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Tumor Biology and Immunology

CCL27/CCL28–CCR10 Chemokine SignalingMediatesMigration of Lymphatic Endothelial CellsTaraKarnezis1,2,3,4, RaeH. Farnsworth1, Nicole C. Harris1,2,3,5, Steven P.Williams1,2,3,5,Carol Caesar1,2, David J. Byrne1, Prad Herle3, Maria L. Macheda1,Ramin Shayan1,2,3,4,5, You-Fang Zhang1,2, Sezer Yazar2, Simon J. Takouridis2,Craig Gerard6, Stephen B. Fox1,7, Marc G. Achen1,2,5,7, and Steven A. Stacker1,2,5,7

Abstract

Metastasis via the lymphatic vasculature is an importantstep in cancer progression. The formation of new lymphaticvessels (lymphangiogenesis), or remodeling of existing lym-phatics, is thought to facilitate the entry and transport of tumorcells into lymphatic vessels and on to distant organs. Themigration of lymphatic endothelial cells (LEC) toward guid-ance cues is critical for lymphangiogenesis. While chemokinesare known to provide directional navigation for migratingimmune cells, their role in mediating LEC migration duringtumor-associated lymphangiogenesis is not well defined.Here, we undertook gene profiling studies to identify chemo-kine–chemokine receptor pairs that are involved in tumorlymphangiogenesis associated with lymph node metastasis.CCL27 and CCL28 were expressed in tumor cells with meta-static potential, while their cognate receptor, CCR10, wasexpressed by LECs and upregulated by the lymphangiogenicgrowth factor VEGFD and the proinflammatory cytokineTNFa. Migration assays demonstrated that LECs are attracted

to both CCL27 and CCL28 in a CCR10-dependent manner,while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 inlymphatic patterning. In vivo analyses showed that LECs arerecruited to a CCL27 or CCL28 source, while VEGFDwas required in combinationwith these chemokines to enableformation of coherent lymphatic vessels. Moreover, tumorxenograft experiments demonstrated that even though CCL27expression by tumors enhanced LEC recruitment, the ability tometastasizewas dependent on the expression of VEGFD. Thesestudies demonstrate that CCL27 andCCL28 signaling throughCCR10 may cooperate with inflammatory mediators andVEGFD during tumor lymphangiogenesis.

Significance: The study shows that the remodeling of lym-phatic vessels in cancer is influenced by CCL27 and CCL28chemokines, which may provide a future target to modulatemetastatic spread.

IntroductionSpread of tumor cells via lymphatic vessels to regional lymph

nodes is a key early step in the metastasis of many humanmalignancies (1, 2). Many of these tumors induce lymphangio-genesis–the formation of new lymphatics–and lymphatic vesselremodeling via specific growth factors, the best-characterized ofwhich are VEGFC and VEGFD (1). These proteins act throughreceptor tyrosine kinases VEGFR2 and VEGFR3 on lymphatic

endothelial cells (LECs) to drive lymphangiogenesis, thus pro-viding tumors with greater access to the lymphatic system andthereby promoting metastasis (1–3). Another mechanism con-tributing to metastasis via the lymphatics involves exploiting anormal physiological function of lymphatics, whereby tumorcells mimic immune cells that traffic from peripheral tissues tolymph nodes and other secondary lymphoid sites, a process forwhich chemokines and their receptors are essential (4–6).

Chemokines are a family of over 50 small chemoattractantcytokines that bind in a nonexclusive manner to over 20 cellsurface G protein–coupled receptors on target cells, allowing thecells tomigrate along chemokine gradients to selected tissues (4).For example, T and B lymphocytes and dendritic cells expresschemokine receptor CCR7 upon activation, causing them tomigrate toward LECs expressing the CCR7 ligand CCL21 (6, 7).Similarly, LECs secreting CCL27 guided entry of activated T cellsexpressing the CCL27 receptor, CCR10, to afferent lymphaticvessels (8). An experimental model in which B16 murine mela-noma cells overexpressed CCR7 showed greatly enhanced metas-tasis to lymph nodes expressing CCL21 (9).

Chemokines have also previously been implicated in tumor-associated angiogenesis (4, 10). CXCL12, through its receptorCXCR4, can induce blood endothelial cell (BEC) migration andpromote angiogenesis both in vitro and in vivo (10). Interestingly,CXCL12 was also shown to act synergistically with the angiogenicfactor, VEGFA, to induce vascularization in ovarian cancer (11). In

1Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 2Ludwig Insti-tute for CancerResearch, RoyalMelbourneHospital, Parkville, Victoria, Australia.3O'Brien Institute Department, St Vincent's Institute of Medical Research,Fitzroy, Victoria, Australia. 4Department of Medicine, St Vincent's Hospital,University of Melbourne, Fitzroy, Victoria, Australia. 5Department of Surgery,Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria, Australia.6Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.7Sir Peter MacCallum Department of Oncology, University of Melbourne, Park-ville, Victoria, Australia.

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

Corresponding Author: Steven A. Stacker, Peter MacCallum Cancer Centre, 305Grattan St, Melbourne 3000, Australia. Phone: 613-8559-7106; Fax: 613-8559-8054; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-1858

�2019 American Association for Cancer Research.

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contrast, the role of chemokines in lymphangiogenesis is onlybeginning to emerge.Metastaticmelanoma cells expressing CCR7migrated toward LECs in a CCL21-dependentmanner (12), whilecoexpression of CCR7 and VEGFC by tumor cells also enhancedtheir migration toward lymphatics and metastasis to lymphnodes (13). Despite this early evidence, the extent of chemokineinvolvement, and their specific roles in tumor-associated lym-phangiogenesis, remain poorly defined.

In this study, we identified the CCL27 and CCL28 signalingthrough CCR10 as a mechanism to induce the recruitment ofLECs during tumor lymphangiogenesis. We confirmed thecapacity of CCL27 and CCL28 to drive LEC migration andshowed a role for CCR10 in lymphatic vessel development andpatterning. We demonstrated that CCL27 and CCL28 cooperatewith VEGFD to promote lymphangiogenesis. These findingsfurther define the molecular mechanisms controlling tumorlymphangiogenesis.

Materials and MethodsAnimals

SCID/NOD mice (RRID:IMSR_JAX:001303; IMVS, Adelaide,Australia), housed in microisolators, and FVB mice (Walter andEliza Hall Institute, Melbourne, Australia) were 6–8 weeks old.Ccr10�/� mice (RRID: MGI:3822166) were obtained fromBoston Children's Hospital, Harvard Medical School (Boston,MA) and genotyped as described previously (14). Mouseexperiments were in accordance with the "Australian code forthe care and use of animals for scientific purposes", 8th edition,and were approved by the Ludwig Institute for Cancer Research/Department of Surgery Animal Ethics Committee and the PeterMacCallum Cancer Centre Animal Experimentation EthicsCommittee.

Cell lines293EBNAcells stably expressingVEGFD(VEGFD-293EBNA)or

empty expression vector (Apex-293EBNA) were passaged asdescribed previously (15). The CCL27-overexpressing 293EBNAand vector control pVITRO3-293EBNA cell lines were created in asimilar manner to VEGFD-293EBNA (15, 16), using humanCCL27 cDNA, cloned by PCR from VEGFD-293EBNA cells (usingprimers: forward 50-GGAAGAGTCTAGGCTGAGCAA-30 andreverse 50-TCCAATGCTGCTTTATTATTTGG-30), which was thenligated into pVITRO3mcs (InvivoGen). Two independent tumorcell lines were generated by cloning CCL27 cDNA into pcDNA 3.1(Invitrogen) and stably transfecting either empty pcDNA 3.1vector or pcDNA 3.1 containing CCL27 cDNA into Apex-293EBNA cells.

