il17a-mediated endothelial breach promotes metastasis ... · b16f10-luc, or 2 510 llc were injected...

Priority Brief

IL17A-Mediated Endothelial Breach PromotesMetastasis FormationPaulina Kulig1, Sara Burkhard1, Joanna Mikita-Geoffroy1, Andrew L. Croxford1,Nadine H€ovelmeyer2, Gabor Gy€ulv�eszi1, Christian Gorzelanny3, Ari Waisman2,Lubor Borsig4, and Burkhard Becher1

Abstract

The role of the IL23/IL17Aaxis in tumor–immune interactions isa matter of controversy. Although some suggest that IL17A-pro-ducingT cells (TH17)cansuppress tumorgrowth,others report thatIL17A and IL23 accelerate tumor growth. Here, we systematicallyassessed the impact of IL17A-secreting lymphocytes in severalmurine models of tumor lung metastasis. Genetic fate mappingrevealed that IL17A was secreted within lung metastases predom-inantly by gd T cells, whereas TH17 cells were virtually absent.Using different tumor models, we found Il17a�/� mice to

consistently develop fewer pulmonary tumor colonies. IL17Aspecifically increased blood vessel permeability and the expres-sion of E-selectin and VCAM-1 by lung endothelial cells in vivo.In transgenic mice, specific targeting of IL17A to the endothe-lium increased the number of tumor foci. Moreover, the directimpact of IL17A on lung endothelial cells resulted in impairedendothelial barrier integrity, showing that IL17A promotesthe formation of lung metastases through tumor-endothelialtransmigration. Cancer Immunol Res; 4(1); 26–32. �2015 AACR.

IntroductionThere is clear evidence that the immune system controls the

development of malignancies, a process termed tumor immunesurveillance (1). Nonetheless, cancers usually subvert antitu-mor immune responses, resulting in progressive tumor growth(2). Given the current limitations of conventional cancer treat-ment, reinforcement of antitumor immune responses remainsan attractive therapeutic strategy (3). Studies on the role of theIL23/IL17A axis and TH17 cells in cancer have yielded contro-versial data (4, 5), because both pro- and antitumorigenicproperties have been proposed for IL17A (6, 7). Nonetheless,the infiltration of IL17A-producing lymphocytes into humantumors suggests a potential function of IL17A in human malig-nancies (8).

The metastasis of tumor cells is the main cause of cancer-related deaths. Metastasis formation requires the invasion ofcancer cells into the lymphatic system or the bloodstream. Todisseminate, tumor cells need to clasp hold in the vascular orlymphatic endothelium, extravasate, and colonize targetorgans (9), a process involving both integrins and selectins

(10, 11). The receptors required to transduce IL17A signals, theIL17RA and IL17RC complex, are expressed predominantly byepithelial and endothelial cells (12, 13). Using transplantableand spontaneous mouse models of tumor metastasis, wefound IL17A to promote the formation of lung metastasis byregulating endothelial barrier function and extravasation oftumor cells from the circulation into the lung parenchyma.

Materials and MethodsMice

C57BL/6micewere purchased from Janvier. Il17a�/�micewereprovided by Y. Iwakura (University of Tokyo, Tokyo, Japan),IL17Aind and IL17F-Cre were described before (14, 15),ROSA26-EYFP mice were provided by A. Diefenbach (Universityof Freiburg, Freiburg, Germany), VE-cadherin-Cre mice wereprovided by C. Halin-Winter (University of Zurich, Zurich, Swit-zerland), and Tcrbd�/� were bred in-house from Tcrb�/� andTcrd�/� mice obtained from The Jackson Laboratory. Mice werekept in house under specific pathogen-free conditions.

Cell linesMouse B16F10 melanoma was purchased from the ATCC

(CRL-6475), B16F10 cells expressing luciferase (B16F10-luc)were purchased from Caliper LifeScience, and Lewis lung carci-noma (LLC) was kindly provided by R. Schwendener (Universityof Zurich, Zurich, Switzerland). All cell lines were tested to bemycoplasma free. No genomic reauthentication was performed.Tumors were established from cryopreserved stocks that havebeen split less then four times. B16F10 andLLC cellswere culturedin DMEM medium supplemented with 10% FCS, 1% penicillin,1% streptomycin, and 1% glutamine. B16F10-luc cells wereselected with a DMEM medium supplemented with 0.2 mg/mLzeocin (Invitrogen), 10% FCS, 1% penicillin, 1% streptomycin,and 1% glutamine.

