The CXCL12 CXCR4 Chemokine Pathway: A Novel...

Cancer Therapy: Preclinical

The CXCL12–CXCR4 Chemokine Pathway: A Novel AxisRegulates Lymphangiogenesis

Wei Zhuo1,2,3, Lin Jia1,2,3, Nan Song1,2,3, Xin-an Lu1,2,3, Yanping Ding1,2,3, Xiaofeng Wang1,2,3,Xiaomin Song1,2,3, Yan Fu1,2,3, and Yongzhang Luo1,2,3

AbstractPurpose: Lymphangiogenesis, the growth of lymphatic vessels, contributes to lymphatic metastasis.

However, the precise mechanism underlying lymphangiogenesis remains poorly understood. This study

aimed to examine chemokine/chemokine receptors that directly contribute to chemoattraction of activated

lymphatic endothelial cells (LEC) and tumor lymphangiogenesis.

Experimental Design: We used quantitative RT-PCR to analyze specifically expressed chemokine

receptors in activated LECs upon stimulation of vascular endothelial growth factor-C (VEGF-C). Subse-

quently, we established in vitro and in vivo models to show lymphangiogenic functions of the chemokine

axis. Effects of targeting the chemokine axis on tumor lymphangiogenesis and lymphatic metastasis were

determined in an orthotopic breast cancer model.

Results: VEGF-C specifically upregulates CXCR4 expression on lymphangiogenic endothelial cells.

Moreover, hypoxia-inducible factor-1a (HIF-1a) mediates the CXCR4 expression induced by VEGF-C.

Subsequent analyses identify the ligandCXCL12 as a chemoattractant for LECs. CXCL12 inducesmigration,

tubule formation of LECs in vitro, and lymphangiogenesis in vivo. CXCL12 also stimulates the phosphor-

ylation of intracellular signaling Akt and Erk, and their specific antagonists impede CXCL12-induced

chemotaxis. In addition, its level is correlated with lymphatic vessel density in multiple cancer tissues

microarray. Furthermore, the CXCL12–CXCR4 axis is independent of the VEGFR-3 pathway in promoting

lymphangiogenesis. Intriguingly, combined treatment with anti-CXCL12 and anti-VEGF-C antibodies

results in additive inhibiting effects on tumor lymphangiogenesis and lymphatic metastasis.

Conclusions:These results show the role of theCXCL12–CXCR4 axis as a novel chemoattractant for LECs

in promoting lymphangiogenesis, and support the potential application of combined targeting of both

chemokines and lymphangiogenic factors in inhibiting lymphatic metastasis. Clin Cancer Res; 18(19);

5387–98. �2012 AACR.

IntroductionThe major cause of cancer mortality is metastasis that

occurs via multiple pathways including lymphatic vascula-ture. A considerable numberof studies has documented thatlymphangiogenesis, the growth of lymphatic vessels, con-tributes to lymphatic metastasis in experimental tumormodels and in clinic (1, 2), although it remains controver-sial in several types of human cancer (3, 4). Tumors canactively induce the growth of lymphatic vessels toward

tumor tissues. Studies over the past decade have identifiedseveral growth factors and receptors, particularly vascularendothelial growth factor-C (VEGF-C), VEGF-D, and theirreceptor vascular endothelial growth factor receptor-3(VEGFR-3) on lymphatic endothelial cells (LEC), contrib-uting to LEC growth, migration, and survival (5, 6).Nevertheless, the precise mechanism underlying lymphan-giogenesis is still a focus of intensive investigation.

The chemokine system, which is known to comprisemore than 40 chemokines and 18 chemokine receptors todate, correlates withmany biologic and pathologic process-es, for instance, inflammation, HIV-infection, angiogenesis,and tumor growth (7–9). Chemokines are smallmonomer-ic cytokines of 8 to15 kDa. On the basis of a cysteine motif,they have been classified into 4 subgroups: CXC, CC, C,and CX3C (10). Chemokines interact with 7 transmem-braneG-protein-coupled cell surface receptors andpromotechemotaxis, a process that induces the directional cellmigration toward a gradient of chemotactic cytokine(10). Though chemokine receptors were initially found onleukocytes, many nonhematopoietic cell types have been

Authors' Affiliations: 1National Engineering Laboratory for Anti-tumorProtein Therapeutics; 2Beijing Key Laboratory for Protein Therapeutics;and 3Cancer Biology Laboratory, School of Life Sciences, Tsinghua Uni-versity, Beijing, China

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Yongzhang Luo, School of Life Sciences, Tsin-ghuaUniversity, Beijing 100084, China. Phone: 86-10-6277-2897; Fax: 86-10-6279-4691; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-0708

�2012 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 5387

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Published OnlineFirst August 29, 2012; DOI: 10.1158/1078-0432.CCR-12-0708

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identified to express chemokine receptors. These chemo-kine/chemokine receptor interactions help coordinate celltrafficking and reorganization within various tissue com-partments (11). Previous studies have documented thatchemokines and their receptors play important roles intumor development and angiogenesis (9), among whichAugustin et al. (12) reported that blood endothelial cellsexpress chemokine receptors CXCR1-4. Considering theabundant levels of chemokines existing in the local tumorand their advantages indirecting cellmigration (10, 13), it isreasonable to hypothesize that LECs also use the chemo-kine-mediated mechanism during the process of lymphan-giogenesis, an event that requires directional migration ofLECs, such as those inducing angiogenesis or regulatingleukocyte trafficking.

In this study, we have analyzed specific chemokine pat-terns that contribute to the chemoattraction of LECs and thelymphangiogenesis process. We report here that mouseLECs (mLEC) express abundant amount of chemokinereceptor CXCR4. CXCL12 executes lymphangiogenic activ-ities via CXCR4. CXCL12, also known as stromal cell-derived factor-1 (SDF-1), is highly conserved with 99%homology between human and mouse, allowing it to actacross species barriers (14). The CXCL12–CXCR4 axis isessential for many biologic processes, including develop-ment, hematopoiesis, organogenesis, as well as vasculari-zation (15–18). Furthermore, CXCR4 is highly expressed ina variety of cancers (19). The interaction between CXCL12and CXCR4 plays a prominent role in tumorigenesis (20)and metastasis (19, 21).