MDA-MB-435, MDA-MB-231 (both from Robin Anderson,Peter MacCallum Cancer Centre, Australia), MIA PaCa-2 (ATCCcatalog no. CRL-1420, RRID:CVCL_0428), SK-MEL-2 (ATCC cat-alog no. HTB-68, RRID:CVCL_0069), and Caco-2 cells (fromAntony Burgess, Ludwig Institute for Cancer Research, Australia)were maintained in DMEM (Life Technologies) with 10% FBS.FEMX-I (from Oystein Fodstad, Norwegian Radium Hospital,Norway), DU 145 (from Andrew Scott, Ludwig Institute forCancer Research, Australia), PC-3 (fromChris Hovens, Universityof Melbourne, Australia), DLD-1, and HT-29 cells (from AntonyBurgess) were maintained in RPMI1640 (Life Technologies) with10% FBS. Dates of cell acquisition, thawing, and Mycoplasmatesting are outlined in Supplementary Table S1. Primary human

dermal lymphatic and blood microvascular endothelial cells(Lonza) were grown on fibronectin-coated plates (5 mg/mL;Sigma-Aldrich), in complete endothelial cell medium (EGM-2-MV; Lonza). Cell lines were incubated at 37�C in 5% CO2, exceptMDA-MB-435 (10% CO2). Growth rate and morphology of celllines weremonitored routinely, and VEGFD or CCL27 expressionby stably transfected cells was verified by Western blot analysisprior to each experiment.

RNA isolation and quantitative RT-PCRRNA was isolated using the RNeasy Mini or RNeasy Plus Mini

Kit (Qiagen). To investigate mRNA expression, total RNA wasreverse transcribed using SuperScript II Reverse Transcriptase orHigh capacity cDNA Reverse Transcription kit with a mixture ofoligo(dT) and random hexamer primers (Life Technologies).Quantitative reverse-transcription PCR (qRT-PCR) analysis wasperformed using TaqMan Gene Expression assays (ABI), withassay IDs: CCR10, Hs00706455_s1; CCL27, Hs00171157_m1;CCL28, Hs00219797_m1; FIGF (VEGFD), Hs01128659_m1;ACTB (b-actin), 4333762T. To assess CCR10 knockdown,qRT-PCR was performed using Sensimix Plus SYBR Green(Quantace) and CCR10 primers (forward: 50-TGCTGGATACT-GCCGATCTACTG-30; reverse: 50- TCTAGATTCGCAGCCCTAG-TTGTC-30). All data were normalized to b-actin.

Chemokine and chemokine receptor miniarraysTo determine chemokine and chemokine receptor expression

in LECs and cancer cell lines, RNA was isolated using the Array-Grade Total RNA Isolation Kit (SABiosciences). Sample RNA wasreverse transcribed into cDNA then transcribed into biotin-labeled cRNA target using the TrueLabeling-AMP 2.0 Kit (SABios-ciences). Labeled target cRNA was then hybridized to an OligoGEArray Human Chemokine and Chemokine Receptor minia-rray, as per manufacturer's instructions (SABiosciences). Theintensity of each spot was quantified using ImageJ software (NIH)and normalized relative to GAPDH. A heatmap with hierarchicalclustering was created using Cluster 3.0 and Java TreeViewsoftware.

Chemokines, growth factors, and ELISARecombinant human CCL27, CCL28, VEGFA, IFNg , TNFa,

IL1b (all from R&D Systems), and mature VEGFC and VEGFD(Opthea Pty Ltd) were used in various assays. Levels of humanCCL27 and CCL28 secreted by cancer cell lines were quantifiedusing Quantikine ELISA Kits (R&D Systems).

siRNA-mediated gene knockdownFlexiTube siRNAs targeting human CCR10 mRNA (Qiagen)

were used for transient CCR10 knockdowns. Negative controlsiRNA (Qiagen) was also used. LECs (2 � 105) were reversetransfected with 40 nmol/L siRNA in 6-well plates, using 10mL/well siPORT Amine Transfection Reagent (Ambion, Life Tech-nologies), and medium was changed 24 hours posttransfection.CCR10 expressionwas assessed 72hours posttransfection byqRT-PCR or flow cytometry.

Tumor establishmentTumors were established in the flanks of female SCID/NOD

mice by subcutaneous injection of 2–2.5 � 107 293EBNA cells,stably transfected with expression vectors cDNA encoding eitherVEGFD or CCL27 or with empty vector control plasmids (pApex,

CCR10 Ligands Mediate Lymphatic Endothelial Cell Migration

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pVITRO3 or pcDNA 3.1), as described previously (15). Othermice were injected with 3� 107 MDA-MB-435 or FEMX-I cells or6� 106 DU 145 cells. Mice were euthanized when tumor volumereached 1,500–2,000 mm3.

In vivo lymphangiogenesis assay and immunofluorescenceFemale FVB mice were injected subcutaneously in the flank

with 500 mL of Matrigel (Growth Factor Reduced BasementMembrane Matrix, Corning/BD Biosciences) containing 2.5 mgVEGFA, VEGFD, CCL28, combined VEGFD and CCL28, or PBS ascontrol. In a second experiment, 200 mL of Matrigel was injectedcontaining 4 mg/mL VEGFA, 4 mg/mL VEGFD, 2 mg/mL CCL27, 4mg/mL VEGFD plus 2 mg/mL CCL27, or PBS þ 0.1% BSA. After 7days,micewere euthanized, andMatrigel plugswere removed andsnap frozen in Tissue-Tek optimal cutting temperature (OCT)compound (Sakura Finetek). Cryosections of Matrigel plugs werestained using antibodies for rabbit anti-mouse LYVE1 (FitzgeraldIndustries International catalog no. 70R-LR003, RRID:AB_1287923), hamster anti-mouse podoplanin (Fitzgerald cata-log no. 10R-P155a, RRID:AB_1288912), or rat anti-mousePECAM1/CD31 (BD Pharmingen catalog no. 553370, RRID:AB_394816) to assess the extent of lymphangiogenesis andangiogenesis, as described previously (17). Staining was quanti-fied over 2–5 fields each from 1–3 sections per Matrigel plug ortumor (up to 15 fields per mouse). F4/80 antigen (macrophagemarker) was detected using a rat anti-mouse antibody (Abcamcatalog no. ab6640, RRID:AB_1140040 or BioLegend catalog no.123102, RRID: AB_893506).

Proliferation and migration assaysLEC proliferation was analyzed using the CellTiter 96 AQueous

One Solution Cell Proliferation Assay (Promega). Approximately2,000 cells were resuspended in proliferation medium [EBM-2medium (Lonza), 2% FBS, without supplements] containingpurified growth factors and incubated for 72 hours before theaddition of the MTS-containing assay reagent.

Migration assays were performed using HTS FluoroBlokmigra-tion chambers with 8-mm pore size membrane inserts (BD Bios-ciences). Cells were incubated overnight in starvation medium(0.2%BSA in EBM-2) and 20,000 or 50,000 cells seeded into a 96-well migration chamber plate that had the underside of themembrane coated with fibronectin (5 mg/mL). Starvation medi-um containing purified growth factors or chemokines was addedto the bottom wells. After 16–24 hours, cells that migrated to theunderside of the membrane were stained with calcein (5 mg/mL;Molecular Probes) or fixed with 4% paraformaldehyde in PBSand stained with DAPI (5 mg/mL). Cells were then visualizedusing a Nikon TE2000-E microscope and quantified using Meta-Morph software (Molecular Devices, LLC). For experimentsinvolving siRNA knockdown of CCR10, cells were transfected48 hours prior to the migration assay (see siRNA-mediated geneknockdown), then migrated cells were visualized with calcein,and counted manually. In other experiments, cells were preincu-bated with functional blocking antibodies to human CCL27(R&D Systems catalog no. MAB376, RRID: AB_2070653) priorto the migration assay.

