Ccl27/Ccl28-Ccr10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

Author Manuscript Published OnlineFirst on February 1, 2019; DOI: 10.1158/0008-5472.CAN-18-1858 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

CCL27/CCL28 CCR10 CHEMOKINE SIGNALING MEDIATES MIGRATION OF

LYMPHATIC ENDOTHELIAL CELLS

Tara Karnezis1,2,3,4, Rae 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

1,2 2 2 6 1,7 Zhang , Sezer Yazar , Simon J. Takouridis , Craig Gerard , Stephen Fox , Marc G.

Achen1,2,5,7 and Steven A. Stacker1,2,5,7

1 Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.

2Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria 3050,

Australia.

3 , Victoria

3065, Australia.

4 Fitzroy,

Victoria 3065, Australia.

5Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville,

Victoria 3050, Australia.

7Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville,

Victoria 3010, Australia.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 1, 2019; DOI: 10.1158/0008-5472.CAN-18-1858 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running title: CCR10 ligands mediate lymphatic endothelial cell migration

Keywords: lymphatic endothelial cells, migration, lymphangiogenesis, CCL27/CCL28,

CCR10

Financial support: This work was funded partly by a Program Grant from the National

Health and Medical Research Council of Australia (NHMRC). SAS and MGA are supported

by NHMRC Senior Research Fellowships. SAS would like to acknowledge the support of the

Pfizer Australia Fellowship. RS is supported by the Raelene Boyle Sporting Chance

Foundation and Royal Australasian College of Surgeons (RACS) Foundation Scholarship,

and the RACS Surgeon Scientist Program.

Corresponding author: Steven Stacker, Peter MacCallum Cancer Centre, 305 Grattan St,

Melbourne 3000. Phone: +61 3 8559 7106; Email: [email protected]

Conflict of interest: MGA and SAS are shareholders of Opthea Ltd, a company involved in

developing therapeutics and diagnostics for vascular targets. All other authors declare no

competing financial interests.

ABSTRACT

Metastasis via the lymphatic vasculature is an important step in cancer progression. The

formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing

lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic

vessels and on to distant organs. The migration of lymphatic endothelial cells (LECs) toward

guidance cues is critical for lymphangiogenesis. While chemokines are known to provide

directional navigation for migrating immune cells, their role in mediating LEC migration

during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene

profiling studies to identify chemokine chemokine receptor pairs that are involved in tumor

lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were

expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was

expressed by LECs and up-regulated by the lymphangiogenic growth factor VEGF-D and the

pro-inflammatory cytokine TNF- . 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 in lymphatic

patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source,

while VEGF-D was required in combination with these chemokines to enable formation of

coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even

though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasise

was dependent on the expression of VEGF-D. These studies demonstrate that CCL27 and

CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGF-D

during tumor lymphangiogenesis.

Statement of Significance

The study shows that the remodeling of lymphatic vessels in cancer is influenced by

chemokines, which may provide a future target to modulate metastatic spread.

INTRODUCTION

Spread of tumor cells via lymphatic vessels to regional lymph nodes is a key early step in the

metastasis of many human malignancies (1,2). Many of these tumors induce

lymphangiogenesis the formation of new lymphatics and lymphatic vessel remodelling

via specific growth factors, the best-characterized of which are vascular endothelial growth

factor (VEGF)-C and VEGF-D (1). These proteins act through receptor tyrosine kinases

VEGFR-2 and VEGFR-3 on lymphatic endothelial cells (LECs) to drive lymphangiogenesis,

thus providing tumors with greater access to the lymphatic system and thereby promoting

metastasis (1-3). Another mechanism contributing to metastasis via the lymphatics involves

exploiting a normal physiological function of lymphatics, whereby tumor cells mimic

immune cells that traffic from peripheral tissues to lymph nodes and other secondary

lymphoid sites, a process for which chemokines and their receptors are essential (4-6).

Chemokines are a family of over 50 small chemoattractant cytokines that bind in a

non-exclusive manner to over 20 cell surface G protein coupled receptors on target cells,

allowing the cells to migrate along chemokine gradients to selected tissues (4). For example,

T and B lymphocytes and dendritic cells express chemokine receptor CCR7 upon activation,

causing them to migrate toward LECs expressing the CCR7 ligand CCL21 (6,7). Similarly,

LECs secreting CCL27 guided entry of activated T cells expressing the CCL27 receptor,

CCR10, to afferent lymphatic vessels (8). An experimental model in which B16 murine

melanoma cells overexpressed CCR7 showed greatly enhanced metastasis to lymph nodes

expressing CCL21 (9).

Chemokines have also previously been implicated in tumor-associated angiogenesis

(4,10). CXCL12, through its receptor CXCR4, can induce blood endothelial cell (BEC)

migration and promote angiogenesis both in vitro and in vivo (10). Interestingly, CXCL12

was also shown to act synergistically with the angiogenic factor, VEGF-A, to induce

vascularization in ovarian cancer (11). In contrast, the role of chemokines in

lymphangiogenesis is only beginning to emerge. Metastatic melanoma cells expressing CCR7

migrated toward LECs in a CCL21-dependent manner (12); while co-expression of CCR7

and VEGF-C by tumor cells also enhanced their migration toward lymphatics and metastasis

to lymph nodes (13). Despite this early evidence, the extent of chemokine involvement, and

their specific roles in tumor-associated lymphangiogenesis, remain poorly defined.

In the current study, we identified the CCL27 and CCL28 signaling through CCR10

as a mechanism to induce the recruitment of LECs during tumor lymphangiogenesis. We

confirmed the capacity of CCL27 and CCL28 to drive LEC migration and showed a role for

CCR10 in lymphatic vessel development and patterning. We demonstrated that CCL27 and

CCL28 cooperate with VEGF-D to promote lymphangiogenesis. These findings further

define the molecular mechanisms controlling tumor lymphangiogenesis.

MATERIALS AND METHODS

Animals

SCID/NOD mice (RRID:IMSR_JAX:001303; IMVS, Adelaide, Australia), housed in

microisolators, and FVB mice (Walter and Eliza Hall Institute, Melbourne, Australia) were

6 8 weeks old. Ccr10 / mice (RRID: MGI:3822166) were obtained from

Hospital, Harvard Medical School, Boston, and genotyped as described previously (14).

Australian code for the 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 Peter MacCallum

Cancer Centre Animal Experimentation Ethics Committee.