We found that among all chemokine receptors expressedinmLECs, VEGF-C stimulationdramatically upregulates theexpression level of CXCR4on lymphangiogenic endothelialcells comparedwith quiescentmLECs. Subsequent analysessuggest that hypoxia-inducible factor-1a (HIF-1a) isresponsible for the upregulation of CXCR4 by VEGF-C. In

addition, chemokine CXCL12 induces migration, tubuleformation of mLECs, activates intracellular signaling ofAkt and Erk, and stimulates lymphangiogenesis in a Matri-gel plug assay in vivo, which can be impeded by blockingor knocking down CXCR4. Additionally, blocking theVEGFR-3 pathway has no effect on CXCL12-induced lym-phangiogenic activities. However, antibodies targeting bothCXCL12 and VEGF-C pathways show a more dramaticinhibitory effect on the tumor lymphangiogenesis andlymphatic metastasis inMDA-MB-231 breast cancermodel.Taken together, our study provides direct evidence that thechemokine system is involved in regulating lymphangio-genesis and shows that the CXCL12–CXCR4 chemotacticaxis plays a critical role in promoting lymphangiogenesis ina VEGFR-3 independent pattern.

Materials and MethodsCell lines, antibodies, and reagents

PrimarymLECswere isolated, characterized, and culturedin endothelial cell culture medium (ECM) as previouslydocumented (2). Human LECs (hLEC) were purchasedfrom Sciencell. B16/F10 and MDA-MB-231 cell lines werepurchased from the American Type Culture Collection.Enhanced green fluorescent protein (eGFP)-labeled MDA-MB-231 cell lines (MDA-MB-231/eGFP) were constructedusing the Kit from Genepharma according to the manufac-turer’s instructions. Hamster antimouse Podoplaninantibody, antibodies against Erk 1/2, phosphorylated Erk1/2 (p-Erk 1/2), Lamin B, and Actin were from SantaCruz Biotechnology. Rat antihuman Podoplanin antibodywas from Biolegend. Akt antibody was from BioworldTechnology. P-Akt antibody was from Cell Signaling Tech-nology. CXCR4 antibody was from Abcam. TRITC- andFITC-conjuncted secondary antibodies were from SantaCruz Biotechnology. Anti-CXCR4, anti-VEGFR-3, anti-CXCL12, anti-VEGF-C-blocking antibodies were fromBioss. AMD3100, U0126, and LY294002 were from Sig-ma-Aldrich. VEGF-C and CXCL12 were from R&D Systems.

Quantitative RT-PCRCultured mLECs were starved overnight, and then stim-

ulated with or without 100 ng/mL of VEGF-C for 24 hours.The total RNA from cultured mLEC was isolated usingTRIZOL Reagent (Invitrogen), and converted into cDNAusing the First Strand cDNA Synthesis Kit (Fermentas).Quantitative RT-PCR (qRT-PCR) was conducted using theBrilliant II SYBRGreenqRT-PCRMasterMixKit (Stratagene)with the standard PCR conditions applied in this study (40cycles). All primers for chemokine receptors were listed insupplementary Table S1.Relative quantitationwas analyzedusing the DDCt method. Glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) was used as an internal control. Inde-pendent experiments were repeated in triplicates.

Flow cytometryFor detection of the cell surface expression level of

CXCR4, mLECs were incubated with CXCR4 antibody

Translational RelevanceThe Chemokine/chemokine receptor has been impli-

cated in tumor growth, metastasis, and angiogenesis.This study, for the first time, points out that the chemo-kine/chemokine receptor system is directly involved inpromoting tumor lymphangiogenesis.Wehave screenedand showed that the CXCL12–CXCR4 axis is a potentpositive-regulator of lymphangiogenesis by directly act-ing on lymphangiogenic endothelial cells in a vascularendothelial growth factor (VEGF) receptor-3 indepen-dent pattern. Targeting both CXCL12 and VEGF-C path-ways results in a more dramatic inhibitory effect ontumor lymphangiogenesis and lymphatic metastasis ina breast cancermodel.Our present studynot only revealsa novel mechanism of tumor lymphangiogenesis, butalso provides a novel route to inhibit lymph nodemetastasis via targeting both chemokines and lymphan-giogenic growth factors.

Zhuo et al.

Clin Cancer Res; 18(19) October 1, 2012 Clinical Cancer Research5388




(Abcam), or rabbit IgG as an isotype control, and subjectedto the standard protocol of cytometry. The data were ana-lyzed using a FACS Calibur flow cytometry system (BectonDickinson).

Chemotaxis assayThe migration efficiency of mLECs was assessed using

8-mm-pore Transwell filter membrane (Costar) as previous-ly described (2). Migrated cells were quantified by countingin 8 fields under an Olympus IX71 optical microscope(Olympus). Experiments were conducted in triplicate andrepeated twice.

Tubule formation assayThe tubule formation assay was conducted as previously

described (2). Tubule structures were imaged by the Olym-pus microscope and quantified by measuring the length ofcords in 6 randomly viewed fields. The tubule length wasdefined as previously described (22). Experiments wereconducted in triplicate and repeated twice.

RNA interferenceThe siRNA for HIF-1a and siRNA (1#) for CXCR4 were

purchased from Santa Cruz Biotechnology. The siRNA forAkt isoforms (Akt1, Akt2, Akt3) were purchased from Gen-ePharma. Scrambled siRNA was purchased from Gene-Pharma (Shanghai, China). The sequence of ineffectivesiRNA (2#) for CXCR4 is 50-gagacuaugacuccaacaatt-30. Thesequence of siRNA for Akt1 is 50-gcaccuuuauuggcuacaaggtt-30, siRNA for Akt2 is 50-aagaguggaugcgggcuaucctt-30, andsiRNA for Akt3 is 50-aaggaugaaguggcacacacutt-30. The siR-NAs were transfected with Lipofectamine 2000 reagent(Invitrogen) according to themanufacturer’s protocol. After72-hour transfection, knock-down efficiency was detectedby Western blot analysis.

Immunoblotting analysisCells were harvested, denatured, and subjected to SDS-

PAGE. Proteins were transferred to polyvinylidene difluor-ide (PVDF) membrane, immunolabeled with appropriatedprimary antibodies overnight at 4�C, incubated with horse-radish peroxidase-conjugated secondary antibodies for 1hour at room temperature, then detected with an enhancedchemiluminescence system (Roche) according to the man-ufacturer’s protocol.