Flow cytometryFor detection of cell-surface CCR10, LECs were incubated with

rat anti-human CCR10 antibody (R&D Systems catalog no.FAB3478A, RRID:AB_573043) for 20–30 minutes on ice, then

stained with fluorescently conjugated anti-rat secondary antibo-dies and propidium iodide (Sigma-Aldrich). Cells were analyzedusing a FACSCalibur flow cytometer (BD Biosciences) and FlowJosoftware (TreeStar Inc.).

Immunoprecipitation and Western blot analysisTo assess CCL27 protein expression levels in 293EBNA cells,

conditioned medium was collected and immunoprecipitationsperformedusingmouse anti-humanCCL27antibody (SantaCruzBiotechnology #sc-390112, RRID:AB_2736849). Immunopreci-pitates were run on 10% or 16% Tricine gels (Novex; Life Tech-nologies) and Western blotted using the same antibody as usedfor immunoprecipitation.

IHC, whole mount staining, and visualization of collectinglymphatic vessels

Mouse and human tissues were harvested and either frozenin OCT compound or fixed in 4% paraformaldehyde or 10%neutral buffered formalin before paraffin embedding andsectioning. Sections were immunostained for LYVE1 (Fitzger-ald anti-mouse catalog no. 70R-LR003, RRID:AB_1287923;anti-human catalog no. 70R-LR004, RRID:AB_1287920), ham-ster anti-mouse podoplanin (Fitzgerald catalog no. 10R-P155a,RRID:AB_1288912), PECAM1/CD31 (BD Pharmingen catalogno. 553370, RRID:AB_394816), CCR10 (Novus Biologicalscatalog no. NB100-56319, RRID:AB_837897), Prox1 (BioLe-gend catalog no. 925201, RRID:AB_291595), F4/80 (Abcamcatalog no. ab6640, RRID:AB_1140040 or BioLegend catalogno. 123102, RRID: AB_893506), and developed with specificsecondary antibodies as described previously (17). To stain forchemokines, paraffin sections underwent optimized antigenretrieval with trypsin for CCL28 (ProteinTech catalog no.18214-1-AP, RRID:AB_2262251); or Tris EDTA pH 9.0 forCCL27 (R&D Systems, #MAB376). After incubation withhorseradish peroxidase–conjugated secondary antibodies, andtyramide signal amplification (Perkin Elmer), the signal wasdeveloped using DAB and sections counterstained with hema-toxylin. Staining was quantified using MetaMorph software.

Wholemount staining andmorphometric analysis of ear tissuefromCcr10�/�micewere performed as described previously (18).Collecting lymphatic vessel visualization based on dye injectionwas performed as described previously (19).

Reanalysis of publicly available microarray dataA publicly available microarray dataset representing normal

human skin, benign nevi, and melanoma (GSE3189; ref. 20) wasdownloaded from Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). Downloaded data had been pre-processed by normalization, background subtraction, and log2transformation and were reanalyzed without further processing.Unsupervised clustering was performed using Cluster 3.0 soft-ware. Another dataset derived fromhumanLECs stimulated for 24hours with 1 ng/mL TNFa (GSE6257; ref. 21) was reanalyzed toidentify differentially expressed genes using the GEO2R webinterface (https://www.ncbi.nlm.nih.gov/geo/geo2r/) based onlimma (Linear Models for Microarray Analysis).

Statistical analysisPairs of conditions were compared using a Student t test

for normally distributed data or a Mann–Whitney test for non-normally distributed data. Multiple conditions were compared

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https://www.ncbi.nlm.nih.gov/geo/

https://www.ncbi.nlm.nih.gov/geo/

https://www.ncbi.nlm.nih.gov/geo/geo2r/


using a one-way ANOVA with Dunnett or Tukey post hoc testfor normally-distributed data, or a Kruskal–Wallis test for non-normally distributed data unless stated otherwise. Sample sizeswere determined as appropriate for each assay based on empiricaldetermination in prior experiments.

ResultsChemokine profiling reveals aCCL27/CCL28–CCR10 signalingaxis in lymphangiogenesis

We and others have shown that the lymphangiogenic factorsVEGFC and VEGFD can induce tumor cell metastasis to regionallymph nodes by promoting tumor-associated lymphangiogen-esis (1). To identify pairs of chemokine receptors and ligandsimportant during this pathologic lymphangiogenesis, expressionof chemokines and chemokine receptors was analyzed using anestablished tumor model of VEGFD–dependent lymphogenousspread (15, 19). In this model, VEGFD overexpression in 293-EBNA cells drives tumor-associated lymphangiogenesis, lymphat-ic remodeling, and metastasis, similarly to other tumor cell linesthat endogenously overexpress VEGFD (e.g., 66cl4 murine breastcarcinoma and MDA-MB-435 human melanoma; ref. 19). As anin vitromodel of the tumormicroenvironment, themRNAprofilesof cultured VEGFD-293EBNA cells and LECs were analyzed usingcDNA miniarrays. VEGFD-293EBNA cells expressed CCL27 andCCL28, while LECs expressed their cognate receptor, CCR10(Fig. 1A; Supplementary Fig. S1). CCR10 was also expressed inVEGFD-293EBNA cells, suggesting there may be some autocrinesignaling in these cells. Other chemokines and their correspond-ing receptors thatwere expressed across the two cell types includedCCL19 inVEGFD-293EBNA cells and the receptor CCRL2 in LECs,as well as the CXCL12–CXCR4 signaling pair, implicated inangiogenesis (4, 10). We chose to focus on CCL27/CCL28 andtheir receptor CCR10 as these chemokines are expressed in tissuessuch as skin (CCL27) and mucosal epithelia (CCL28), which arecommon sites of cancer (22, 23).

To validate the miniarray data, qRT-PCR was performed onhuman LECs and VEGFD-293EBNA cells, which confirmed thatthey expressed CCR10 or CCL27 and CCL28 mRNA, respectively(Fig. 1B and C). Interestingly, examination of CCR10 mRNAshowed expression was higher in LECs compared with BECs invitro, and further increased in LECs (but not BECs) stimulatedwithVEGFD (Fig. 1C). Flow cytometry was used to confirm CCR10expression on the surface of LECs (Fig. 1D). Although the amountof cell-surface CCR10 was low, the specificity of the signal wasconfirmed by targeting CCR10 with siRNA: siCCR10_4 reducedCCR10mRNA expression to less than 50% relative to the negativecontrol (Fig. 1E) and completely abrogated the CCR10 antibodysignal in flow cytometry (Fig. 1D). Furthermore, CCR10 stainingin LYVE1-positive lymphatic vessels was confirmed by IHC ofhuman breast cancer (ductal carcinoma in situ; DCIS) and normal

Figure 1.

