL10-induced RANKL mRNAexpression by PD98059 (PD, 10mmol/L) or AKT inhibitor VIII (AKT I,5 mmol/L; �, P < 0.001). DMSO,dimethyl sulfoxide.

Lee et al.





growth is dependent on angiogenesis (36), and CXCL10 hasbeen suggested to inhibit tumor angiogenesis (27, 28). How-ever, host-deficiency of CXCL10 did not affect tumor angio-genesis by intrafemoral injection of B16F10 melanoma. Giventhe importance of CXCR3 expression in metastatic outgrowthin lymph nodes (12), it is likely that augmented CXCL10facilitates cancer outgrowth by directly acting on cancer cells.It has been shown that bonemetastatic prostate cancer cells

preferentially adhere to type I collagen comparedwith prostatecancer cells that formonly visceralmetastases (24). In addition,selection for collagen adhesion confers outgrowth within thebone to prostate cancer (LNCaP) cells, which routinely fail tobind to collagen and do not have bone metastatic potential(24). Thus, our finding that CXCL10 stimulates adhesion ofcancer cells to ECM including type I collagen might provide apossible mechanism underlying CXCL10-induced cancer out-growth within the bone. Interestingly, it has been shown thatblockage of CXCR3 on cancer cells inhibits lung colonization ofmurine mammary cancer cells, which is compromised in micedepleted of NK cells or with mutation in IFN-g (11, 15). Theseprevious observations imply that CXCR3 activation conferscancer cells resistance to the antitumor effects of NK cells andIFN-g during cancer metastasis. Therefore, this might provideanother possible mechanism for CXCL10-induced cancer out-growth within the bone.In this study, host deficiency of CXCL10markedly decreased

bonemetastasis-induced osteoclastic bone destruction. Our invitro study revealed that CXCL10 stimulates osteoclast forma-tion in coculture of osteoblasts and BMCs by inducing RANKLexpression in osteoblasts, which is reminiscent of our previousobservations about the role of CXCL10 in osteoclast differen-tiation in coculture of CD4þ T cells and BMCs (18). Here weadditionally found that CXCL10 increases RANKL expressionthrough TLR4-mediated activation of ERK and AKT in osteo-

blasts. These results suggest that CXCL10 mediates bonemetastasis-induced osteoclastic bone destruction via stimu-lating RANKL expression.

In summary, our findings show that CXCL10 facilitatestrafficking of CXCR3-expressing cancer cells to bone, whichaugments its own production in bone and then promotescancer outgrowth and osteoclastic differentiation. Theseresults underscore the importance of CXCL10 in mediatingosteolytic bone metastasis and suggest that targeting CXCL10may provide a novel therapeutic opportunity for osteolyticbone metastasis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.-H. Lee, H. Ha, Z.H. LeeDevelopment of methodology: H. HaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-H. Lee, W.J. Jin, H. HaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-H. Lee, H.-N. Kim, K.-O. Kim, H. Ha, Z.H. LeeWriting, review, and/or revision of the manuscript: J.-H. Lee, K.-O. Kim, S.Lee, H.-H. Kim, H. Ha, Z.H. LeeAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.-O. Kim, H. HaStudy supervision: J.-H. Lee, H. Ha, Z.H. Lee

Grant SupportThisworkwas supported by theNational Research Foundation ofKorea (NRF,

Grant No. 2010-0015131) and a Science Research Center grant through the BoneMetabolism Research Center funded by the Korea government (MEST, Grant No.2011-0001023).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received February 7, 2012; revised April 16, 2012; accepted April 30, 2012;published OnlineFirst May 4, 2012.

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Lee et al.





2012;72:3175-3186. Published OnlineFirst May 4, 2012.Cancer Res Jong-Ho Lee, Ha-Neui Kim, Kyung-Ok Kim, et al. Cancer Outgrowth and OsteoclastogenesisCXCL10 Promotes Osteolytic Bone Metastasis by Enhancing

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CXCL10PromotesOsteolyticBoneMetastasisbyEnhancing …Corresponding Authors: Hyunil Ha, Traditional Korean Medicine-Based Herbal Drug Research Group, Korea Institute of Oriental Medicine,