ImmunofluorescenceProliferating mLECs were cultured on coverslips in 12-

well plates in serum-containing ECM. After serum-starvedovernight, the cells were treated with or without 100 ng/mLof VEGF-C for 24 hours. The cells were further fixed by 4%paraformaldehyde. According to the previous study (23),Podoplanin andCXCR4were immunostained and detectedbyNikonA1 laser scanning confocalmicroscope using 60X/1.49 Oil DIC objectives.For the immunofluorescence of tissue samples, fluores-

cence images were detected by Nikon A1 laser scanningconfocal microscope using Plan Apo 20X/0.75 objectives.

Images were captured and analyzed with Nikon imagesoftware (NIS-Elements AR 3.0).

Matrigel plug assayLymphatic vessel formation in vivo was evaluated by the

Matrigel plug assay. Briefly, 0.5 mL of Matrigel (Becton-Dickinson Labware) containing CXCL12, VEGF-C (500 ng/mL), antibodies (10 mg/mL), or AMD3100 (a CXCR4 antag-onist that can block CXCL12 signaling, 50 mg/mL) at indi-cated concentrations was injected subcutaneously into theabdominalmidline of BALB/cmouse (female, 5weeks old, 5per group).After8days, plugsweredissectedand subjected tothe immunofluorescent analysis. Evaluationof the lymphaticvessel densitywas assessed in5 independent fields imagedbythe Nikon A1 laser scanning confocal microscope usingNikon image software (NIS-Elements AR 3.0).

Tissue microarrayMultitumor tissue microarray was purchased from Xi’an

Aomei (Aomei) that contained54 clinical specimens (medi-an age 55.6 years, range 15–81,male and femalewere about31–23, including brain glioma, esophagus, stomach, liver,colon, rectum, lung, bladder, heart, kidney, thyroid, pan-creas, cervix, skin, breast, ovarian, prostate, and testis, 3specimens for each). Tissuemicroarraywas immunostainedwith antihuman Podoplanin (Biolegend) and CXCL12(Bioss) antibodies according to the protocol of immuno-fluorescent staining.

Orthotopic breast cancer modelAll animal studies were approved by the Institutional

Animal Care and Use Committee of Tsinghua University.ConstructedMDA-MB-231/eGFP cells (3� 106 in 100 mL ofMatrigel solution) were inoculated into the mammary fatpad of nude mice (female, 6–8 weeks). The mice weredivided randomly into4 groups (6mice per group). Controlrabbit IgG (2 mg/kg), rabbit anti-CXCL12 antibody (2 mg/kg), rabbit anti-VEGF-C antibody (2 mg/kg), or anti-CXCL12 antibody (1 mg/kg) plus anti-VEGF-C antibody(1 mg/kg) were administered intraperitoneally into themice every other day, respectively. After 3 weeks, primarytumor, axillary lymph nodes were dissected, photographed,and applied to immunofluorescent staining. MetastasizedMDA-MB-231/eGFP cells in lymph node were detected byNikon A1 laser scanning confocal microscope. Area ofeGFP-positive signals in lymph nodes was assessed in atleast 6 independent fields in different sections using Nikonimage software (NIS-Elements AR 3.0).

Statistical analysisData are presented as mean. Statistical analyses were

assessed by a 2-tailed Student’s t-test.

ResultsVEGF-C upregulates chemokine receptor CXCR4 inlymphangiogenic endothelial cells via HIF-1a in vitro

To determine whether the chemokine attractant/chemokine receptor interaction participates in the

Chemokine Axis Directly Regulates Tumor Lymphangiogenesis

www.aacrjournals.org Clin Cancer Res; 18(19) October 1, 2012 5389




lymphangiogenesis process, we systematically analyzedthe mRNA levels of well-known chemokine receptors(CCR1–CCR10, CXCR1–CXCR7, and CX3CR1) in isolat-ed primary mLECs. PCR results showed that primarymLECs prominently express chemokine receptors CCR5,CCR9, CXCR4, CXCR6, and CXCR7, however, weaklyexpress chemokine receptors CCR4, CCR6, CCR8,CCR10, CXCR3, and CX3CR1 (Supplementary Fig. S1).VEGF-C, which is secreted by tumor cells and mast cells intumor microenvironment, has been reported to be apredominant pro-lymphangiogenesis factor (5). We won-dered whether VEGF-C stimulation can regulate theexpression of chemokine receptors on LECs. Intriguingly,qRT-PCR showed that only CXCR4 mRNA level wasdramatically increased upon VEGF-C treatment, amongthose chemokine receptors (CCR4-6, CCR8-10, CXCR3,CXCR4, CXCR6, CXCR7, and CX3CR1) expressed onmLECs (Fig. 1A), indicating that LECs selectively expresscertain chemokine receptors, and VEGF-C stimulationcan specifically upregulate CXCR4 expression in lym-phangiogenic endothelial cells.

To verify this result, cultured mLECs were starved ofendothelial cell growth supplement overnight, and thenstimulated by VEGF-C, followed by flow cytometry analy-ses. As expected, the level of cell surface CXCR4 protein wasmuch higher after 24-hour culture with VEGF-C (Fig. 1B).Consistent observation was obtained from immunofluo-rescence assay showing that CXCR4 expression is associatedwith the proliferative status of mLECs and hLECs (Supple-mentary Fig. S2A). The regulation of CXCR4 surface expres-sion was also analyzed by immunoblotting. VEGF-C coulddramatically upregulateCXCR4 expression, as shown in Fig.1C. Because CXCR4 is one of the target genes of HIF-1a(24), we hypothesized that VEGF-C upregulates CXCR4 viaHIF-1a. Indeed, VEGF-C treatment increased the expressionlevel of HIF-1a (Fig. 1C). HIF-1a accumulation in mLECsinduced by cobalt chloride treatment and hypoxia exposureincreased the expression of CXCR4 (Supplementary Fig.S2B). When HIF-1a was knocked down in mLECs bytransfection of HIF-1a siRNA, VEGF-C failed to increasethe expression of CXCR4 in comparison with scrambledsiRNA (Fig. 1D). Collectively, these results provide com-pelling evidence that VEGF-C is a positive-regulator ofCXCR4 in LECs, in which HIF-1a is involved.