Cell lines

293EBNA cells stably expressing VEGF-D (VEGF-D-293EBNA) or empty expression

vector (Apex-293EBNA) were passaged as previously described (15). The CCL27-

overexpressing 293EBNA and vector control pVITRO3-293EBNA cell lines were created in

a similar manner to VEGF-D-293EBNA (15,16), using human CCL27 cDNA, cloned by

PCR from VEGF-D-293EBNA cells (using primers: forward 5 -

GGAAGAGTCTAGGCTGAGCAA-3 and reverse 5 -TCCAATGCTGCTTTATTATTTGG-

3 ), which was then ligated into pVITRO3mcs (InvivoGen). Two independent tumor cell

lines were generated by cloning CCL27 cDNA into pcDNA 3.1 (Invitrogen) and stably

transfecting either empty pcDNA 3.1 vector 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 (ATCC Cat# CRL-1420, RRID:CVCL_0428), SK-MEL-2

(ATCC Cat# HTB-68, RRID:CVCL_0069) and Caco-2 cells (from Antony 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 for Cancer Research, Australia), PC-3 (from Chris

Hovens, University of Melbourne, Australia), DLD-1 and HT-29 cells (from Antony

Burgess) were maintained in RPMI-1640 (Life Technologies) with 10% FBS. Dates of cell

acquisition, thawing and mycoplasma testing are outlined in Supplementary Table S1.

Primary human dermal lymphatic and blood microvascular endothelial cells (Lonza) were

grown on fibronectin-coated plates (5 g/ml; Sigma-Aldrich), in complete endothelial cell

medium (EGM-2-MV; Lonza). Cell lines were incubated at 37 C in 5% CO2, except MDA-

MB-435 (10% CO2). Growth rate and morphology of cell lines were monitored routinely, and

VEGF-D or CCL27 expression by stably transfected cells was verified by Western blot prior

to each experiment.

RNA isolation and quantitative RT-PCR

RNA was isolated using the RNeasy Mini or RNeasy Plus Mini Kit (Qiagen). To investigate

mRNA expression, total RNA was reverse transcribed using SuperScript II Reverse

Transcriptase or High capacity cDNA Reverse Transcription kit with a mixture of oligo(dT)

and random hexamer primers (Life Technologies). Quantitative reverse-transcription PCR

(qRT-PCR) analysis was performed using TaqMan Gene Expression assays (ABI), with assay

IDs: CCR10, Hs00706455_s1; CCL27, Hs00171157_m1; CCL28, Hs00219797_m1; VEGF-

D (FIGF), Hs01128659_m1; beta-actin, 4333762T. To assess CCR10 knockdown, qPCR was

performed using Sensimix Plus SYBR Green (Quantace) and CCR10 primers (forward: 5 -

TGCTGGATACTGCCGATCTACTG-3 ; reverse: 5 -

TCTAGATTCGCAGCCCTAGTTGTC-3 ). All data were normalized to -actin.

Chemokine and chemokine receptor miniarrays

To determine chemokine and chemokine receptor expression in LECs and cancer cell lines,

RNA was isolated using the ArrayGrade Total RNA Isolation Kit (SABiosciences). Sample

RNA was reverse transcribed into cDNA then transcribed into biotin-labelled cRNA target

using the TrueLabeling- (SABiosciences). Labelled target cRNA was then

hybridized to an Oligo GEArray® Human Chemokine and Chemokine Receptor miniarray,

The intensity of each spot was quantified

using ImageJ software (NIH) and normalized relative to GAPDH. A heat map with

hierarchical clustering was created using Cluster 3.0 and Java TreeView software.

Chemokines, growth factors and ELISA

Recombinant human CCL27, CCL28, VEGF-A, interferon (IFN- ), tumor necrosis factor-

(TNF- ) - (all from R&D Systems), and mature VEGF-C and VEGF-D

(Opthea Pty Ltd) were used in various assays. Levels of human CCL27 and CCL28 secreted

by cancer cell lines were quantified using Quantikine ELISA Kits (R&D Systems).

siRNA-mediated gene knockdown

FlexiTube siRNAs targeting human CCR10 mRNA (Qiagen) were used for transient CCR10

knockdowns. Negative Control siRNA (Qiagen) was also used. LECs (2 105) were reverse

transfected with 40 nM siRNA in 6-well plates, using 10 l/well siPORT Amine Transfection

Reagent (Ambion, Life Technologies), and medium was changed 24 h post-transfection.

CCR10 expression was assessed 72 h post-transfection by qRT-PCR or flow cytometry.

Tumor establishment

Tumors were established in the flanks of female SCID/NOD mice by subcutaneous injection

107 293EBNA cells, stably transfected with expression vectors cDNA encoding

either VEGF-D or CCL27 or with empty vector control plasmids (pApex, pVITRO3 or

pcDNA 3.1), as described (15). Other mice were injected with 3 107 MDA-MB-435 or

FEMX-I cells, or 6 106 DU 145 cells. Mice were euthanized when tumor volume reached

1500 2000 mm3.

In vivo lymphangiogenesis assay and immunofluorescence

Female FVB mice were injected subcutaneously in the flank with 500 l of Matrigel (Growth

Factor Reduced Basement Membrane Matrix, Corning/BD Biosciences) containing 2.5 g

VEGF-A, VEGF-D, CCL28, combined VEGF-D and CCL28, or PBS as control. In a second

experiment, 200 l of Matrigel was injected containing 4 g/ml VEGF-A, 4 g/ml VEGF-D,

2 g/ml CCL27, 4 g/ml VEGF-D plus 2 g/ml CCL27, or PBS + 0.1% BSA. After seven

days mice were euthanized, and Matrigel plugs were removed and snap frozen in Tissue-Tek

OCT compound (Sakura Finetek). Cryosections of Matrigel plugs were stained using

antibodies for rabbit anti-mouse LYVE1 (Fitzgerald Industries International Cat# 70R-LR003,

RRID:AB_1287923), hamster anti-mouse podoplanin (Fitzgerald Cat# 10R-P155a,

RRID:AB_1288912) or rat anti-mouse PECAM1/CD31 (BD Pharmingen Cat# 553370,

RRID:AB_394816) to assess the extent of lymphangiogenesis and angiogenesis, as described

(17). Staining was quantified over 2-5 fields each from 1-3 sections per Matrigel plug or

tumor (up to 15 fields per mouse). F4/80 antigen (macrophage marker) was detected using a

rat anti-mouse antibody (Abcam Cat# ab6640, RRID:AB_1140040 or BioLegend Cat#

123102, RRID: AB_893506).

Proliferation and migration assays

LEC proliferation was analysed using the CellTiter 96 AQueous One Solution Cell

Proliferation Assay (Promega). Approximately 2,000 cells were resuspended in proliferation

medium (EBM-2 medium (Lonza), 2% FBS, without supplements) containing purified

growth factors and incubated for 72 h before the addition of the MTS-containing assay

reagent.