1Institute of Experimental Immunology, University of Zurich, Zurich,Switzerland. 2Institute for Molecular Medicine, University MedicalCenter of the Johannes Gutenberg, University of Mainz, Mainz,Germany. 3Department of Dermatology, Experimental Dermatology,Medical Faculty Mannheim, Heidelberg University, Mannheim,Germany. 4Institute of Physiology, University of Zurich, Zurich,Switzerland.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Burkhard Becher, University of Zurich, Winterthurer-strasse 190, 8057 Zurich, Switzerland. Phone: 41-44-635-3701; Fax: 41-44-635-6883; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-15-0154

�2015 American Association for Cancer Research.

CancerImmunologyResearch

Cancer Immunol Res; 4(1) January 201626

on December 14, 2020. © 2016 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst November 19, 2015; DOI: 10.1158/2326-6066.CIR-15-0154

http://cancerimmunolres.aacrjournals.org/

Experimental and spontaneous tumor metastasis modelSix- to 10-week-old mice were used for all the experiments.

For subcutaneous tumor challenge, 5 � 105 LLC cells were s.c.injected into the lateral flank of the mouse. The tumor area wascalculated from two perpendicular diameters of the tumormeasured with a digital caliper. Once the tumor size reached80 to 100 mm2 (days 10–14 after injection), mice were anes-thetized and the tumor was surgically removed. Animals werekept for an additional 4 weeks before being sacrificed for tumorcolony counts in the lung.

For the induction of lung metastasis, 2 � 105 B16F10, 3 � 105

B16F10-luc, or 2 � 105 LLC were injected i.v. After 6, 12, or 24hours or at days 14 or 16 after tumor inoculation, mice weresacrificed and perfused, followed by lung extraction for tumorcolony counts, flow cytometry analysis, or RNA isolation.

In vivo bioluminescence imagingTumor-bearing mice were injected with D-Luciferin (50 mg/kg

body weight; Caliper Lifesciences). Luminescence signal wasmeasured using the Xenogen IVIS 100 (Caliper Lifesciences)imaging system. Recorded data were analyzed using Living Image2.5 software (Caliper Lifesciences). A region of interest wasdefined around the tumor site and photon flux from that areawas read out.

Electric cell-substrate impedance sensingImpact of 1,000 ng/mL of IL17A (PeproTech) on transendothe-

lial electrical resistance was measured using electric cell-substrateimpedance sensing (ECIS). Endothelial cells were seeded intogelatine-coated 8-well ECIS slides (8W1E PET; ibidi GmbH) at aconcentration of 1 � 105 cells/well. Impedance at a frequency of4,000 Hz was measured every 48 seconds (ECIS-zeta system;Applied BioPhysics Inc.) while cells were continuously main-tained in a humidified atmosphere at 37�C and 5% CO2.

Evans Blue vascular permeability testMice were injected i.v. with 2 � 105 tumor cells and 24 hours

later, 2 mg of Evans Blue (Sigma-Aldrich) was i.v. applied. After30 minutes, animals were euthanized and lungs were perfusedwith PBS and homogenized in 1 mL of 50% trichloroacetic acid(Sigma-Aldrich). Evans Blue fluorescence was measured at anexcitation of 620 nm and an emission wavelength of 680 nm.

Study approvalAnimal experiments were approved by the Swiss Cantonal

Veterinary Office (16–2009 and 147/2012; Zurich, Switzerland).See the Supplementary Material and Methods for additionaldetails.

Results and DiscussionIL17A promotes the formation of lung tumor foci

To determine the role of IL17A in lung tumor development, weinjected B16F10 cells i.v. into Il17a�/– and wild-type C57Bl/6(WT) control mice. Mice lacking IL17A showed a significantdecrease in tumor burden compared with control mice (Fig.1A). Injection of luciferase-expressing B16F10 cells (B16F10-luc)permitted in vivo monitoring of tumor growth (Fig. 1B). Tumor-free mice (control) were used to set up background detectionsignal of luminescence. Again, tumor cells transplanted intoIl17a�/� mice showed a significant delay in accumulation in the

lungs, suggesting a tumor-promoting function of IL17A in thelung microenvironment (Fig. 1C).