CXCR4 colocalizes with lymphangiogenic vesselsin vivo

Wewere therefore prompted to explore the expression ofCXCR4 on lymphatic vessels in vivo. Stained tissue sectionsfromMatrigel plugs, dissected 8days after injected intomicesubcutaneously, revealed that VEGF-C-induced lymphan-giogenic vessels express higher levels of CXCR4, as com-pared with Matrigels without VEGF-C (Fig. 2A). Then wefurther investigated the distribution of CXCR4 on lymphat-ic vessels in both normal tissues and tumor tissues. Inves-tigationonmousenormal intestine, colon, and lymphnodeconfirmed that matured lymphatic vessels do not expressdetectable level of CXCR4. In comparison, strong expres-

sion of CXCR4 was observed in melanoma tissues and inlymph nodes from tumor-bearing mice (Fig. 2B). We alsoobserved high expression of CXCR4 on lymphatic vessels inhuman rectal cancer, colon cancer, and skin squamous cellcarcinoma (Supplementary Fig. S2C). These immunofluo-rescence studies in vivo confirmed that CXCR4 expression isupregulated on lymphangiogenic vessels.

CXCL12 promotes lymphangiogenesisBecause CXCL12 is one of the major chemokines corre-

sponding to CXCR4 (14), we speculated that CXCL12 hasthe potential to attract CXCR4-positive lymphangiogenicendothelial cells. In a chemotaxis assay,mLECswere seeded

Figure 1. VEGF-C upregulates chemokine receptor CXCR4 inlymphangiogenic endothelial cells via HIF-1a in vitro. A, primary mLECswere starved overnight, and then stimulatedwith or without 100 ng/mL ofVEGF-C for 24 hours. qRT-PCR was conducted to analyze theexpression of chemokine receptors in VEGF-C-stimulated mLECs, ascomparedwith untreated quiescent cells. ThemRNA levels of chemokinereceptors were normalized by GAPDH mRNA level. B, cultured mLECswere serum-starvedovernight, then culturedwithorwithout 100ng/mLofVEGF-C for 24 hours. Expression level of CXCR4 on serum-starved cellsand VEGF-C-rescued cells was detected by Flow cytometry. MFI, meanfluorescence intensity; N.C., isotype control IgG. C, CXCR4 and HIF-1aexpression in mLECs exposed to 100 ng/mL of VEGF-C at the indicatedtimes. The Western blot analysis was conducted using lamin B as aloading control. D, mLECs transfected with scrambled siRNA (N.C.) orHIF-1a siRNA were treated with or without 100 ng/mL of VEGF-C.Expression levels of CXCR4 and HIF-1a were detected by Western blotanalysis using actin as a loading control. The experiments wereconducted in 3 independent series.

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in the upper chamber of a Transwell insert, andCXCL12wasadded in the lower chamber. The results showed thatCXCL12 induced a dose-dependent increase in cell migra-tion (Fig. 3A). We also observed that CXCL12 promotes themigration of hLECs, which could be blocked by AMD3100,a well-known antagonist of CXCR4 (Supplementary Fig.S3). In addition, CXCL12 also had an effect on mLECstubule formation in a dose-dependent manner, as assessedby measuring the length of cords formed by mLECs (Fig.3B). To further confirm this result, we conducted a Matrigelplug assay in vivo. Plugs supplemented with different dosesofCXCL12were injected intomice subcutaneously. VEGF-Cserved as a positive control. In agreement with the findingsin vitro, Matrigels with CXCL12 showed significantlyenhanced lymphatic vessel formation with a tubule-likestructure compared with control group (Fig. 3C). To test

the relationship between lymphatic vessel density andCXCL12 level in human cancers, expression levels ofCXCL12 and Podoplanin, a specific marker for lymphaticendothelium, were examined in a multitumor tissuemicroarray containing a total of 54 clinical tissue samples.Immunostaining analyses revealed that the density oftumor-associated lymphatic vessels was positively correlat-ed with the expression level of CXCL12 in 50 samplesamong 54 specimens detected (Fig. 3D and SupplementaryTable S2). Taken together, these results show thatCXCL12 isa positive regulator of lymphangiogenesis.

CXCL12 activates intracellular signaling pathways ofmLECs

Stimulation of mLECs motility by CXCL12 suggests thatthis chemokine can activate intracellular signaling path-ways. Previous studies have documented a direct involve-ment of PI3K/Akt and Erk signaling pathway in cell migra-tion mediated by the CXCL12–CXCR4 axis (25, 26). Asexpected, increased phosphorylation of Erk 1/2 andAktwasdetected in mLECs upon CXCL12 stimulation (Fig. 4A).When CXCR4 was blocked by its neutralizing antibody, thephosphorylation of Erk 1/2 and Akt in response to CXCL12was significantly decreased, as compared with control IgG(Fig. 4B). Similarly, knocking down of CXCR4 using effec-tive siRNA eliminated the effect of CXCL12 on activation ofErk 1/2 comparing to scramble siRNA or ineffective CXCR4siRNA (Fig. 4C). To evaluate the contributions of the Aktand/or Erk 1/2 pathways to CXCL12-induced chemotaxis,the inhibitors of PI3k/Akt (LY294002) or Erk 1/2 (U0126)were used to pretreatmLECs,whichdramatically attenuatedthe migrated mLEC number induced by CXCL12 (Fig. 4D).Because Akt has 3 different isoforms and mediates distinctfunctions, we thus used siRNAs to knock down Akt1, Akt2,and Akt3, respectively. Chemotaxis assay revealed thatknocking down Akt1 dramatically inhibits the function ofCXCL12, whereas either Akt2 siRNA or Akt3 siRNA hasslight effect, showing that Akt1, other than Akt2 or Akt3,plays a principal role in the CXCL12-induced migration ofmLECs (Supplementary Fig. S4). In summary, these resultsshow that activation of Akt1 and Erk 1/2 signaling compo-nents is essential for the chemotactic activity of CXCL12 onlymphangiogenic endothelial cells.