Migration assays were performed using HTS FluoroBlok migration chambers with 8

m pore size membrane inserts (BD Biosciences). 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 the membrane coated with fibronectin (5

g/ml). Starvation medium containing purified growth factors or chemokines was added to

the lower wells. After 16 24 h, cells that migrated to the underside of the membrane were

stained with calcein (5 g/ml; Molecular Probes) or fixed with 4% paraformaldehyde in PBS

and stained with DAPI (5 g/ml). Cells were then visualized using a Nikon TE2000-E

microscope and quantified using MetaMorph software (Molecular Devices, LLC). For

experiments involving siRNA knockdown of CCR10, cells were transfected 48 hours prior to

the migration assay (see siRNA mediated gene knockdown), migrated cells visualized with

calcein and counted manually. In other experiments, cells were pre-incubated with functional

blocking antibodies to human CCL27 (R&D Systems Cat# MAB376, RRID: AB_2070653)

prior to the migration assay.

Flow cytometry

For detection of cell-surface CCR10, LECs were incubated with rat anti-human CCR10

antibody (R&D Systems Cat# FAB3478A, RRID:AB_573043) for 20 30 min on ice, then

stained with fluorescently-conjugated anti-rat secondary antibodies and propidium iodide

(Sigma-Aldrich). Cells were analyzed using a FACSCalibur flow cytometer (BD

Biosciences) and FlowJo software (TreeStar Inc.).

Immunoprecipitation and Western blot analysis

To assess CCL27 protein expression levels in 293EBNA cells, conditioned medium was

collected and immunoprecipitations performed using mouse anti-human CCL27 antibody

(Santa Cruz Biotechnology #sc-390112, RRID:AB_2736849). Immunoprecipitates were run

on 10% or 16% Tricine gels (Novex; Life Technologies) and Western blotted using the same

antibody as used for immunoprecipitation.

Immunohistochemistry, whole mount staining and visualization of collecting lymphatic

vessels

Mouse and human tissues were harvested and either frozen in OCT or fixed in 4%

paraformaldehyde or 10% neutral buffered formalin before paraffin embedding and

sectioning. Sections were immunostained for LYVE1 (Fitzgerald anti-mouse Cat# 70R-

LR003; anti-human Cat# 70R-LR004, RRID:AB_1287920), hamster anti-mouse podoplanin

(Fitzgerald Cat# 10R-P155a, RRID:AB_1288912), PECAM1/CD31 (BD Pharmingen Cat#

553370, RRID:AB_394816), CCR10 (Novus Biologicals Cat# NB100-56319,

RRID:AB_837897), Prox1 (BioLegend Cat# 925201, RRID:AB_291595), F4/80 (Abcam

Cat# ab6640, RRID:AB_1140040 or BioLegend Cat# 123102, RRID: AB_893506), and

developed with specific secondary antibodies as described (17). To stain for chemokines,

paraffin sections underwent optimized antigen retrieval with trypsin for CCL28 (Proteintech

Cat# 18214-1-AP, RRID:AB_2262251); or Tris EDTA pH 9.0 (skin) for CCL27 (R&D

Systems, #MAB376). After incubation with HRP-conjugated secondary antibodies, and

tyramide signal amplification (Perkin Elmer), the signal was developed using DAB and

sections counterstained with haematoxylin. Staining was quantified using MetaMorph

software.

Whole mount staining and morphometric analysis of ear tissue from Ccr10 / mice

were performed as described previously (18). Collecting lymphatic vessel visualization based

on dye injection was performed as described (19).

Reanalysis of publically-available microarray data

A publically-available microarray dataset representing normal human skin, benign nevi and

melanoma (GSE3189; 20) was downloaded from Gene Expression Omnibus (GEO;

https://www.ncbi.nlm.nih.gov/geo/). Downloaded data had been preprocessed by

normalization, background subtraction and log2 transformation and were reanalysed without

further processing. Unsupervised clustering was performed using Cluster 3.0 software.

Another dataset derived from human LECs stimulated for 24 h with 1 ng/ml TNF-

(GSE6257; 21) was reanalyzed to identify differentially-expressed genes using the GEO2R

web interface (https://www.ncbi.nlm.nih.gov/geo/geo2r/) based on limma (Linear Models for

Microarray Analysis).

Statistical analysis

Pairs of conditions were compared using a -test for normally-distributed data or a

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

using a one-way ANOVA with post-hoc test for normally-distributed

data, or a Kruskall-Wallis test for non-normally distributed data. Sample sizes were

determined as appropriate for each assay based on empirical determination in prior

experiments.

RESULTS

Chemokine profiling reveals a CCL27/CCL28 CCR10 signaling axis in

lymphangiogenesis

We and others have shown that the lymphangiogenic factors VEGF-C and VEGF-D can

induce tumor cell metastasis to regional lymph nodes by promoting tumor-associated

lymphangiogenesis (1). To identify pairs of chemokine receptors and ligands important

during this pathological lymphangiogenesis, expression of chemokines and chemokine

receptors was analysed using an established tumor model of VEGF-D-dependent

lymphogenous spread (15,19). In this model, VEGF-D overexpression in 293-EBNA cells

drives tumor-associated lymphangiogenesis, lymphatic remodeling and metastasis, similarly

to other tumor cell lines that endogenously overexpress VEGF-D (e.g. 66cl4 murine breast

carcinoma and MDA-MB-435 human melanoma (19)). As an in vitro model of the tumor

microenvironment, the mRNA profiles of cultured VEGF-D-293EBNA cells and LECs were

analysed using cDNA miniarrays. VEGF-D-293EBNA cells expressed CCL27 and CCL28,

while LECs expressed their cognate receptor, CCR10 (Figure 1A; Figure S1). CCR10 was

also expressed in VEGF-D-293EBNA cells, suggesting there may be some autocrine

signaling in these cells. Other chemokines and their corresponding receptors that were

expressed across the two cell types included CCL19 in VEGF-D-293EBNA cells and the

receptor CCRL2 in LECs, as well as the CXCL12-CXCR4 signaling pair, implicated in

angiogenesis (4,10). We chose to focus on CCL27/CCL28 and their receptor CCR10 as these

chemokines are expressed in tissues such as skin (CCL27) and mucosal epithelia (CCL28)

which are common sites of cancer (22,23).

To validate the miniarray data, qRT-PCR was performed on human LECs and VEGF-

D-293EBNA cells, which confirmed that they expressed CCR10 or CCL27 and CCL28

mRNA, respectively (Figure 1B, C). Interestingly, examination of CCR10 mRNA showed

expression was higher in LECs compared to BECs in vitro, and further increased in LECs

(but not BECs) stimulated with VEGF-D (Figure 1C). Flow cytometry was used to confirm

CCR10 expression on the surface of LECs (Figure 1D). Although the amount of cell-surface

CCR10 was low, the specificity of the signal was confirmed by targeting CCR10 with

siRNA: siCCR10_4 reduced CCR10 mRNA expression to less than 50% relative to the

negative control (Figure 1E) and completely abrogated the CCR10 antibody signal in flow

cytometry (Figure 1D). Furthermore, CCR10 staining in LYVE1-positive lymphatic vessels

was confirmed by immunohistochemistry of human breast cancer (ductal carcinoma in situ;

DCIS) and normal colon tissue specimens (Figure 1F). CCR10 staining appeared to be

restricted to a subset of cells comprising lymphatic vessels, implying that its expression may

become upregulated in a polarized or locally restricted manner in response to secreted

guidance cues. VEGF-D is known to be expressed in DCIS, which often exhibits

lymphangiogenesis (24,25). Our results suggest that focal upregulation of CCR10 by VEGF-

D and/or other factors may be involved in regulating lymphangiogenesis in diverse tissues.