Analysis of chemokine and chemokine receptor mRNA levels in VEGFD-293EBNA cells and VEGFD–treated LECs. A,Miniarrays containingoligonucleotides for chemokine and chemokine receptor genes were probedwith biotin-labeled cRNA derived from VEGFD-293EBNA cells and humanLECs. Signals for CCL27, CCL28, and CCR10 are indicated. B,Quantification ofmRNA levels for CCL27 and CCL28 in VEGFD-293EBNA cells using qRT-PCR.Chemokine expression was normalized to b-actin. Data represent mean�SEM of three replicates. C, qRT-PCR quantification of CCR10 expression inhuman LECs or BECs treated with 500 ng/mL VEGFD for 24 hours.Expressionwas normalized to b-actin. Data represent mean� SEM of threereplicates. � , P < 0.05 by Student t test. D, Flow cytometry of LEC CCR10 cellsurface expression using an anti-CCR10 antibody (dashed line) and isotypecontrol (solid line). LECs were transfected with negative control (left) orCCR10_4 siRNA (right). E, Knockdown of CCR10 mRNA by four differentsiRNAs was assessed by qRT-PCR, compared with a negative control(normalized to 100%). Data represent mean� SEM of three experiments. � , P< 0.05 by Student t test. F, Serial tissue sections of human breast cancer(ductal carcinoma in situ, DCIS) and normal colon were immunostained for

CCR10 in parallel with LYVE-1, a lymphatic vessel marker. Arrows, colocalizedLYVE1 and CCR10 staining. G, Ear tissue from Ccr10�/� and wild-type (WT)littermate control mice were stained as whole mounts with an antibody toLYVE-1. Scale bars, 100 mm. H, Analysis of mouse ear tissue sections forparameters that describe lymphatic vessel morphology: number of branches,number of loops, number of blind-ending sacs, average width of the vessel,average spacing distance of the vessels, and overall vessel density.Parameters are displayed as mean� SEM number of events for WT (n¼ 5)and Ccr10�/� (n¼ 5) mice. � , P < 0.05 by Student t test.






colon tissue specimens (Fig. 1F). CCR10 staining appeared to berestricted to a subset of cells comprising lymphatic vessels, imply-ing that its expression may become upregulated in a polarized orlocally restricted manner in response to secreted guidance cues.VEGFD is known to be expressed in DCIS, which often exhibitslymphangiogenesis (24, 25). Our results suggest that focal upre-gulation of CCR10 by VEGFD and/or other factors may beinvolved in regulating lymphangiogenesis in diverse tissues.

To confirm the role of CCR10 in lymphangiogenesis,lymphatic vessel patterning was examined in CCR10-deficientmice.Ccr10�/�mice showdefective homing and accumulation ofspecific lymphocyte subsets in vivo (14). Homing of specificCCR10þ T-cell subsets to skin is particularly affected, due to thehighly selective expression of CCL27 in keratinocytes (26, 27).CCR10 is also expressed in cutaneous melanocytes, fibroblasts,and blood vascular endothelial cells (22, 28). As the role ofCCR10 has not previously been investigated in lymphatics, der-mal lymphatic vessels in the ears of Ccr10�/� mice (14) wereexamined and compared with the characteristic pattern found inwild-type mice using LYVE1 wholemount staining of ear skin(Fig. 1G).

Quantification of defined parameters of lymphatic pattern-ing (18) demonstrated thatCcr10�/�mice had significantly lowerlymphatic vessel density, with lymphatics spaced further apartand having fewer branches than wild-type vessels, but withnormal vessel width (Fig. 1H). This pattern of decreased branch-ing resembled that seen when mice are treated in the earlypostnatal period with inhibitors of Dll4 or Notch1 signaling,which regulate lymphatic vessel sprouting (29). This hints thatCCR10 may be important for lymphatic vessel sprout formationand branching. Interestingly, comparison of lymphatic vessels inlate embryonic dorsal skin of Ccr10�/� mice to wild-type litter-mates revealed increased cross-sectional area of individual lym-phatic vessels, while the overall stained area of lymphatic endo-thelium was unchanged (Supplementary Fig. S2A–S2G). Com-bined with the observation of less lymphatic branching in theadult ear, these results may indicate a defect in the sprouting ofinitial lymphatics from the primitive dermal lymphatic plexustoward epidermalCCL27 expressed by keratinocytes (22, 27), andtherefore a relative paucity of smaller-caliber lymphatics in theembryonic dermis ofCcr10�/�mice. Alternatively or additionally,these defects in lymphatic vessel patterning may be contributedto by tissue-specific microenvironmental factors, such as changesin the distribution of leukocyte subsets that normally expressCCR10 (e.g., plasmacytoid dendritic cells, Langerhans cells, skin-resident or -homing T cells, plasma cells) and that may influencelymphatic development (14, 23, 30). Overall, these results sup-port the involvement of CCR10 in patterning the dermal lym-phatic network.

Distribution of CCL27 and CCL28 chemokines during tumor-associated lymphangiogenesis

CCL27 is overexpressed in certain types of squamous cellcarcinoma (31), while CCL28 is expressed in ovarian carcinoma,lung adenocarcinoma, and pancreatic adenocarcinoma (32–34).Expression of CCL27 and CCL28 was further assessed in meta-static cancer cell lines expressing endogenous VEGFD (Supple-mentary Fig. S3A). CCL27 and CCL28 were expressed at variouslevels in metastatic prostate, breast, colorectal and pancreaticcancer and melanoma cell lines (Supplementary Fig. S3B andS3C). CCL27 and CCL28 expression in a context of pathologic

lymphangiogenesis in vivo was analyzed using several humancancer cell lines in mouse xenograft experiments. These celllines included VEGFD-293EBNA, and endogenous VEGFD–

expressing melanoma (MDA-MB-435 and FEMX-I) and prostatecancer (DU 145) cells, which also showed the highest levelsof CCL27 and CCL28 mRNA (Supplementary Fig. S3A–S3C).IHC analysis of VEGFD-293EBNA, DU 145, MDA-MB-435, andFEMX-I tumors revealed lymphatic vessels (using LYVE1 stain-ing), as well as expression of both CCL27 and CCL28 (Fig. 2A;Supplementary Fig. S3D). CCL27 and CCL28 proteins weredetected in surrounding stroma as well as tumor cells, and insome cases were in close proximity to lymphatic vessels. Expectedstaining patterns of CCL27 and CCL28 were observed in positivecontrol tissues (human skin and colonic mucosa, respectively),validating the specificity of these antibodies (SupplementaryFig. S3E and S3F).

Coimmunofluorescence staining or IHC on serial sectionsshowed that both CCL27 and CCL28 localized to LYVE1þ endo-thelial cells that comprise lymphatic vessels (Fig. 2B and C).CCL27 expression has been detected in the precollecting subtypeof LECs in vivo (8); however, this subtype represents a higher-order, more complex vessel type than the newly formed initiallymphatics most likely present within and around tumors. Giventhe lack of detectable CCL27 and CCL28 transcripts in LECs(Fig. 1A), it is possible that CCL27/CCL28 released from tumorcells into the surrounding stroma may bind to CCR10 that ispresent on the surface of LECs (30).

To further validate expression of CCL27/CCL28 and theirreceptorCCR10 inhuman tumors, a publicly availablemicroarraydataset derived from normal skin, benign nevi, and malignantmelanomas was analyzed (Fig. 2D; Supplementary Fig. S4A andS4B; ref. 20). Melanoma specimens were predominantly preme-tastatic, while the majority (78%) of the nevi was resected fromthe trunk and lower limbs; notably, melanomas that have pro-gressed from nevi are most commonly associated with theseanatomic locations (35). While CCL28 was not represented onthe microarray, both CCL27 and CCR10 were elevated in nevirelative to both normal skin and malignant melanoma (Fig. 2D).This observationmay indicate a transient role for this chemokine–receptor pair in regulating melanocyte proliferation or recruit-ment of immune cells in nevi that is lost in melanomas that haveacquired more potent driver mutations and immune evasionmechanisms (36, 37). Overall, our data emphasize that CCL27andCCL28are expressed in a variety of tumor cell types and canbecoexpressed with the lymphangiogenic growth factor VEGFD.