The CXCL12–CXCR4 pathway is independent of theVEGF-C/VEGFR-3 pathway in promotinglymphangiogenesis

The above-mentioned evidences showed that CXCL12 isa chemoattractant for lymphangiogenic endothelial cells.However, whether CXCL12 stimulates lymphangiogenesisdirectly via CXCR4 or indirectly via other pathways is stillunknown. Among the known prolymphangiogenic factors,VEGF-C and -D are the most specific and potent growthfactors which directly bind to VEGFR-3 on LECs (5, 6). Inaddition, it was also reported that the abilities of basicfibroblast growth factor (bFGF), VEGF-A, and hepatocytegrowth factor to induce lymphangiogenesis are mediated atleast in part via the VEGF-C/VEGF-D/VEGFR-3 pathway

Figure 2. CXCR4 colocalizes with lymphangiogenic vessels in vivo. A,detection of CXCR4 on newly formed lymphatic vessels in aMatrigel plugassay. Matrigel mixedwith or without 500 ng/mL of VEGF-Cwas injectedsubcutaneously into BALB/c mouse and dissected after 8 days.Immunofluorescent detection of lymphatic vessels (red) and CXCR4(green) was conducted. B, immunostaining of lymphatic vessels (red) andCXCR4 (green) in the intestines, colons, and lymph nodes from normaladult mice (n ¼ 3), melanoma tissue (B16/F10), and lymph nodes fromtumor-bearing mice (n ¼ 5). Images were observed by Nikon A1 laserscanning confocal microscope using Plan Apo 20X/0.75 objectives. A,scale bar, 50 mm; B, scale bar, 20 mm. Data are representative of3 independent series.






(27–29). To investigate whether CXCL12 may employ thesimilar pathway to execute its prolymphangiogenic func-tions, we treated mLECs with CXCL12 in the presence orabsence of VEGFR-3-neutralizing antibody. Surprisingly,incubation of mLECs with VEGFR-3-neutralizing antibodydid not affect CXCL12-induced mLECs migration, whereasVEGF-C-induced mLECsmigration was dramatically inhib-ited (Fig. 5A). Therefore, we detected the effect of inhibitingCXCL12–CXCR4 pathway on the function of VEGF-C/VEGFR-3 pathway. When we used AMD3100 or CXCR4-neutralizing antibody to treat mLECs in a chemotaxis assay,the CXCL12-mediated migration of mLECs was effectivelyblocked, but did not exert any significant inhibitory effectagainst VEGF-C-induced cell migration (Fig. 5B). In agree-ment with the in vitro results, Matrigel plug assay in vivofurther confirmed that blocking CXCR4 by CXCR4-neutral-izing antibody or AMD3100 inhibited the function of

CXCL12 without affecting the VEGF-C activity. Moreover,blocking VEGFR-3 dramatically inhibited the function ofVEGF-C, whereas it did not have any effect on CXCL12-induced lymphangiogenesis (Fig. 5C). Taken together,these results show that the CXCL12–CXCR4 pathway isindependent of the VEGF-C/VEGFR-3 pathway in LECchemoattraction and lymphangiogenesis. This conclusionmeans that in addition to the VEGF-C/VEGFR-3 pathway,we discovered a novel CXCL12–CXCR4 pathway regulatinglymphangiogenesis.

CXCL12 and VEGF-C pathways have additive effects inpromoting tumor lymphangiogenesis and metastasis

On the basis of our findings, it is naturally to speculatethat simultaneous inhibition of CXCL12–CXCR4 andVEGF-C/VEGFR-3 pathways should result in more potentblockade of tumor lymphangiogenesis and lymphatic

Figure 3. CXCL12 promotes lymphangiogenesis. A, chemotactic migration of mLECs toward CXCL12 in the Transwell assay. mLECs were seeded toeach upperwell with freshmedium.CXCL12 at the indicated concentrationswas added to the lower chamber. VEGF-Cwas used as a positive control.mLECswere allowed to migrate for 6 hours. Cell chemoattraction was assessed by counting the number of migrated mLECs. B, histogram showing quantitationof tubule formation ability of mLECs. Cells were cultured on the Matrigel-coated 24-well plates with different dosage of CXCL12 or 100 ng/mLof VEGF-C, followed by incubation for 6 hours. Tubule structure was quantified by measuring the length of cords. C, representative photomicrographsshowing CXCL12-induced lymphatic vessel formation in a Matrigel plug assay. Matrigels containing indicated reagents were injected subcutaneously intoabdominal midline of mice. After 8 days, plugs were dissected and applied to immunofluorescent analysis for lymphatic vessel density. Podoplanin (red)represents lymphatic vessels (top). Quantified results were shown (bottom; n ¼ 5; 2 independent series). D, expression levels of CXCL12 (green)and the density of lymphatic vessels (red) in humanmultiple organ cancer tissues microarray. Tissue microarray containing 54 clinical tissue specimens fromdifferent organs was applied for immuno-staining. Micrographs were taken by Nikon A1 laser scanning confocal microscope using Plan Apo 20X/0.75objectives. We analyzed the intensity of positive signals of CXCL12 and podoplanin in independent fields from each specimen using Nikon imagesoftware (NIS-Elements AR 3.0). Specimens were subsequently classified into 4 groups: high expression level of CXCL12 with high density of lymphaticvessels (LV), low expression level of CXCL12 with low density of LV, high expression level of CXCL12 with low density of LV, and low expression level ofCXCL12with highdensity of LV.Representativemicrographsof the 4groupswere shown in the top. Thenumber of specimens indifferent groupswas countedand presented in the bottom. C and D, scale bar, 50 mm. ��, P < 0.01; ��, P < 0.001.

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metastasis. First, chemotaxis assay in vitro (SupplementaryFig. S5) andMatrigel plug assay in vivo (Fig. 6A) showed thatCXCL12 and VEGF-C have additive effect on promotingmigration ofmLECs. Second, in an orthotopic breast cancermodel in immunodeficient mice, eGFP-labeled humanbreast carcinoma cell line MDA-MB-231 (MDA-MB-231/eGFP) was injected orthotopically into the mammary fatpad of nude mice. Intraperitoneally treatment every otherday with either CXCL12-neutralizing antibody, VEGF-C-neutralizing antibody, or together for 3 weeks inhibited theprimary tumor growth (Supplementary Fig. S6). A signifi-cant decrease in lymphatic vessel density in primary tumortissue was observed in either anti-CXCL12- or anti-VEGF-C-treated mice (Fig. 6B). Furthermore, combination of bothanti-CXCL12 and anti-VEGF-C blocking antibodies showedan evenmore efficient inhibition in tumor lymphangiogen-esis than each antibody alone (Fig. 6B). Because tumorlymphangiogenesis actively contributes to cancer dissemi-nation (30), and because it was reported that CXCL12 isassociated with lymph node metastasis (20, 21), thus axil-

lary lymph nodes were dissected from tumor-bearing mice.Our results showed that treatment with both anti-CXCL12and anti-VEGF-C antibodies led to a significant reduction inlymph node metastasis (Fig. 6C). As MDA-MB-231 cell linewas labeled with eGFP, disseminated tumor cells werefurther analyzed by confocal fluorescence microscopy (Fig.6D). Quantified results showed that blockade of CXCL12dramatically impedes the tumor cell metastasis to the lym-phatic system, and targeting both CXCL12 and VEGF-Cpathways shows an additive effect on prevention of lym-phatic metastasis (Fig. 6D).