To confirm the role of CCR10 in lymphangiogenesis, lymphatic vessel patterning was

examined in CCR10-deficient mice. Ccr10 / mice show defective homing and accumulation

of specific lymphocyte subsets in vivo (14). Homing of specific CCR10+ T cell subsets to

skin is particularly affected, due to the highly-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 of CCR10 has not previously been investigated in

lymphatics, dermal lymphatic vessels in the ears of Ccr10 / mice (14) were examined and

compared to the characteristic pattern found in wild-type mice using LYVE1 wholemount

staining of ear skin (Figure 1G).

Quantification of defined parameters of lymphatic patterning (18) demonstrated that

Ccr10 / mice had significantly lower lymphatic vessel density, with lymphatics spaced

further apart and having fewer branches than wild-type vessels, but with normal vessel width

(Figure 1H). This pattern of decreased branching resembled that seen when mice are treated

in the early postnatal period with inhibitors of Dll4 or Notch1 signaling, which regulate

lymphatic vessel sprouting (29). This hints that CCR10 may be important for lymphatic

vessel sprout formation and branching. Interestingly, comparison of lymphatic vessels in late

embryonic dorsal skin of Ccr10 / mice to wild-type littermates revealed increased cross-

sectional area of individual lymphatic vessels, while the overall stained area of lymphatic

endothelium was unchanged (Figure S2A G). Combined with the observation of less

lymphatic branching in the adult ear, these results may indicate a defect in the sprouting of

initial lymphatics from the primitive dermal lymphatic plexus towards epidermal CCL27

expressed by keratinocytes (22,27), and therefore a relative paucity of smaller-calibre

lymphatics in the embryonic dermis of Ccr10-/- mice. Alternatively or additionally, these

defects in lymphatic vessel patterning may be contributed to by tissue-specific

microenvironmental factors, such as changes in the distribution of leukocyte subsets that

normally express CCR10 (e.g. plasmacytoid dendritic cells, Langerhans cells, skin-resident or

-homing T cells, plasma cells) and which may influence lymphatic development (14,23,30).

Overall, these results support the involvement of CCR10 in patterning the dermal lymphatic

network.

Distribution of CCL27 and CCL28 chemokines during tumor-associated

lymphangiogenesis

CCL27 is overexpressed in certain types of squamous cell carcinoma (31), whilst CCL28 is

expressed in ovarian carcinoma, lung adenocarcinoma and pancreatic adenocarcinoma (32-

34). Expression of CCL27 and CCL28 was further assessed in metastatic cancer cell lines

expressing endogenous VEGF-D (Figure S3A). CCL27 and CCL28 were expressed at

various levels in metastatic prostate, breast, colorectal and pancreatic cancer and melanoma

cell lines (Figure S3B C). CCL27 and CCL28 expression in a context of pathological

lymphangiogenesis in vivo was analysed using several human cancer cell lines in mouse

xenograft experiments. These cell lines included VEGF-D-293EBNA, and endogenous

VEGF-D-expressing melanoma (MDA-MB-435 and FEMX-I) and prostate cancer (DU 145)

cells, which also showed the highest levels of CCL27 and CCL28 mRNA (Figure S3A C).

Immunohistochemical analysis of VEGF-D-293EBNA, DU 145, MDA-MB-435 and FEMX-I

tumors revealed lymphatic vessels (using LYVE1 staining), as well as expression of both

CCL27 and CCL28 (Figure 2A, Figure S3D). CCL27 and CCL28 proteins were detected in

surrounding stroma as well as tumor cells, and in some cases were in close proximity to

lymphatic vessels. Expected staining patterns of CCL27 and CCL28 were observed in

positive-control tissues (human skin and colonic mucosa, respectively), validating the

specificity of these antibodies (Figure S3E F).

Co-immunofluorescence staining or immunohistochemistry on serial sections showed

that both CCL27 and CCL28 localized to LYVE1+ endothelial cells that comprise lymphatic

vessels (Figure 2B C). CCL27 expression has been detected in the precollecting subtype of

LECs in vivo (8); however, this subtype represents a higher-order, more complex vessel type

than the newly-formed initial lymphatics most likely present within and around tumors.

Given the lack of detectable CCL27 and CCL28 transcripts in LECs (Figure 1A), it is

possible that CCL27/CCL28 released from tumor cells into the surrounding stroma may bind

to CCR10 that is present on the surface of LECs (30).

To further validate expression of CCL27/CCL28 and their receptor CCR10 in human

tumors, a publically-available microarray dataset derived from normal skin, benign nevi and

malignant melanomas was analyzed (20) (Figure 2D, Figure S4 A-B). Melanoma specimens

were predominantly pre-metastatic, while the majority (78%) of the nevi were resected from

the trunk and lower limbs; notably, melanomas that have progressed from nevi are most

commonly associated with these anatomical locations (35). While CCL28 was not

represented on the microarray, both CCL27 and CCR10 were elevated in nevi relative to both

normal skin and malignant melanoma (Figure 2D). This observation may indicate a transient

role for this chemokine-receptor pair in regulating melanocyte proliferation or recruitment of

immune cells in nevi that is lost in melanomas that have acquired more potent driver

mutations and immune evasion mechanisms (36,37). Overall, our data emphasise that CCL27

and CCL28 are expressed in a variety of tumor cell types and can be co-expressed with the

lymphangiogenic growth factor VEGF-D.

Pro-inflammatory stimuli promote expression of CCR10 and its ligands

Inflammation is a hallmark of cancer. Furthermore, CCL27 or CCL28 expression in various

cancer cells or in skin can be upregulated by inflammatory cytokines (30,38,39). To test

whether this may be applicable to a model of pathological lymphangiogenesis, we assessed

whether expression of CCL27/CCL28 in tumor cells or CCR10 in LECs was altered by

inflammatory cytokines. Expression of CCR10 mRNA in LECs was increased by TNF-

stimulation, peaking at 16 h (Figure 3A). Upregulation of CCR10 protein by TNF- was also

detected (Figure S5 A-B). These data confirm a previous observation of CCR10 mRNA

upregulation in LECs by TNF- , detected by microarray (21) (Figure S5C). In VEGF-D-

293EBNA cells, IFN- , TNF- and IL-

over unstimulated cells, as detected by ELISA (Figure S5D). qRT-PCR indicated that CCL28

mRNA was consistently upregulated in response to TNF- ; although the fold increase varied,

the trend supported the increased expression observed by ELISA (Figure S5E). These data

confirm previous observations that CCR10 and its ligands can be upregulated by pro-

inflammatory cytokines.