These findings contrast with those from a report in which thesame B16F10 cell line was shown to be moremetastatic in IL17A-deficient animals (6). In that study, TH17 cells promoted CD8þ T-cell activation, which was necessary for lung tumor eradication.Althoughwehavenoobvious explanation for this discrepancy,wefound that, similar to B16F10 cells, LLC tumor colonies also werereduced in Il17a�/� mice (Fig. 1D).

gd T cells are the main source of IL17A in tumor-bearing lungsIn previous reports, both ab T cells and gd T cells (4, 16) were

found to produce IL17A in tumor tissues. To delineate the sourceof IL17A in the metastatic lung tissue, on day 16 after tumorchallenge, lung-invading leukocytes were isolated, stimulated for4 hours with phorbol myristate acetate (PMA) and ionomycin,andnext analyzedby intracellular cytokine staining.Of the IL17A-producing cell types, approximately 50% could be attributed toCD3þ T cells. Among CD3þIL17Aþ T cells, only small fractions ofthem were CD4þ TH17 cells. IL17Aþgd TCRþ T cells were signif-icantly more abundant (Fig. 2A and B). The CD3� lymphocytes,but not natural killer cells, accounted for the remaining IL17A-expressing cell types. We found that the majority of CD3�IL17Aþ

cells were CD5þ positive (Fig. 2C), showing that this populationconsists of T cells that downregulate the CD3/TCR complex afterPMA activation.

To unambiguously identify the source of IL17A in the tumormicroenvironment, we used the IL17F-CreEYFP fate mappingstrain, where IL17-expressing cells are irreversibly labeled withenhanced yellow fluorescent protein (EYFP) after IL17F promoteractivity (15). IL17A and IL17F are juxtaposed in the genome, andboth genes are usually coexpressed (17–19). Thus, IL17F-CreEYFP

mice can be used to faithfully track IL17A-producing lymphocytes(20). On day 16 after tumor challenge, tumor-infiltrating leuko-cytes were isolated and directly analyzed. A clear majority ofEYFPþ cells in the lung tissue were CD3þ positive, among which80%were gd TCRþ (Fig. 2D), confirming that gd T cells andnotabT cells are the major IL17-producing tumor-infiltrating lympho-cyte subset in tumor-bearing lungs.

In line with a recent study in which IL17A-secreting gd T cellswere shown to promote tumor cell dissemination in a mousemodel of spontaneous breast cancer metastasis (21), we con-firmed the tumor-promoting role of IL17-producing lympho-cytes, using Tcrbd�/� mice, which lack both ab and gd T cells.These mice formed significantly fewer tumor lung colonies (Sup-plementary Fig. S1).

IL17A promotes the formation of lung metastasisTumor cell arrest in the lung vasculature and subsequent tissue

extravasation requires interaction between cell surface receptorsand their ligands on tumor cells, leukocytes, and endothelial cells(22). Selectins, which belong to the family of vascular adhesionmolecules, mediate tumor cell interactions with the endothelium(23). Because epithelial and endothelial cells are the main targetsof IL17A, we tested the impact of IL17A on E-selectin expressionby injecting B16F10 cells i.v. into Il17a�/� and WTmice. First, weconfirmed the source of IL17A in tumor-bearing lungs 24 hoursafter tumor injection. Again, gd T cells were the main producersof IL17A (Supplementary Fig. S2A and S2B). Next, after 6, 12, and24 hours, lungs were isolated and E-selectin expression was

Role of IL17A in Tumor Control

www.aacrjournals.org Cancer Immunol Res; 4(1) January 2016 27




quantified by qPCR. Six hours after cell transfer, we found thatWTmice displayed significantly higher levels of the E-selectin tran-scripts compared with lungs of Il17a�/�mice (Fig. 3A), suggestingthat IL17A induces the expression of adhesion molecules by theendothelium when challenged with tumor cells. As with E-selec-tin, expression of integrin ligands, such as vascular cell adhesionmolecule 1 (VCAM-1), was also reduced in Il17a�/� mice (Fig.3B). When we concomitantly injected mice with recombinantIL17A and B16F10 cells, we found that IL17A alone slightlyinduced E-selectin expression, which was further augmented bythe presence of tumor cells (Fig. 3C). This was apparently not dueto upregulation of the IL17R expression in lung tissues afterencountering B16 melanoma cells and/or IL17A (Fig. 3D).

Because IL17A regulated adhesion molecule expression, we de-termined the impact of IL17A on vessel permeability by measuringEvans Blue dye uptake. Lungs of Il17a�/� mice were significantly

less permeable compared with those from WT mice (Fig. 3E). Tofunctionally confirm the impact of IL17A on vascular integrity, weperformed electrical resistancemeasurements of primary lung endo-thelial monolayers. IL17A caused decrease of the relative electricalresistance already at 1 hour after treatment (Fig. 3F). Lastly, IL17Aengagement of primary lung endothelial cells increased B16F10melanoma cell transmigration in vitro (Fig. 3G).