DiscussionChemokines in tumor-lymphatic microenvironment

Chemokines and their receptors have been documentedin tumor cell growth, angiogenesis and metastasis (9, 31).However, few studies have explored the chemokine systemin the tumor-lymphatic microenvironment. Tumor cells,when expressing receptors for lymphatic-derived chemo-kines, may gain access to lymphatic vessels via chemoat-traction. Kim and coworkers (32) reported that LECs pro-mote the migration of CXCR4-positive tumor cells bysecretion of CXCL12. Shields and coworkers (33) reportedthat tumor cells utilize interstitial flow and CCR7 signalingto access lymphatics. They found that VEGF-C expressed bytumor cells induces upregulation of chemokine CCL21, aligand of CCR7, by lymphatic vessels, which in turn guidesthe migration of tumor cells toward lymphatics (34). Here-in, our study provides strong evidence that VEGF-C stim-ulation can also induce upregulation of chemokine receptorCXCR4, not only just chemokines such as CCL21, on LECs,and the chemokine/chemokine receptor interaction pro-motes lymphangiogenesis by directly acting on LECchemotaxis.

The lymphatic vasculature is a network that transportsinterstitial fluid from tissues to the blood. Previousstudies have shown that lymphangiogenesis, growth oflymphatic vessels in the solid tumors, correlates withlymphatic metastasis. However, the exact mechanism forlymphangiogenesis remains unclear. One of the majordirections related to lymphangiogenesis is how LECsbeing regulated. Both Alitalo group (35) and Detmargroup (36) have investigated the differences of geneprofile between LECs and blood endothelial cells. Theirresults indicate that LECs express a large number ofdistinct genes involved in multiple endothelial cell func-tions such as inflammatory processes and cell–cell inter-actions. In addition, tumor-related LECs have also beenshowed having distinct profile from that of normal LECs(37). Given the critical role of the chemokine system inguiding cells migration, as the foregoing analyses, wefocus on the chemokine system and have identified sev-eral chemokine receptors expressed on LECs. The presentstudy highlights the variation of gene profile, chemokinereceptors in particular, in LECs in response to the stim-ulation of VEGF-C. It is possible that lymphangiogenicfactors can activate the lymphatic endothelium to express

Figure 4. CXCL12 activates intracellular signaling pathways of mLECs. A,CXCL12 induces the phosphorylation of Akt and Erk. mLECs werestarved in serum-free medium for 12 hours, incubated with 100 ng/mL ofCXCL12, andharvested at different timepoints. Equal amounts ofmLECslysates were analyzed by Western blot analysis and probed withantibodies against Akt, Erk, and active forms of Akt, Erk. Data arerepresentative of 3 independent experiments. B, Western blot analysisdetection of the CXCL12-induced phosphorylation of Akt and Erk in thepresence or absence of CXCR4-neutralizing antibody (5 mg/mL). Cellswere pretreated with blocking antibodies for 30 minutes, then incubatedwith CXCL12 (100 ng/mL) for 10 minutes. Isotype rabbit IgG was used asa control. C, in the presence or absence of 100 ng/mLofCXCL12,mLECstransfected with 2 CXCR4 siRNA (1#, 2#), or scrambled siRNA (N.C.),respectively, were incubated for 10 minutes. CXCR4 siRNA (1#) iseffective siRNA. CXCR4 siRNA (2#) is ineffective siRNA. Activation of Erkwas detected by Western blot analysis (bottom). The expression ofCXCR4 was shown (top). D, in the presence or absence of 100 ng/mL ofCXCL12, freshmediumcontaining dimethyl sulfoxide (DMSO), U0126 (aninhibitor of Erk signaling, 10 mmol/L), or LY294002 (an inhibitor of Aktsignaling, 10 mmol/L) was added to the lower chambers, respectively.mLECs were seeded into the upper wells. After 6 hours, migrated cellswere quantified (n ¼ 8). ��, P < 0.001.






increased amounts of chemotactic factors and receptors,which contribute to the migration of LECs toward tumortissues and LEC-tumor cell interactions. Indeed, we haveshown that, among all chemokine receptors expressed inLECs, VEGF-C specifically upregulates CXCR4. Becauseother chemokine receptors, such as CCR5, CCR9, CXCR6,and CXCR7, are also expressed by LECs, we can notexclude the possibility that other growth factors orinflammatory factors may regulate the expression of thosechemokine receptors, which further mediate the che-moattraction of activated LECs. Therefore, exploringthe role of the chemokine system on the properties oflymphangiogenic endothelial cells and the lymphangio-genesis process will be of considerable interest in theimmediate future.

The CXCL12–CXCR7 axis in lymphangiogenesisIt has been reported that tumor cells and carcinoma-

associated fibroblasts express high levels of CXCL12(13, 38), one of the major chemokines corresponding toCXCR4. Here, we emphasize that the CXCL12–CXCR4 axisplays a pivotal role in promoting the chemoattraction ofLECs. Additionally, we found that LECs also express highlevel of CXCR7, which has been reported to be a novelreceptor for CXCL12 by Bachelerie group (39) and Schallgroup (40), respectively. However, unlike CXCR4, CXCL12activation of CXCR7 results in a proliferative effect but notcell migration. Further, CXCR7 expression usually endorsestumor cells with a growth and survival advantage andincreased adhesion properties (40, 41). In this study,VEGF-C stimulation exclusively upregulates CXCR4 but not