Proinflammatory stimuli promote expression of CCR10 andits ligands

Inflammation is a hallmark of cancer. Furthermore, CCL27 orCCL28 expression in various cancer cells or in skin can beupregulated by inflammatory cytokines (30, 38, 39). To testwhether this may be applicable to a model of pathologic lym-phangiogenesis, we assessed whether expression of CCL27/CCL28 in tumor cells or CCR10 in LECs was altered by inflam-matory cytokines. Expression of CCR10 mRNA in LECs wasincreased by TNFa stimulation, peaking at 16 hours (Fig. 3A).Upregulation of CCR10 protein by TNFa was also detected(Supplementary Fig. S5A and S5B). These data confirm a previousobservation of CCR10 mRNA upregulation in LECs by TNFa,detected by microarray (21) (Supplementary Fig. S5C). InVEGFD-293EBNA cells, IFNg , TNFa, and IL1b enabled increased

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production of CCL28 protein over unstimulated cells, asdetected by ELISA (Supplementary Fig. S5D). qRT-PCR indi-cated that CCL28 mRNA was consistently upregulated inresponse to TNFa; although the fold increase varied, the trendsupported the increased protein expression observed byELISA (Supplementary Fig. S5E). These data confirm previous

observations that CCR10 and its ligands can be upregulated byproinflammatory cytokines.

Tumors frommouse xenograft experiments were subsequentlyexamined for infiltrating macrophages, which are known tosecrete proinflammatory/proangiogenic factors such as TNFa.VEGFD-293EBNA tumors, which have a greater density of

Figure 2.

CCL27 and CCL28 protein localization in tumor-associated lymphangiogenesis. A, Apex-293EBNA, VEGFD-293EBNA, and FEMX-I xenograft tumors were stainedvia IHC with LYVE1, CCL27, or CCL28 antibodies. Top insets show the position of the main image (black box) in a lower magnification overview of the tumorsection. Lower insets show a serial field of the main image at the samemagnification stained with a corresponding isotype-matched negative control antibody.Scale bar, 100 mm inmain; 200 mm in overview. B, Immunofluorescence staining of VEGFD-293EBNA tumors with LYVE1 and CCL27 antibodies. Scale bar, 100mm. C, CCL28 staining on a lymphatic vessel in a FEMX-I tumor section. Scale bar, 100 mm. D, Relative expression of CCR10 and CCL27mRNA in samples of humanmelanoma (n¼ 45), benign nevi (n¼ 18), and normal skin (n¼ 7) from the publicly available microarray expression dataset GSE3189 (20). Data are presented asindividual points with mean� SEM (� , P < 0.05; �� , P < 0.001; �� , P < 0.0001 by Mann–Whitney test).






lymphatic vessels than their nonmetastatic counterparts, showedincreasedmacrophage recruitment, as determined by F4/80 stain-ing (Fig. 3B–E). Although a small subset ofmacrophages is knownto express LYVE1, of all LYVE1þ structures in experimentalVEGFD-293EBNA tumors, on average only approximately 6.5%were costained for the macrophage marker F4/80 (Fig. 3F). Colo-calization of the lymphatic markers Prox1 and podoplanin inthese LYVE1þ vessel structures further validated their lymphaticidentity (Supplementary Fig. S5F and S5G). These results suggestthat tumor cells secrete CCL27 and CCL28 in response to inflam-matory mediators that are typically produced by macrophagesand other cells in the tumor microenvironment, which may thenattract CCR10-expressing LECs.

CCL27 and CCL28 promote CCR10-dependent LEC migrationTo define the role(s) of CCL27 and CCL28 in lymphangiogen-

esis, in vitro LEC responses toward these chemokines were eval-

uated. Unlike VEGFD, which had a dose-dependent effect on LECproliferation, CCL27 and CCL28 had no significant proliferativeeffects (Fig. 4A). The effects of CCL27 and CCL28 on LECmigration were then assessed alongside VEGFC and VEGFD,known inducers of LEC migration. LECs had statistically signif-icant migratory responses to titrations of CCL27 and CCL28,demonstrating characteristic bell-shape curves typical of chemo-kine-induced migration (Fig. 4B; ref. 40). LEC migration towardCCL27 could be blocked using an inhibitory antibody (Fig. 4C;Supplementary Fig. S6). Interestingly, CCL27 and CCL28 did notsignificantly promote migration of BECs in these assays, indicat-ing that LECs may be particularly responsive to the promigratoryeffect of these chemokines (Fig. 4C).

To confirm that CCL27/CCL28-induced LEC migration occursvia CCR10-specific binding, gene knockdown studies were per-formed. CCR10 levels were reduced using siRNA (Fig. 1D and E).Migration of LECs toward the CCR10 ligands CCL27 and CCL28

Figure 3.

Proinflammatory cytokines stimulate CCR10 expression in LECs. A, qRT-PCR analysis of CCR10 mRNA levels in LECs after stimulation with 10 ng/mL TNFa. Datarepresent mean� SEM of three experiments. � , P < 0.05 by ANOVAwith Dunnett posttest compared with unstimulated (Unstim). B and C, Immunofluorescencestaining with antibodies to the macrophage marker F4/80 (red) and the LECmarker LYVE1 (green) demonstrates the infiltration of macrophages into VEGFD–expressing tumors (C), with empty vector (Apex)-293EBNA tumors (not expressing VEGFD) showing fewer macrophages and lymphatic vessels (B). Filledarrows, macrophages; open arrows, lymphatics. Scale bars, 100 mm. D and E,Quantification of the F4/80–stained area (D) or LYVE1-stained area (E) in VEGFD-293EBNA tumors versus Apex-293EBNA tumors. Data represent mean� SEM of 5 fields each from 10 tumors. � , P < 0.05 by Student t test. F,Quantificationshowing minimal costaining of F4/80 with LYVE1 in VEGFD-293EBNA tumors. Data represent mean� SEM, n¼ 5 tumor-bearing mice, 2 sections per tumor.

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was diminished when CCR10 levels were reduced by siCCR10_4,whereas CCR10 knockdown had no effect on LEC migrationtoward VEGFA (Fig. 4D). These results indicate that CCR10specifically mediates CCL27/CCL28–induced LEC migration.

CCR10 ligands promote lymphatic recruitment in vivoCCL28 and CCL27 were assessed for their ability to promote

in vivo LEC migration, which is necessary for the formation ofnascent lymphatic vessels. Matrigel plugs containing CCL27,

CCL28, VEGFD, VEGFA, CCL27, or CCL28 together with VEGFDor PBS alone were injected intomouse flanks. Approximately oneweek after implantation, plugs were removed and immunofluo-rescence was performed to detect (Fig. 5A–E; Supplementary Fig.S7A–S7E) and quantify (Fig. 5F–I) lymphatic and blood vesselsusing antibodies to LYVE1 and PECAM1, respectively.

Formation of LYVE1þ structures was evident in VEGFD–

containing positive control Matrigel plugs, while minimalstaining was observed in PBS negative control plugs (Fig. 5A,

Figure 4.