IL17A regulates tumor dissemination in a spontaneous lungmetastasis model

To confirm that IL17A promotes vessel activation and perme-ability and thereby contributes to tumor dissemination, we chosea different model in which lung metastases arise from a primarytumor mass located at a distant site. WT and Il17a�/� mice wereinjected s.c. with LLC cells, and the tumor mass was resected afterreaching a size of 80 to 100 mm2. On the day of resection, no

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Figure 1.IL17A deficiency regulates tumor growth in the lung. WT and Il17a�/� mice were challenged i.v. with B16F10 melanoma cells, LLC, or B16F10-luc cells. A, 14days after B16F10 inoculation, lungs of the mice were harvested and photographed and tumor colonies were counted. Pooled data from more than threeindependent experiments (n ¼ 23 WT, n ¼ 28 Il17a�/�, average � SEM). B, representative picture of luminescence imaging. C, kinetics of B16F10-luc tumorgrowth in the lungs. The gray dotted line represents luminescence detection threshold. Pooled data from two independent experiments (n ¼ 8 WT, n ¼ 7Il17a�/�, average � SEM). D, 14 days after LLC inoculation, lung tumor colonies were counted. Pooled data from two independent experiments (n ¼ 12 WT, n ¼ 11Il17a�/�, average � SEM). A, C, and D, each data point represents an individual mouse. � , P < 0.05;

��, P < 0.01;

��, P < 0.001 (unpaired two tailed t-test).

Kulig et al.

Cancer Immunol Res; 4(1) January 2016 Cancer Immunology Research28




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Figure 2.IL17A production by lung-infiltrating leukocytes. WT or IL17F-CreEYFP mice were challenged i.v. with B16F10 cells. On day 16, lungs were resected and processed forflow cytometry analysis. A, representative dot plots display IL17A secretion among CD45þCD11b� leukocytes in WT mice. B, percentage distribution of IL17A-producing cells in WT mice. Pooled data from two independent experiments (n ¼ 7, average � SEM). C, representative dot plots display IL17A secretion amongCD45þ lineage negative (CD11b�CD11c�B220�) leukocytes in WT mice. D, representative dot plots and percentages of IL17F/EYFP-positive cells among CD45þ

CD11b� leukocytes. Pooled data from two independent experiments (n ¼ 3–7, average � SEM). B and D, each data point represents an individual mouse.






significant difference in primary tumor size was observedbetween the twomouse strains (Fig. 4A). Four weeks after surgery,Il17a�/� mice showed a reduced number of lung metastases

compared with WT controls (Fig. 4B). Of note, primary tumorsize at the time of resection did not correlate with the numberof metastases (not shown).

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Figure 3.IL17A impact on lung endothelial activity and permeability. A and B,WT and Il17a�/�mice were challenged i.v. with B16F10 cells. After 6, 12, and 24 hours, lungswereresected and quantitative PCR was performed using whole lung tissue. Depicted data are from one experiment (n ¼ 3 per group, average � SEM). C and D,WT mice were challenged i.v. with B16F10 cells, 500 ng IL17A, or in combination. Six hours later, lungs were resected and quantitative PCR was performedon the whole lung tissue. Pooled data from three independent experiments (n ¼ 9 per group, average � SEM). E, spectrophotometric quantification of EBextracted from whole lungs of WT and Il17a�/– mice 24 hours after B16F10 challenge. Pooled data from two independent experiments (n ¼ 7 per na€�ve group,n ¼ 9–10 per B16F10 group, average � SEM). F, representative transendothelial electrical resistance. Primary lung endothelial cell monolayers were stimulatedwith 1,000 ng/mL of IL17A at 0 hour. The loss of electrical resistance was quantified 1 hour later. Experiment performed independently three times. G,transendothelial migration assay. Primary lung endothelial cells grown to confluence on 8-mm pore sized transwell inserts were stimulated with 100 ng/mL ofIL17A for 10 hours. Next, media were changed and cells were incubated with red fluorescent–labeled B16F10 tumor cells for the next 16 hours. Results showthe number of transmigrated tumor cells per field view. Pooled data from two independent experiments (n¼ 6 permedium group, n¼ 9 per IL17A group, average�SEM). A–E, each data point represents individual mouse; G, each data point represents individual insert; � , P < 0.05;

��, P < 0.01;

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ANOVA with Bonferroni posttest for A–D or unpaired two tailed t-test for E–G). n.s., not statistically significant.

Kulig et al.