Tumors from mouse xenograft experiments were subsequently examined for

infiltrating macrophages, which are known to secrete pro-inflammatory/pro-angiogenic

factors such as TNF- . VEGF-D-293EBNA tumors, which have a greater density of

lymphatic vessels than their non-metastatic counterparts, showed increased macrophage

recruitment, as determined by F4/80 staining (Figure 3B E). Although a small subset of

macrophages is known to express LYVE1, of all LYVE1+ structures in experimental VEGF-

D-293EBNA tumors, on average only ~6.5% were co-stained for the macrophage marker

F4/80 (Figure 3F). Co-localization of the lymphatic markers Prox1 and podoplanin in these

LYVE1+ vessel structures further validated their lymphatic identity (Figure S5F-G). These

results suggest that tumor cells secrete CCL27 and CCL28 in response to inflammatory

mediators that are typically produced by macrophages and other cells in the tumor

microenvironment, which may then attract CCR10-expressing LECs.

CCL27 and CCL28 promote CCR10-dependent LEC migration

To define the role(s) of CCL27 and CCL28 in lymphangiogenesis, in vitro LEC responses

toward these chemokines were evaluated. Unlike VEGF-D, which had a dose-dependent

effect on LEC proliferation, CCL27 and CCL28 had no significant proliferative effects

(Figure 4A). The effects of CCL27 and CCL28 on LEC migration were then assessed

alongside VEGF-C and VEGF-D, known inducers of LEC migration. LECs had statistically

significant migratory responses to titrations of CCL27 and CCL28, demonstrating

characteristic bell-shape curves typical of chemokine-induced migration (Figure 4B) (40).

LEC migration towards CCL27 could be blocked using an inhibitory antibody (Figure 4C;

Figure S6). Interestingly, CCL27 and CCL28 did not significantly promote migration of

BECs in these assays, indicating that LECs may be particularly responsive to the pro-

migratory effect of these chemokines (Figure 4C).

To confirm that CCL27/CCL28-induced LEC migration occurs via CCR10-specific

binding, gene knockdown studies were performed. CCR10 levels were reduced using siRNA

(Figure 1D E). Migration of LECs toward the CCR10 ligands CCL27 and CCL28 was

diminished when CCR10 levels were reduced by siCCR10_4, whereas CCR10 knockdown

had no effect on LEC migration toward VEGF-A (Figure 4D). These results indicate that

CCR10 specifically mediates CCL27/CCL28-induced LEC migration.

CCR10 ligands promote lymphatic recruitment in vivo

CCL28 and CCL27 were assessed for their ability to promote in vivo LEC migration, which

is necessary for the formation of nascent lymphatic vessels. Matrigel plugs containing CCL27,

CCL28, VEGF-D, VEGF-A, CCL27 or CCL28 together with VEGF-D, or PBS alone were

injected into mouse flanks. Approximately one week after implantation, plugs were removed

and immunofluorescence was performed to detect (Figure 5 -E, Figure S7A-E) and quantify

(Figure 5F-I) lymphatic and blood vessels using antibodies to LYVE1 and PECAM1

respectively.

Formation of LYVE1+ structures was evident in VEGF-D-containing positive control

Matrigel plugs, while minimal staining was observed in PBS negative control plugs (Figure

5A, C, F, H; Figure S7A, C). Matrigel plugs containing CCL28 or CCL27 showed more

LYVE1 staining compared to PBS controls (Figure 5A B, F, H; Figure S7A B). LYVE1+

structures in these plugs were small, often appearing to be single cells. LYVE1 staining

within Matrigel plugs co-localized predominately with staining for podoplanin, another LEC

marker (Figure 5J, Figure S7F-G). F4/80+ macrophages were detected in the Matrigel plugs

to varying degrees, with some co-staining for LYVE1 (Figure 5K, Figure S7H-I). Some

elongated LYVE1+ structures that resembled microvessels were weak or negative for F4/80,

while others exhibited associated F4/80 staining (Figure S7H-I). This possibly reflected

LYVE1+ macrophages or myeloid-derived lymphatic progenitors closely associating with or

incorporating into nascent lymphatic vessels in the Matrigel plugs, as has been observed in

experimental models of cancer and inflammation and some human cancers (41,42). These

data suggest that at least in Matrigel plugs, CCL27 and/or CCL28 may recruit both locally-

derived LECs and pro-lymphangiogenic myeloid or LEC progenitor cells from circulation.

Matrigel plugs containing CCL28 or CCL27 together with VEGF-D showed a

different pattern of LYVE1 staining compared to the plugs containing either factor alone. In

these plugs, LECs commonly formed more coherent vessel-like structures (Figure 5D, Figure

S7D). VEGF-D in combination with CCL27 or CCL28 showed a trend indicating an additive

effect on LYVE1 staining compared to the chemokines alone (Figure 5F, H). These results

suggest that VEGF-D works together with chemokines to promote LEC migration and

lymphatic vessel formation. The presence of VEGF-D in Matrigel plugs may enhance

lymphatic vessel formation from LECs recruited to the plug by CCL28 or CCL27 by

inducing LEC proliferation and vessel remodeling (19).

PECAM1 staining was next used to assess blood vessel density. Although PECAM1

is also present in low amounts on LECs, its relatively much higher abundance in BECs (43)

allows its use to measure blood vessel density when appropriate intensity thresholds are

applied. Blood vessel density was slightly higher in plugs containing CCL27 or CCL28 than

those containing PBS alone, although this difference did not reach significance (Figure 5A B,

G, I; Figure S7 A B). In contrast, the angiogenic positive control VEGF-A plugs showed

strong angiogenic responses. Matrigel plugs containing both CCL28 and VEGF-D or CCL27

and VEGF-D also showed an angiogenic response (Figure 5D, G, I; Figure S7D). These

results suggest that the chemotactic effect of CCL27 or CCL28 in Matrigel plugs is biased

more towards a lymphangiogenic than an angiogenic response, corroborating the results of

the in vitro chemotaxis assays (Figure 4C).

CCL27 promotes tumor-associated recruitment of lymphatic endothelial cells

To study the role of CCR10 signaling in promoting tumor lymphangiogenesis, two

independent CCL27-overexpressing tumor cell lines were created, based on the 293EBNA

model of lymphogenous spread driven by exogenous lymphangiogenic growth factors (15,19).

Xenograft tumors formed from CCL27-overexpressing cells were less vascularized than

VEGF-D-293EBNA tumors, but grew readily (Figure 6A D, Figure S8A). Tumors expressed

CCL27, as shown by CCL27 immunoprecipitation and Western blotting from conditioned

media and tumor lysates (Figure 6A, Figure S8B).