CCL27, CCL28, and CCR10 mediate LECmigration butnot proliferation in vitro. A, Proliferation assay of LECsstimulated with 0–500 ng/mL of recombinant CCL27,CCL28, or VEGFD. Proliferation was compared withunstimulated controls. Data represents mean� SEM ofthree replicates. � , P < 0.05 by Student t test. B, LECswere assessed for their ability to migrate towardtitrated concentrations of CCL27, CCL28, VEGFC, orVEGFD in a transwell migration chamber over 21–24hours. Migrated cells were quantified and comparedwith migration toward basal medium alone (Starve).Complete growth medium (Complete) was used as apositive control. Data represent mean� SEM of threereplicates; concentrations of each chemokine or VEGF(0.001–500 ng/mL) were compared with Starvecontrol using a one-way ANOVAwith Dunnett post hoctest. � , P < 0.05; �� , P < 0.001; ��, P < 0.0001. C, BECs(left) and LECs (right) were assessed for chemotacticmigration toward the indicated factors over 21–24hours, as compared with migration toward starvationmedium alone. Data represents mean� SEM of threereplicates; results compared using one-way ANOVAwith Tukey's post hoc test; chemokines and VEGFs(positive controls) analyzed separately. � , P < 0.05;�� , P < 0.01. D,Migration of LECs toward CCL27, CCL28,or VEGFA after CCR10 knockdownwith siRNA. Datarepresents mean� SEM of two technical replicates.� , P < 0.05 by Student t test.






Figure 5.

CCL28 and CCL27 cooperate with VEGFD to attract LECs in vivo. A–E, FVBmice were injected subcutaneously with 200 mL Matrigel containing PBS (A) or2 mg/mL of recombinant human CCL27 (B), 4 mg/mL VEGFD (C), 2 mg/mL CCL27 and 4 mg/mL VEGFD (D), or 4 mg/mL VEGFA (E). Matrigel plugs were harvestedafter one week and sections thereof stained with antibodies to LYVE1 (green) and PECAM1 (red) and nuclei counterstained with DAPI (blue). Scale bars, 100 mm.F and G,Quantification of LYVE1-positive vessels (F) or PECAM1-positive vessels (G) in Matrigel plugs. Data points represent average % stained area of <15 fieldsper mouse with mean� SEM of n¼ 6mice per group (5 for PBS group); P values from Kruskal–Wallis test with uncorrected Dunn test. � , P < 0.05; �� , P < 0.01;�� , P < 0.001. H and I,A similar experiment was conducted in which 2.5 mg CCL28 and VEGFD, alone or in combination as shown, were injected subcutaneouslyinto FVBmice in 500 mL of Matrigel. LYVE1 staining was quantified in H and PECAM1 staining in I. Data points represent mean average % stained area of <15 fieldsper mouse with mean� SEM of n¼ 4mice (3 for PBS group); P values from Kruskal–Wallis test with uncorrected Dunn test; � , P < 0.05; �� , P < 0.01. J, CCL28Matrigel plug costained with antibodies to LYVE1 (green) and podoplanin (red). Right, magnified view of the boxed area. Scale bars, 50 mm. K, CCL28 Matrigelplug costained with LYVE1 (green) and the macrophagemarker F4/80 (red) showing infiltration of macrophages into the plug. Right, a magnified view of theboxed area. Open arrows, LYVE1þ LECs; closed arrows, F4/80þmacrophages. Scale bars, 50 mm.

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C, F, and H; Supplementary Fig. S7A and S7C). Matrigel plugscontaining CCL28 or CCL27 showed more LYVE1 stainingcompared with PBS controls (Fig. 5A, B, F, and H; Supplemen-tary Fig. S7A and S7B). LYVE1þ structures in these plugs weresmall, often appearing to be single cells. LYVE1 stainingwithin Matrigel plugs colocalized predominately with stainingfor podoplanin, another LEC marker (Fig. 5J; SupplementaryFig. S7F and S7G). F4/80þ macrophages were detected in theMatrigel plugs to varying degrees, with some costaining forLYVE1 (Fig. 5K; Supplementary Fig. S7H and S7I). Someelongated LYVE1þ structures that resembled microvessels wereweak or negative for F4/80, while others exhibited associatedF4/80 staining (Supplementary Fig. S7H and S7I). This possiblyreflected LYVE1þ macrophages or myeloid-derived lymphaticprogenitors closely associating with or incorporating intonascent lymphatic vessels in the Matrigel plugs, as has beenobserved in experimental models of cancer and inflammationand some human cancers (41, 42). These data suggest that atleast in Matrigel plugs, CCL27 and/or CCL28 may recruit bothlocally derived LECs and prolymphangiogenic myeloid or LECprogenitor cells from circulation.

Matrigel plugs containing CCL28 or CCL27 together withVEGFD showed a different pattern of LYVE1 staining comparedwith the plugs containing either factor alone. In these plugs,LECs commonly formed more coherent vessel-like structures(Fig. 5D; Supplementary Fig. S7D). VEGFD in combinationwith CCL27 or CCL28 showed a trend indicating an additiveeffect on LYVE1 staining compared with the chemokines alone(Fig. 5F and H). These results suggest that VEGFD workstogether with chemokines to promote LEC migration andlymphatic vessel formation. The presence of VEGFD in Matrigelplugs may enhance lymphatic vessel formation from LECsrecruited to the plug by CCL28 or CCL27 by inducing LECproliferation and vessel remodeling (19).

PECAM1 staining was next used to assess blood vessel density.Although PECAM1 is also present in low amounts on LECs, itsrelatively much higher abundance in BECs (43) allows its use tomeasure blood vessel density when appropriate intensity thresh-olds are applied. Blood vessel density was slightly higher in plugscontaining CCL27 or CCL28 than those containing PBS alone,although this difference did not reach significance (Fig. 5A–B,G, I;Supplementary Fig. S7A and S7B). In contrast, the angiogenicpositive control VEGFA plugs showed strong angiogenicresponses. Matrigel plugs containing both CCL28 and VEGFD orCCL27 and VEGFD also showed an angiogenic response (Fig. 5D,G, and I; Supplementary Fig. S7D). These results suggest that thechemotactic effect of CCL27 or CCL28 in Matrigel plugs is biasedmore toward a lymphangiogenic than an angiogenic response,corroborating the results of the in vitro chemotaxis assays(Fig. 4C).

CCL27 promotes tumor-associated recruitment of lymphaticendothelial cells

To study the role of CCR10 signaling in promoting tumorlymphangiogenesis, two independent CCL27-overexpressingtumor cell lines were created, based on the 293EBNA modelof lymphogenous spread driven by exogenous lymphangio-genic growth factors (15, 19). Xenograft tumors formed fromCCL27-overexpressing cells were less vascularized thanVEGFD-293EBNA tumors, but grew readily (Fig. 6A–D; Sup-plementary Fig. S8A). Tumors expressed CCL27, as shown by

CCL27 immunoprecipitation and Western blotting from con-ditioned media and tumor lysates (Fig. 6A; SupplementaryFig. S8B).

Immunostaining for LYVE1 and PECAM1was used to visualizelymphatics and blood vessels within these tumors. Quantificationof LYVE1þ microvessels revealed a statistically significant differ-ence between CCL27-overexpressing 293EBNA tumors and con-trol tumors (Fig. 6E–H; Supplementary Fig. S8C). LYVE1þ struc-tures identified in CCL27-overexpressing tumors were often ofsmall size, in some cases resembling single migrating LYVE1þ

LECs, and were distributed predominantly around the tumorperiphery (Fig. 6F). LYVE1þ microvessel structures tended not tocolocalize with F4/80 staining, although some single cells cost-ained for LYVE1 and F4/80 were observed (Supplementary Fig.S8D). This was in contrast to the extensive distribution of dilatedlymphatics both centrally within and peripheral to VEGFD–over-expressing tumors (Fig. 2A and 6G). In further contrast withVEGFD-293EBNA tumors, blood vessel density in CCL27-293EBNA tumors was similar to that of control tumors(Fig. 6E–G and I). The observation that VEGFD promotes angio-genesis (through VEGFR3 and VEGFR2 expressed on blood vas-cular endothelium) is consistent with previous findings (15).These results provide further support that CCL27 preferentiallyfacilitates migration of LECs and other prolymphangiogenic cellsrather than BECs.