Figure 5. The CXCL12–CXCR4 axisis independent of the VEGF-C/VEGFR-3 pathway in promotinglymphangiogenesis. A,chemotactic assay was carried outto evaluate the effect of VEGFR-3-neutralizing antibody on theCXCL12-induced cell migration.CXCL12 (100 ng/mL) or VEGF-C(100 ng/mL) was added alone, ortogether with VEGFR-3-neutralizing antibody (5 mg/mL) orcontrol IgG (5mg/mL). After 6 hours,migrated cells were quantified(n¼8). B, in the chemotactic assay,CXCL12 (100 ng/mL) or VEGF-C(100 ng/mL) was added alone, ortogether with AMD3100 (a CXCR4antagonist that can block CXCL12signaling, 25 mg/mL), VEGFR-3-neutralizing antibody (5 mg/mL), orcontrol IgG (5 mg/mL), respectively.After 6 hours, migrated cells wereexamined (top) and quantified(bottom; n ¼ 8). C, Matrigel plugassay was conducted to analyzethe CXCL12- or VEGF-C-inducedlymphangiogenesis in thepresence of indicated reagents.Matrigels containing CXCL12 (500ng/mL) or VEGF-C (500 ng/mL)alone, or together with CXCR4-neutralizing antibody (10 mg/mL),VEGFR-3-neutralizing antibody(10 mg/mL), control IgG (10 mg/mL),or AMD3100 (50 mg/mL) weresubcutaneously injected intoBALB/c mice. After 8 days, plugswere dissected and subjected toimmunostaining. Podoplanin (red)represents lymphatic vessels.Newly formed lymphatic vesselswere presented (top) andquantified (bottom; n ¼ 8). B, scalebar, 100 mm; C, scale bar, 50 mm.��, P < 0.01; ��, P < 0.001.

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CXCR7 on LECs. Thus, we focus on the role of the CXCL12–CXCR4 axis in the chemoattraction of LECs. Nevertheless,the function of CXCR7 on LECs remains to be furtherexplored. As the expression of CXCR7 can be upregulatedon the activated blood endothelial cells after stimulationwith the cytokines TNF-a and IL-1b (40), it is possible thatlymphangiogenic endothelial cells express high levels ofCXCR7 in the tumor microenvironment, which in turnsupports lymphangiogenic endothelial cells growth andlymphangiogenesis.

CXCR4 expression and its role in lymphatic metastasisAnother major concern is the regulation mechanism of

CXCR4 expression in LECs. Many studies have stated thatCXCR4 is expressed in a variety of tumors, supporting its rolein cell survival, proliferation, adhesion, and migration (19,20). In addition, CXCR4 also localizes on vascular endothe-lial cells and mediates the angiogenic activity of CXCL12(42). It has been described that angiogenic factors VEGF andbFGF can upregulate the expression of CXCR4 on vascular

endothelial cells (12, 43). Consistently, our result showsinducible expression of CXCR4 in cultured mLECs byVEGF-C, whereas the expression level of CXCR4 in maturedlymphatic vessels is much lower. Though we only chooseVEGF-C as the stimulator, it is still possible that activation ofthe lymphatic system by other lymphangiogenic factors likeVEGF-A, platelet-derived growth factor BB (PDGF-BB), hepa-tocyte growth factor,mayalso increase theCXCR4expressionon LECs. Our group (44) has reported that PDGF-BB canstimulate the CXCL12–CXCR4 axis during tumor angiogen-esis. Moreover, we have also observed that CXCL12 stimu-lation led to an upregulation of CXCR4 on LECs (Data notshown), indicating the possibility that CXCR4 expression is,at least in part, regulated under an autocrine manner. Thisautocrine fashion might explain the fact that CXCL12 alonecan also induce considerable number of newly formed lym-phatic vessels in aMatrigel plug assay.On theother hand, it isknown that HIF-1a, a central mediator of hypoxia, caninduce a significant increase in the expression of CXCR4 onnormal andmalignant cells through the vonHippel–Lindau-

Figure 6. CXCL12 and VEGF-Cpathways have additive effects in promoting tumor lymphangiogenesis andmetastasis. A, Matrigel plug assaywas conductedto analyze the additive effect of CXCL12 and VEGF-C in lymphangiogenesis. Matrigels containing CXCL12 (500 ng/mL) or VEGF-C (500 ng/mL)alone or together were subcutaneously injected into BALB/c mice. After 8 days, plugs were dissected and subjected to immunostaining of podoplanin.Micrographs were taken byNikon A1 laser scanning confocal microscope. Newly formed lymphatic vessels in plugs were evaluated by Nikon image software(NIS-Elements AR 3.0; n¼ 8). B, the effects of anti-CXCL12 and anti-VEGF-C blocking antibodies on tumor lymphangiogenesis in the orthotopic MDA-MB-231/eGFP human breast cancer model. Constructed MDA-MB-231/eGFP cells were implanted into the mammary fat pad of nude mice. Mice wereadministrated intraperitoneally with control IgG (2mg/kg), CXCL12-neutralizing antibody (2mg/Kg), VEGF-C-neutralizing antibody (2mg/kg), or anti-CXCL12antibody (1mg/kg) plus anti-VEGF-Cantibody (1mg/kg), respectively. Threeweeks after the first injection, the lymphatic vessel density in primary tumor tissue(mammary fat pad) was analyzed and evaluated by Nikon image software (n ¼ 6). C, axillary lymph nodes were removed from MDA-MB-231/eGFP tumor-bearing BALB/c mice. Dissected lymph nodes were photographed. D, dissected lymph nodes in the MDA-MB-231/eGFP tumor model were applied foranalysis of the disseminated tumor cells. Images were observed by Nikon A1 laser scanning confocal microscope using Plan Apo 20X/0.75 objectives (left).Blue,DAPI; green,MDA-MB-231/eGFP (231/eGFP). The signals of 231/eGFPcells in lymphnodes from tumor-bearingmicewere quantified (right;n¼6 lymphnodes). A, scale bar, 100 mm; C, scale bar, 2 cm; D, scale bar, 50 mm, magnified picture (20 mm). ��, P < 0.01; ��, P < 0.001.






HIF-1a pathway (24). In our study, both cobalt chloridetreatment and hypoxia exposure increase the expression ofCXCR4 on LECs. It is possible that the CXCL12–CXCR4 axisis related to lymphangiogenesis under tissue repair of hyp-oxic–ischemic injury. Therefore, the regulation of CXCR4expression in LECs is sophisticated in that it might beregulated in both a paracrine and an autocrine manner,involving lymphangiogenic factors and/or hypoxia factorsunder different pathologic conditions.