Immunostaining for LYVE1 and PECAM1 was used to visualize lymphatics and

blood vessels within these tumors. Quantification of LYVE1+ microvessels revealed a

statistically significant difference between CCL27-overexpressing 293EBNA tumors and

control tumors (Figure 6E H, Figure S8C). LYVE1+ structures identified in CCL27-

overexpressing tumors were often of small size, in some cases resembling single migrating

LYVE1+ LECs, and were distributed predominantly around the tumor periphery (Figure 6F).

LYVE1+ microvessel structures tended not to colocalise with F4/80 staining, although some

single cells co-stained for LYVE1 and F4/80 were observed (Figure S8 D). This was in

contrast to the extensive distribution of dilated lymphatics both centrally within and

peripheral to VEGF-D-overexpressing tumors (see Figure 2A). In further contrast with

VEGF-D-293EBNA tumors, blood vessel density in CCL27-293EBNA tumors was similar to

that of control tumors (Figure 6E G, I). The observation that VEGF-D promotes

angiogenesis (through VEGFR-3 and VEGFR-2 expressed on blood vascular endothelium), is

consistent with previous findings (15). These results provide further support that CCL27

preferentially facilitates migration of LECs and other pro-lymphangiogenic cells rather than

BECs.

Having demonstrated that CCL27 could induce the formation of LYVE1+ structures

within CCL27-overexpressing tumors, it was explored whether these structures were

functionally sufficient to promote tumor metastasis in the absence of VEGF-D. Draining

lymph nodes from mice bearing CCL27-293EBNA tumors assessed for the presence of tumor

cells had no detectable metastasis, in contrast with those bearing VEGF-D-293EBNA tumors

(Table S2). The inability of CCL27-overexpressing tumors to promote lymph node metastasis

despite increased LYVE1+ structures may be due to growth factors or chemokines that are

required for metastasis not being expressed in CCL27-293EBNA cells, such as VEGF-D.

In addition to local effects, we and others have shown that secreted lymphangiogenic

growth factors can enhance metastasis by remodeling collecting lymphatic vessels distal to

the tumor, promoting lymphatic vessel dilation and increased lymph transport rate (19,44).

Therefore, we assessed the diameter of collecting lymphatic vessels in the CCL27-293EBNA

tumors. Unlike VEGF-D, CCL27 secretion had no effect on dilation of draining collecting

lymphatic vessels (Figure 6J M), potentially contributing to the observed lack of metastasis.

Interestingly, we also observed accelerated growth of CCL27-overexpressing tumors

relative to control 293EBNA tumors (Figure 6N; Figure S8A). This may result from pro-

survival autocrine signaling through CCR10 expressed in 293EBNA tumor cells (Figure 1A).

CCL27 or CCL28 signaling through CCR10 is known to promote tumor growth and tumor

cell survival or proliferation in several tumor types including melanoma, glioblastoma, and

hepatocellular carcinoma, chiefly mediated by PI3K/Akt signaling (39,45,46). Some

contribution of secreted factors from recruited CCR10+ leukocytes or stromal cells to

lymphangiogenesis and tumor growth is also conceivable. For example, in syngeneic models

of ovarian cancer, CCR10+ regulatory T cells recruited by tumor-expressed CCL28 promoted

tumor angiogenesis by secreting VEGF-A (32). However, the 293EBNA tumor models were

conducted in SCID/NOD mice lacking mature T and B lymphocytes, which suggests a more

direct effect of CCL27 on lymphangiogenesis, and may explain the lack of angiogenesis

observed in CCL27-293EBNA tumors.

Collectively, these findings provide evidence that while CCR10-specific ligands can

act as lymphangiogenic effectors, providing navigational cues for migration of LECs, the

resultant structures may not be functional and therefore cannot promote lymphogenous

spread.

DISCUSSION

Several previous studies of chemokines and endothelial cells investigated the

attraction of a mobile cell population (immune cells or tumor cells) toward a chemokine

gradient established by a stationary endothelial cell population (7,13). The current study

suggests a different model, in which chemokines (CCL27 or CCL28) secreted by the primary

tumor may act in concert with lymphangiogenic factors (VEGF-C or VEGF-D) to promote

LEC migration and lymphatic vessel remodelling. In the early stages of endothelial cell

movement, CCL27 or CCL28 may orient and attract LECs towards the tumor through CCR10.

VEGF-D enhances CCR10 expression, thus potentiating migration, and is required to drive

subsequent proliferation, remodeling, and formation of patent lymphatic vessels that can

support metastasis (Figure 7).

This model is concordant with a similar mechanism described previously, in which

VEGF-C upregulated expression of chemokine receptor CXCR4 in LECs, and

CXCL12/CXCR4 signaling operated in an additive manner with VEGF-C/VEGFR-3

signaling to promote LEC migration and lymphangiogenesis (47). The prior study showed

that blockade of CXCL12/CXCR4 signaling could inhibit tumor lymphangiogenesis and LN

metastasis of MDA-MB-231 cells. However, it did not directly address whether CXCL12

alone, in the absence of VEGF-C, was sufficient to generate patent lymphatic vessels that are

competent to transport metastatic tumor cells. Another recent study showed that CXCL5

signaling through LEC-expressed CXCR2 could induce lymphatic sprouting in vitro and

lymphangiogenesis in vivo, and promoted metastasis in vivo through the contribution of

recruited neutrophils (48). The present study indicates that, at least for CCL27 and CCL28,

co-expression of VEGFs (as we observed in xenografts of human tumor cell lines) may be

required for the generation of functional lymphatic vessels.

Previous studies have indicated that CCL27 and CCL28 and their receptor CCR10

play pleiotropic roles in tumor biology, influencing tumor cell proliferation (39,45,46),

migration and metastasis (28), angiogenesis (32,33) and recruitment of T cells (36,37). The

specific function of these chemokines in a tumor is likely influenced by tissue type and the

associated immune context. Our data indicated expression of CCL27 and CCL28 along with

VEGF-D in a prostate cancer cell line and two human melanoma lines. Furthermore we

detected upregulation of CCR10 and CCL27 in nevi relative to both skin and malignant

melanoma in a publically-available microarray dataset. Upregulation of CCL27 enhances

recruitment of T lymphocytes in inflammatory or hyperproliferative skin conditions such as

psoriasis (30), but its expression was progressively lost during progression of keratinocyte-

derived skin tumors, suggesting a mechanism for immune evasion (36). Perhaps accordingly,

higher expression of CCL27 in the epidermis covering melanomas was found to be correlated

with positive clinical outcome (49). Little is known about CCL28 in melanoma, although one

study observed specific upregulation of CCL28 in cutaneous metastases relative to other

metastatic sites (50). Our data add to the understanding of the multiplicity of roles for these

chemokines in cancer.