Having demonstrated that CCL27 could induce the forma-tion of LYVE1þ structures within CCL27-overexpressingtumors, it was explored whether these structures were func-tionally sufficient to promote tumor metastasis in the absenceof VEGFD. Draining lymph nodes from mice bearing CCL27-293EBNA tumors assessed for the presence of tumor cells hadno detectable metastasis, in contrast with those bearingVEGFD-293EBNA tumors (Supplementary Table S2). Theinability of CCL27-overexpressing tumors to promote lymphnode metastasis despite increased LYVE1þ structures may bedue to growth factors or chemokines that are required formetastasis not being expressed in CCL27-293EBNA cells, suchas VEGFD.

In addition to local effects, we and others have shown thatsecreted lymphangiogenic growth factors can enhance metas-tasis by remodeling collecting lymphatic vessels distal to thetumor, promoting lymphatic vessel dilation and increasedlymph transport rate (19, 44). Therefore, we assessed thediameter of collecting lymphatic vessels in the CCL27-293EBNA tumors. Unlike VEGFD, CCL27 secretion had noeffect on dilation of draining collecting lymphatic vessels(Fig. 6J–M), potentially contributing to the observed lack ofmetastasis.

Interestingly, we also observed accelerated growth of CCL27-overexpressing tumors relative to control 293EBNA tumors(Fig. 6N; Supplementary Fig. S8A). This may result from pro-survival autocrine signaling through CCR10 expressed in293EBNA tumor cells (Fig. 1A). CCL27 or CCL28 signalingthrough CCR10 is known to promote tumor growth and tumorcell survival or proliferation in several tumor types includingmelanoma, glioblastoma, and hepatocellular carcinoma, chief-ly mediated by PI3K/Akt signaling (39, 45, 46). Some contri-bution of secreted factors from recruited CCR10þ leukocytes orstromal cells to lymphangiogenesis and tumor growth is alsoconceivable. For example, in syngeneic models of ovariancancer, CCR10þ regulatory T cells recruited by tumor-expressed






Figure 6.

CCL27 promotes tumor-associated lymphangiogenesis. A,Western blot (WB) for immunoprecipitated (IP) CCL27 from conditioned medium of tumor cells.Positions of molecular weight markers (kDa) are shown on the left. B–D,Mouse xenograft tumors produced from subcutaneous flank injection of pVITRO3-293EBNA (control; B), CCL27-293EBNA (C), or VEGFD-293EBNA (D) cells. E–G, LYVE1 (green) and PECAM1 (red) immunofluorescence of pVITRO3-293EBNA(E), CCL27-293EBNA (F), or VEGFD-293EBNA (G) tumors. Right, magnified view of the boxed area. Scale bars, 50 mm. H and I,Quantification of LYVE1-positivevessels (H) or PECAM1-positive vessels (I) in xenograft tumors. Data points represent the average % stained area of <15 fields with mean� SEM of n¼ 5(pVITRO3), n¼ 10 (CCL27), or n¼ 4 (VEGFD) tumors. � , P < 0.05; �� , P < 0.001 by Kruskal–Wallis test with uncorrected Dunn test. J–L, Collecting lymphaticvessels filled with Patent Blue V frommice with pVITRO3-293EBNA (J), CCL27-293EBNA (K), or VEGFD-293EBNA (L) tumors. Arrowheads demarcate thevessel.M, Collecting lymphatic vessel diameter in mice with subcutaneous tumors. Data represent mean� SEM of 5 mice. �� , P < 0.01 by Student t test.N, Volumes of CCL27-293EBNA, VEGFD-293EBNA, or pVITRO3-293EBNA tumors plotted over time. Data represents mean tumor volume� SEM of twoindependent experiments, each with 5–10 mice per group.

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CCL28 promoted tumor angiogenesis by secreting VEGFA (32).However, the 293EBNA tumor models were conducted inSCID/NOD mice lacking mature T and B lymphocytes, whichsuggests a more direct effect of CCL27 on lymphangiogenesis,and may explain the lack of angiogenesis observed in CCL27-293EBNA tumors.

Collectively, thesefindings provide evidence thatwhileCCR10-specific ligands can act as lymphangiogenic effectors, providingnavigational cues for migration of LECs, the resultant structuresmay not be functional and therefore cannot promote lympho-genous spread.

DiscussionSeveral previous studies of chemokines and endothelial cells

investigated the attraction of a mobile cell population(immune cells or tumor cells) toward a chemokine gradientestablished by a "stationary" endothelial cell population (7,13). This study suggests a different model, in which chemo-kines (CCL27 or CCL28) secreted by the primary tumor may actin concert with lymphangiogenic factors (VEGFC or VEGFD) topromote LEC migration and lymphatic vessel remodeling.In the early stages of endothelial cell movement, CCL27 orCCL28 may orient and attract LECs toward the tumor throughCCR10. VEGFD enhances CCR10 expression, thus potentiatingmigration, and is required to drive subsequent proliferation,

remodeling, and formation of patent lymphatic vessels that cansupport metastasis (Fig. 7).

This model is concordant with a similar mechanismdescribed previously, in which VEGFC upregulated expressionof chemokine receptor CXCR4 in LECs, and CXCL12/CXCR4signaling operated in an additive manner with VEGFC/VEGFR3signaling to promote LEC migration and lymphangiogen-esis (47). The prior study showed that blockade of CXCL12/CXCR4 signaling could inhibit tumor lymphangiogenesis andlymph node metastasis of MDA-MB-231 cells. However, it didnot directly address whether CXCL12 alone, in the absence ofVEGFC, was sufficient to generate patent lymphatic vessels thatare competent to transport metastatic tumor cells. Anotherrecent study showed that CXCL5 signaling through LEC-expressed CXCR2 could induce lymphatic sprouting in vitroand lymphangiogenesis in vivo, and promoted metastasis in vivothrough the contribution of recruited neutrophils (48). Thisstudy indicates that, at least for CCL27 and CCL28, coexpres-sion of VEGFs (as we observed in xenografts of human tumorcell lines) may be required for the generation of functionallymphatic vessels.

Previous studies have indicated that CCL27 and CCL28 andtheir receptor CCR10 play pleiotropic roles in tumor biology,influencing tumor cell proliferation (39, 45, 46), migration andmetastasis (28), angiogenesis (32, 33), and recruitment of Tcells (36, 37). The specific function of these chemokines in atumor is likely influenced by tissue type and the associated

Figure 7.