Initial studies characterizedCXCL12 as a pre-B cell survivalfactor (45). Considerable clinical-pathologic studies haveshowed thatCXCL12andCXCR4expressionare significantlyassociated with lymph node metastasis (20, 21). However,the mechanism that how the CXCL12–CXCR4 axis regulateslymphatic metastasis remains unclear. CXCR4-positivetumor cells may metastasize to areas with high CXCL12expression (46).Hirakawaand coworkers (47) have reportedthat tumor associated LECs express CXCL12 and attractinvasive Paget cells via the CXCL12–CXCR4 axis. In turn,the present study shows that functionally activated LECs alsoexpress increased level of CXCR4. The CXCL12–CXCR4 axisis involved in promoting the chemoattraction of LECs andtheir subsequent lymphangiogenesis, which contributes tolymph node metastasis. Therefore, in addition to its angio-genic activity (38), the CXCL12–CXCR4 axis should play amore pivotal role in regulating tumor growth andmetastasisthan previously thought.

The relationship between the CXCL12–CXCR4 axis andthe VEGF-C/VEGFR-3 pathway

Another intriguing aspect of this study is the observationthat the CXCL12–CXCR4 axis is independent of VEGFR-3pathway in promoting lymphangiogenesis, even thoughVEGF-C can induce the expression of CXCR4. Among theknown lymphangiogenic factors, VEGF-C, which exerts itsfunctions via VEGFR-3, is the most potent and specificgrowth factor acting directly on LECs. Although VEGF-A,bFGF, angiopoient-1/2, insulin-like growth factor-1/2 (IGF-1/2), hepatocyte growth factor, PDGF-BB have been shownto be prolymphangiogenesis factors, the VEGF-C-VEGFR-3signaling is a common pathway for several lymphangio-genic factors. The bFGF was reported to upregulate VEGF-Cexpression in endothelial cells, and its lymphangiogenicproperty is mediated by VEGF-C (27). The VEGF-A-inducedlymphangiogenesismaypartiallymediatedby theVEGF-C/-D-VEGFR-3 signaling (28). Tie-2, IGF-1/2 receptor, andPDGF receptor have been detected on LECs, and theirrespective ligands are shown to promote lymphangiogen-esis (48–50). Intriguingly, blockage of VEGF-C/-D/VEGFR-3 inhibits the angiopoient-1-induced lymphatic sproutingbut does not affect the lymphangiogenic activities of IGF-1/2 and PDGF-BB, indicating that IGF-1/2 and PDGF-BBactivate a signaling pathway independent of that triggeredby the VEGF-C/-D/VEGFR-3 axis. Our present study showsthat CXCL12 is a direct chemoattractant for LECs. In con-trast to the antibody of CXCR4, the VEGFR-3-neutralizingantibody has no effect on the cell chemoattraction of LECsstimulated by CXCL12 as well as CXCL12-induced lym-

phangiogenesis in vivo. Although we can not exclude thepossibility that CXCL12 might also have indirect effects inpromoting lymphangiogenesis via inducing other factors,the observed effects of the CXCL12–CXCR4 axis on LECschemotaxis show that it at leasthas adirect impacton certainsteps of lymphangiogenesis. Importantly, we are moreinterested in the additive effect of CXCL12 and VEGF-C inpromoting cellmigrationof LECs. It seems thatCXCL12 is atleast as potent as, if notmore than, VEGF-C in chemoattrac-tion of LECs according to our results, whereas VEGF-C hasbeen shown to execute a potent mitogenic effect on LECs.We assume that these 2 factors may act in a synergisticmanner by conducting different functions in lymphangio-genesis. Having shown that a combination of anti-CXCL12and anti-VEGF-C antibodies more efficiently inhibit tumorlymphangiogenesis and lymph node metastasis than indi-vidual antibody alone, therefore, the development ofantagonists for both CXCR4 and VEGFR-3 pathway shouldbe a very promising approach for the control of tumormetastasis and lymphangiogenesis-related diseases.

In conclusion, our study shows that the CXCL12–CXCR4axis is a potent positive-regulator of lymphangiogenesis bydirectly acting on LECs. Of all chemokine receptors ana-lyzed in LECs, only CXCR4 is significantly upregulated byVEGF-C compared with that in quiescent LECs. CXCL12induces chemotaxis of LECs in vitro and lymphangiogenesisin vivomediated via CXCR4. Interestingly, lymphangiogen-esis triggered by the CXCL12–CXCR4 axis is independent oftheVEGF-C-VEGFR-3 pathway. Targeting bothCXCL12 andVEGF-C by neutralizing-antibodies results in an additiveinhibitory effect on both tumor lymphangiogenesis andlymphatic metastasis. This study, for the first time, pointsout that the chemokine/chemokine receptor system isdirectly involved in the process of lymphangiogenesis, andprovides a novel route to inhibit lymph nodemetastasis viatargeting both chemokines and lymphangiogenic growthfactors, which must attract intensive investigation in thisdirection in the future.

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

Authors' ContributionsConception and design: W. Zhuo, Y. LuoDevelopment of methodology: W. Zhuo, N. Song, X. Song, Y. Fu, Y. LuoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): W. Zhuo, L. Jia, X. Lu, Y. LuoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):W. Zhuo, L. Jia, N. Song, X. Lu, X. Wang,X. Song, Y. Fu, Y. LuoWriting, review, and/or revision of the manuscript: W. Zhuo, L. Jia, N.Song, Y. Ding, X. Wang, Y. LuoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W. Zhuo, Y. Fu, Y. LuoStudy supervision: W. Zhuo, Y. Luo

AcknowledgmentsThe authors thank the members of the Luo laboratory for their kind

suggestions on this work.

Grant SupportThis work was supported in part by the General Programs of

the National Natural Science Foundation of China (No. 81071742,

Zhuo et al.





No. 81171998 and No. 81171999) and the Doctoral Fund of theNew Teacher Programe of Ministry of Education of China (No.20110002120039).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 3, 2012; revised July 20, 2012; accepted July 23, 2012;published OnlineFirst August 29, 2012.

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2012;18:5387-5398. Published OnlineFirst August 29, 2012.Clin Cancer Res Wei Zhuo, Lin Jia, Nan Song, et al. Lymphangiogenesis

CXCR4 Chemokine Pathway: A Novel Axis Regulates−The CXCL12

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