The involvement of immune cells also needs to be considered in understanding the

influence of CCL27 and CCL28 on tumor lymphangiogenesis. VEGFD recruits macrophages

to tumors (17), and we also observed recruitment of LYVE1+/F4/80+ cells to Matrigel plugs

and (to a lesser extent) tumors containing CCL27 or CCL28. Macrophages and other myeloid

cells can support tumor lymphangiogenesis by secreting pro-lymphangiogenic factors and/or

by incorporating directly into lymphatic vessels, and also play important roles in

developmental lymphatic patterning (41,42). Our data suggest that macrophages recruited by

VEGFD or other factors can further shape tumor lymphangiogenesis by enhancing expression

of CCR10 in LECs and its ligands in tumor cells via their secretion of pro-inflammatory

cytokines such as TNF-

While our in vitro data showed that CCL27 and CCL28 are chemotactic for LECs

through CCR10, the morphological alterations to lymphatic patterning observed in the skin of

CCR10-/- mice further suggest a role for these chemokines in forming new lymphatic sprouts

or branches (29,51). CXCL5 signaling through CXCR2 in LECs promoted lymphatic

sprouting in vitro (48), while lymphatic branching frequency was negatively regulated by the

decoy chemokine receptor ACKR2 expressed in lymphatics (52). The chemokine receptor

CXCR4 is enriched in blood vascular tip cells and together with Dll4-Notch signaling

regulates vascular sprouting and patterning (51,53,54), but the potential role of chemokines

and their receptors in lymphatic tip cells remains unexplored. The restricted expression of

CCR10 in a subset of LECs in vitro and in vivo would also be consistent with a tightly

regulated role in formation and guidance of new lymphatic sprouts, where CCR10 is

upregulated in selected LECs in response to environmental cues. CCR10 has also been

observed co-expressed with CCL27 in some pre-collecting lymphatics (8). Further work is

required to confirm precisely how CCR10 contributes to lymphatic patterning.

The functional consequences of the developmental lymphatic defects observed in

Ccr10-/- mice also warrant further investigation. We did not observe overt edema, a common

consequence of lymphatic dysfunction, in Ccr10-/- mice or embryos. However, other studies

have reported that CCR10 deficiency leads to exacerbated edema and delayed resolution of

tissue swelling in inflamed ear skin during immune responses (55,56). Deficient localisation

of CCR10+ regulatory T cells into skin contributed to this hyperinflammatory phenotype,

which included an influx of myeloid cells. Although these changes in leukocyte recruitment

may indirectly influence inflammation-associated lymphangiogenesis, the prolonged edema

could also be due in part to a primary lowered density of skin lymphatics, and/or a deficient

lymphangiogenic response from LECs lacking CCR10. Tumor studies in Ccr10-/- mice would

help to further clarify the importance of CCR10 in pathological lymphangiogenesis.

Specific regulators of LEC migration represent potential therapeutic targets to

modulate pathological lymphangiogenesis in settings such as cancer (47). However, the

diversity of signaling pathways involved in this process is only beginning to be uncovered

(57). The current study identifies CCL27/CCL28 signaling through CCR10 on LECs as a

novel chemokine signaling axis that can promote LEC migration and potentially influence

lymphatic patterning, and which can contribute to lymphangiogenesis in vivo with the

cooperation of VEGF-D.

ACKNOWLEDGEMENTS

The authors thank Sally Roufail for expert technical assistance and Andrew Naughton,

Jacinta Carter and Animal Facility staff at the Ludwig Institute for Cancer Research

(Melbourne) and Peter MacCallum Cancer Centre for assistance with mouse experiments;

Janna Taylor for assistance in generating figures; Jason Li for bioinformatics assistance; and

Ccr10-/-

mouse tissues. The authors also acknowledge the support and resources of the Centre for

Advanced Histology and Microscopy at the Peter MacCallum Cancer Centre (A/Prof. Sarah

Ellis, Marne Prinsloo, Thu Ming Noc Nguyen, Ethan Passantino, Metta Jana, Jill Danne,

Cameron Skinner, Dhanya Menon), along with imaging assistance from Stephen Cody,

Cameron Nowell, and Naomi Campanale.

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FIGURE LEGENDS

Figure 1. Analysis of chemokine and chemokine receptor mRNA levels in VEGF-D-

293EBNA cells and VEGF-D-treated LECs. A Miniarrays containing oligonucleotides for

chemokine and chemokine receptor genes were probed with biotin-labeled cRNA derived

from VEGF-D-293EBNA cells and human LECs. Signals for CCL27, CCL28 and CCR10 are

indicated. B Quantification of mRNA levels for CCL27 and CCL28 in VEGF-D-293EBNA

cells, using qRT-PCR. Chemokine expression was normalized to -actin. Data represent

mean SEM of 3 replicates. C qRT-PCR quantification of CCR10 expression in human

LECs or BECs treated with 500 ng/ml VEGF-D for 24 h. Expression was normalized to -

actin. Data represent mean SEM of 3 replicates. * p < 0.05 by Student t test. D Flow

cytometry of LEC CCR10 cell surface expression using an anti-CCR10 antibody (dashed

line) and isotype control (solid line). LECs were transfected with negative control (left panel)

or CCR10_4 siRNA (right panel). E Knockdown of CCR10 mRNA by 4 different siRNAs

was assessed by qRT-PCR, compared to a negative control (normalized to 100%). Data

represent mean ± SEM of 3 experiments. * p < 0.05 by Student t test. 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. G Ear tissue

from Ccr10-/- and wild-type (WT) littermate control mice were stained as whole mounts with

an antibody to LYVE-1. Scale bars: 100 m. H Analysis of mouse ear tissue sections for

parameters 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 (n = 5) and Ccr10 / (n = 5) mice. * p < 0.05 -test.

Figure 2. CCL27 and CCL28 protein localization in tumor-associated

lymphangiogenesis. A Apex-293EBNA, VEGF-D-293EBNA, and FEMX-I xenograft

tumors were stained via immunohistochemistry with LYVE1, CCL27 or CCL28 antibodies.

Upper insets show the position of the main image (black box) in a lower-magnification

overview of the tumor section. Lower insets show a serial field of the main image at the same

magnification stained with a corresponding isotype-matched negative control antibody. Scale

bar: 100 m in main, 200 m in overview. B Immunofluorescence staining of VEGF-D-

293EBNA tumors with LYVE1 and CCL27 antibodies. Scale bar 100 m. C CCL28 staining

on a lymphatic vessel in a FEMX-I tumor section. Scale bar 100 m. D Relative expression

of CCR10 and CCL27 mRNA in samples of human melanoma (n = 45), benign nevi (n = 18)

and normal skin (n = 7) from the publically-available microarray expression dataset

GSE3189 (20). Data are presented as individual points with mean ± SEM, * p < 0.05, *** p <

0.001, **** p < 0.0001 by Mann-Whitney test.