Schematic diagram of mechanismsby which lymphangiogenic growthfactors and chemokines maypromote tumor spread. We andothers have shown that CCL28and/or CCL27 expression in tumorcells (blue cells) and CCR10 in LECsmay be upregulated by TNFa(green arrows). TNFa and otherinflammatory cytokines may bederived frommacrophagesrecruited by tumor-secretedVEGFD, or by other cells in thetumor microenvironment. CCR10can also be upregulated in LECs byVEGFD signaling via VEGFreceptors VEGFR3 or VEGFR2(green arrow), thus potentiatingmigration of LECs toward CCL27 orCCL28. Our data suggest a modelwhereby CCR10 upregulation maybe polarized in particular migratoryLECs within sprouting lymphaticvessels, with CCR10 and VEGFR3signaling potentially operating inthe same cells or adjacent cells.While CCL27 and CCL28 can guideLECmigration, cooperation withVEGFD signaling through VEGFreceptors is required to support LECproliferation and remodeling togenerate lymphatic vessels that cansupport metastatic spread of tumorcells.






immune context. Our data indicated expression of CCL27 andCCL28 along with VEGFD in a prostate cancer cell line and twohuman melanoma lines. Furthermore, we detected upregulationof CCR10 and CCL27 in nevi relative to both skin and malignantmelanoma in a publicly available microarray dataset. Upregula-tion of CCL27 enhances recruitment of T lymphocytes in inflam-matory or hyperproliferative skin conditions such as psoria-sis (30), but its expression was progressively lost during progres-sion of keratinocyte-derived skin tumors, suggesting amechanismfor immune evasion (36). Perhaps accordingly, higher expressionof CCL27 in the epidermis covering melanomas was found to becorrelated with positive clinical outcome (49). Little is knownabout CCL28 inmelanoma, although one study observed specificupregulation of CCL28 in cutaneous metastases relative to othermetastatic sites (50). Our data add to the understanding of themultiplicity of roles for these chemokines in cancer.

The involvement of immune cells also needs to be consideredin understanding the influence of CCL27 and CCL28 on tumorlymphangiogenesis. VEGFD recruitsmacrophages to tumors (17),and we also observed recruitment of LYVE1þ/F4/80þ cells toMatrigel plugs and (to a lesser extent) tumors containing CCL27or CCL28. Macrophages and other myeloid cells can supporttumor lymphangiogenesis by secreting prolymphangiogenic fac-tors and/or by incorporating directly into lymphatic vessels, andalso play important roles in developmental lymphatic pattern-ing (41, 42). Our data suggest that macrophages recruited byVEGFD or other factors can further shape tumor lymphangiogen-esis by enhancing expression of CCR10 in LECs and its ligands intumor cells via their secretion of proinflammatory cytokines suchas TNFa.

While our in vitro data showed that CCL27 and CCL28 arechemotactic for LECs through CCR10, the morphologic altera-tions to lymphatic patterning observed in the skin of Ccr10�/�

mice further suggest a role for these chemokines in forming newlymphatic sprouts or branches (29, 51). CXCL5 signaling throughCXCR2 in LECs promoted lymphatic sprouting in vitro (48), whilelymphatic branching frequency was negatively regulated by thedecoy chemokine receptor ACKR2 expressed in lymphatics (52).The chemokine receptor CXCR4 is enriched in blood vascular tipcells and together with Dll4-Notch signaling regulates vascularsprouting and patterning (51, 53, 54), but the potential role ofchemokines and their receptors in lymphatic tip cells remainsunexplored. The restricted expression of CCR10 in a subset ofLECs in vitro and in vivo would also be consistent with a tightlyregulated role in formation and guidance of new lymphaticsprouts, where CCR10 is upregulated in selected LECs in responseto environmental cues. CCR10 has also been observed coex-pressedwithCCL27 in someprecollecting lymphatics (8). Furtherwork is required to confirm precisely how CCR10 contributes tolymphatic patterning.

The functional consequences of the developmental lymphaticdefects observed in Ccr10�/� mice also warrant further investiga-tion.We did not observe overt edema, a common consequence oflymphatic dysfunction, in Ccr10�/� mice or embryos. However,other studies have reported that CCR10 deficiency leads to exac-erbated edema and delayed resolution of tissue swelling ininflamed ear skin during immune responses (55, 56). Deficientlocalization of CCR10þ regulatory T cells into skin contributed tothis hyperinflammatory phenotype, which included an influx ofmyeloid cells. Although these changes in leukocyte recruitmentmay indirectly influence inflammation-associated lymphangio-

genesis, the prolonged edema could also be due in part to aprimary lowered density of skin lymphatics, and/or a deficientlymphangiogenic response from LECs lacking CCR10. Tumorstudies in Ccr10�/� mice would help to further clarify the impor-tance of CCR10 in pathologic lymphangiogenesis.

Specific regulators of LEC migration represent potential ther-apeutic targets to modulate pathologic lymphangiogenesis insettings such as cancer (47). However, the diversity of signalingpathways involved in this process is only beginning to be uncov-ered (57). The current study identifies CCL27/CCL28 signalingthrough CCR10 on LECs as a novel chemokine signaling axisthat can promote LEC migration and potentially influence lym-phatic patterning, and that can contribute to lymphangiogenesisin vivo with the cooperation of VEGFD.

Disclosure of Potential Conflicts of InterestM.G. Achen and S.A. Stacker have ownership interest (including stock,

patents, etc.) in Opthea Ltd. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: T. Karnezis, N.C. Harris, R. Shayan, S. Yazar,M.G. Achen, S.A. StackerDevelopment of methodology: T. Karnezis, N.C. Harris, Y.-F. Zhang, S. YazarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Karnezis, R.H. Farnsworth, N.C. Harris, S.P.Williams, C. Caesar, D.J. Byrne, M.L. Macheda, Y.-F. Zhang, S.J. Takouridis,S.B. Fox, M.G. Achen, S.A. StackerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Karnezis, R.H. Farnsworth, N.C. Harris,S.P. Williams, C. Caesar, D.J. Byrne, P. Herle, R. Shayan, Y.-F. Zhang,S. Yazar, M.G. Achen, S.A. StackerWriting, review, and/or revision of the manuscript: T. Karnezis,R.H. Farnsworth, N.C. Harris, C. Caesar, M.L. Macheda, R. Shayan, S.B. Fox,M.G. Achen, S.A. StackerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.C. Harris, C. Caesar, D.J. Byrne, C. Gerard,S.B. Fox, S.A. StackerStudy supervision: T. Karnezis, M.G. Achen, S.A. Stacker

AcknowledgmentsThe authors thank Sally Roufail for expert technical assistance and Andrew

Naughton, Jacinta Carter and Animal Facility staff at the Ludwig Institute forCancer Research (Melbourne) and Peter MacCallum Cancer Centre for assis-tance withmouse experiments; Janna Taylor for assistance in generating figures;Jason Li for bioinformatics assistance; and Bao Lu of the Boston Children'sHospital, Harvard Medical School, for providing Ccr10�/�mouse tissues. Theauthors also acknowledge the support and resources of the Centre for AdvancedHistology andMicroscopy at the PeterMacCallumCancer Centre (A/Prof. SarahEllis, Marne Prinsloo, Thu Ming Noc Nguyen, Ethan Passantino, Metta Jana, JillDanne, Cameron Skinner, DhanyaMenon), alongwith imaging assistance fromStephenCody, CameronNowell, andNaomiCampanale. Thisworkwas fundedpartly by a Program Grant from the National Health and Medical ResearchCouncil of Australia (NHMRC). S.A. Stacker and M.G. Achen are supported byNHMRC Senior Research Fellowships. S.A. Stacker would like to acknowledgethe support of the Pfizer Australia Fellowship. R. Shayan is supported by theRaelene Boyle Sporting Chance Foundation and Royal Australasian College ofSurgeons (RACS) Foundation Scholarship, and the RACS Surgeon ScientistProgram.

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 indicatethis fact.

Received June 19, 2018; revised November 1, 2018; accepted January 29,2019; published first February 1, 2019.

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Karnezis et al.




2019;79:1558-1572. Published OnlineFirst February 1, 2019.Cancer Res Tara Karnezis, Rae H. Farnsworth, Nicole C. Harris, et al. Lymphatic Endothelial Cells

CCR10 Chemokine Signaling Mediates Migration of−CCL27/CCL28

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