Figure 3. Pro-inflammatory cytokines stimulate CCR10 expression in LECs. A qRT-

PCR analysis of CCR10 mRNA levels in LECs after stimulation with 10 ng/ml TNF- . Data

represent mean ± SEM of three experiments, * p < 0.05 by ANOVA with Dunnet post-test

compared to unstimulated (Unstim). B, C Immunofluorescence staining with antibodies to

the macrophage marker F4/80 (red) and the LEC marker LYVE1 (green) demonstrates the

infiltration of macrophages into VEGF-D-expressing tumors (C), with empty vector (Apex)-

293EBNA tumors (not expressing VEGF-D) showing fewer macrophages and lymphatic

vessels (B). Filled arrows: macrophages; open arrows: lymphatics. Scale bars: 100 m. D, E

Quantification of the F4/80 stained area (D) or LYVE1 stained area (E) in VEGF-D-

293EBNA tumors versus Apex-293EBNA tumors. Data represent mean ± SEM of 5 fields

each from 10 tumors. * p < 0.05 -test. F Quantification showing minimal co-

staining of F4/80 with LYVE1 in VEGF-D-293EBNA tumors. Data represent mean ± SEM,

n = 5 tumour-bearing mice, 2 sections per tumour.

Figure 4. CCL27, CCL28 and CCR10 mediate LEC migration but not proliferation in

vitro. A Proliferation assay of LECs stimulated with 0 500 ng/ml of recombinant CCL27,

CCL28 or VEGF-D. Proliferation was compared to unstimulated controls. Data represents

mean ± SEM of 3 replicates. * p < 0.05 by Student t test. B LECs were assessed for their

ability to migrate toward titrated concentrations of CCL27, CCL28, VEGF-C or VEGF-D in

a transwell migration chamber over 21 24 h. Migrated cells were quantified and compared to

migration toward basal medium alone (Starve). Complete growth medium (Complete) was

used as a positive control. Data represent mean ± SEM of 3 replicates; concentrations of each

chemokine or VEGF (0.001 500 ng/ml) were compared to Starve control using a one-way

ANOVA with Dunnet post-hoc test. * p < 0.05, *** p < 0.001, **** p < 0.0001. C BECs

(left panel) and LECs (right panel) were assessed for chemotactic migration toward the

indicated factors over 21-24 h, as compared to migration toward starvation medium alone.

Data represents mean ± SEM of 3 replicates; results compared using one-way ANOVA with

Tukey post-hoc test; chemokines and VEGFs (positive controls) analysed separately. * p <

0.05, ** p < 0.01. D Migration of LECs toward CCL27, CCL28 or VEGF-A after CCR10

knockdown with siRNA. Data represents mean SEM of 2 technical replicates. * p < 0.05 by

Student t test.

Figure 5. CCL28 and CCL27 cooperate with VEGF-D to attract LECs in vivo. A E FVB

mice were injected subcutaneously with 200 l Matrigel containing PBS (A) or 2 g/ml of

recombinant human CCL27 (B), 4 g/ml VEGF-D (C), 2 g/ml CCL27 and 4 g/ml VEGF-

D (D) or 4 g/ml VEGF-A (E). Matrigel plugs were harvested after one week and sections

thereof stained with antibodies to LYVE1 (green) and PECAM-1 (red) and nuclei

counterstained with DAPI (blue). Scale bars 100 µm. F, G Quantification of LYVE1-positive

vessels (F) or PECAM-1-positive vessels (G) in Matrigel plugs. Data points represent

average % stained area of <15 fields per mouse with mean ± SEM of n = 6 mice per group (5

for PBS group); p values from Kruskall-Wallis test with , * p < 0.05,

** p < 0.01, *** p < 0.001. H, I A similar experiment was conducted in which 2.5 g CCL28

and VEGF-D, alone or in combination as shown, were injected subcutaneously into FVB

mice in 500 l of Matrigel. LYVE1 staining was quantified in (H) and PECAM-1 staining in

(I). Data points represent mean average % stained area of <15 fields per mouse with mean ±

SEM of n = 4 mice (3 for PBS group); p values from Kruskall-Wallis test with uncorrected

0.01. J CCL28 Matrigel plug co-stained with antibodies to

LYVE1 (green) and podoplanin (red). Right panel: magnified view of the boxed area. Scale

bars: 50 m. K CCL28 Matrigel plug co-stained with LYVE1 (green) and the macrophage

marker F4/80 (red), showing infiltration of macrophages into the plug. The right panel

represents a magnified view of the boxed area. Open arrows LYVE1+ LECs, closed arrows

F4/80+ macrophages. Scale bars: 50 m.

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 VEGF-D-293EBNA (D) cells. E G LYVE1 (green) and PECAM1 (red)

immunofluorescence of pVITRO3-293EBNA (E), CCL27-293EBNA (F) or VEGF-D-

293EBNA (G) tumors. Right panel: magnified view of the boxed area. Scale bars: 50 m. H,

I Quantification of LYVE1-positive vessels (H) or PECAM1-positive vessels (I) in xenograft

tumors. Data points represent the average % stained area of <15 fields with mean ± SEM of n

= 5 (pVITRO3), n = 10 (CCL27) or n = 4 (VEGF-D) tumors. * p < 0.05; *** p < 0.001 by

Kruskall- . J L Collecting lymphatic vessels filled

with Patent Blue V from mice with pVITRO3-293EBNA (J), CCL27-293EBNA (K) or

VEGF-D-293EBNA (L) tumors. Arrowheads demarcate the vessel. 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, VEGF-D-293EBNA or

pVITRO3-293EBNA tumors plotted over time. Data represents mean tumor volume ± SEM

of two independent experiments, each with 5 10 mice per group.

Figure 7. Schematic diagram of mechanisms by which lymphangiogenic growth factors

and chemokines may promote tumor spread. We and others have shown that CCL28

and/or CCL27 expression in tumor cells (blue cells) and CCR10 in LECs may be upregulated

by TNF- (green arrows). TNF- and other inflammatory cytokines may be derived from

macrophages recruited by tumour-secreted VEGF-D, or by other cells in the tumor

microenvironment. CCR10 can also be up-regulated in LECs by VEGF-D signaling via

VEGF receptors VEGFR-3 or VEGFR-2 (green arrow), thus potentiating migration of LECs

towards CCL27 or CCL28. Our data suggest a model whereby CCR10 upregulation may be

polarised in particular migratory LECs within sprouting lymphatic vessels, with CCR10 and

VEGFR-3 signaling potentially operating in the same cells or adjacent cells. While CCL27

and CCL28 can guide LEC migration, cooperation with VEGF-D signaling through VEGF

receptors is required to support LEC proliferation and remodeling in order to generate

lymphatic vessels that can support metastatic spread of tumor cells.

CCL27/CCL28-CCR10 CHEMOKINE SIGNALING MEDIATES MIGRATION OF LYMPHATIC ENDOTHELIAL CELLS

Tara Karnezis, Rae Farnsworth, Nicole C. Harris, et al.

Cancer Res Published OnlineFirst February 1, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-1858

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/02/01/0008-5472.CAN-18-1858.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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