To validate the protumorigenic function of IL17A, we tookadvantage of a mouse strain in which IL17A expression can beconditionally regulated (IL17Aind; ref. 14). Crossing theIL17Aind mouse with VE-cadherin-Cre–expressing mice led toexpression of IL17A (and EGFP) by endothelial cells (Fig. 4C),allowing us to directly target IL17A to the endothelium, inwhich IL17A acts in an auto/paracrine manner. Serum fromna€�ve VE-cadherin-CreþIL17Aind/þ mice had elevated levels ofIL17A (Fig. 4D). VE-cadherin-CreþIL17Aind/þ mice and litter-mate controls were challenged with LLC cells as describedabove. Whereas transgenic expression of IL17A again did nothave a significant impact on the primary tumor size (Fig. 4E), asignificantly higher number of metastases were found in the

lungs of VE-cadherin-CreþIL17Aind/þ mice compared with lit-termate controls (Fig. 4F).

Whereas we present here one tangible mechanism by whichIL17A promotes the formation of lungmetastasis, namely thoughaltering vessel permeability, it is likely that IL17A additionallyaffects tumor dissemination through other means, such as theinduction of survival or angiogenic genes (24). Several lines ofevidence suggest that IL17A may play a protective role in tumorsurveillance in the context of T-cell responses (25). In vitro–generated IL17A-secreting TH17 cells for instance were able toeradicate established tumors in an IFNg-dependentmanner upontransfer into tumor-bearing mice (5). Conversely, a growingbody of data support the protumorigenic function of IL17A,

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Figure 4.IL17A regulates tumor dissemination in spontaneous lung metastasis model. A and B, WT and Il17a�/� mice were challenged s.c. with LLC cells. Once the tumorreached average size of 80 to 100 mm2, tumors were removed, and 4 weeks later, lungs were resected and spontaneously arising tumor colonies werecounted. Pooled data from three independent experiments (n ¼ 13 WT, n ¼ 10 Il17a�/�, average � SEM). C, na€�ve lungs from VE-cadherin-CreþIL17Aind/þ andIL17Aind/þ control littermates were resected and processed for flow cytometry analysis. Cells were stained for CD45, CD31, and CD34 markers. EGFP expression isdisplayed as a histogram plot after indicated gating. The frequency of gated cells is shown. Depicted results are from a representative experiment performedindependently two times. D, IL17A expression in a serum isolated from na€�ve VE-cadherin-CreþIL17Aind/þ and IL17Aind/þ littermate controls. Pooled data from twoindependent experiments (n ¼ 5 IL17Aind/þ, n ¼ 6 VE-cadherin-CreþIL17Aind/þ, average � SEM). E and F, VE-cadherin-CreþIL17Aind/þ and IL17Aind/þ controllittermates were challenged with LLC cells as described above. Pooled data from three independent experiments (n ¼ 11 VE-cadherin-CreþIL17Aind/þ,n ¼ 10 IL17Aind/þ, average � SEM). A, B, D, E, and F, each data point represents individual mouse; � , P < 0.05;

��, P < 0.01;

��, P < 0.001 (unpaired two tailed t-test

for A, B, D, E, and F). n.s., not statistically significant.






which is associated with its impact on the nonhematopoieticcompartment (16, 24, 26). Here, we found gd T cells to be themain source of IL17A in the context of lung metastasis usingseveral preclinicalmodels. IL17Amodulates endothelial integrity,leading to enhanced tumor cell extravasation into the lung paren-chyma. By combining an array of IL17-transgenic mice withseveral models of tumor metastasis, we propose a function ofIL17A in promoting tumor cell–endothelial interactions.

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

Authors' ContributionsConception and design: P. Kulig, J. Mikita-Geoffroy, A.L. Croxford, B. BecherDevelopment of methodology: P. Kulig, S. Burkhard, J. Mikita-Geoffroy,A.L. Croxford, G. Gy€ulv�eszi, A. Waisman, L. BorsigAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): P. Kulig, S. Burkhard, J. Mikita-Geoffroy, N. H€ovelmeyer,C. Gorzelanny, L. BorsigAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Kulig, S. Burkhard, J. Mikita-Geoffroy,C. Gorzelanny

Writing, review, and/or revision of the manuscript: P. Kulig, A.L. Croxford,N. H€ovelmeyer, B. BecherAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. BurkhardStudy supervision: B. Becher

AcknowledgmentsThe authors thank S. Hasler and J. Jaberg for their excellent technical

assistance.

Grant SupportThis study was supported by The Swiss National Science Foundation

310030_146130 (B. Becher) and 316030_150768 (B. Becher), Swiss CancerLeague (B. Becher), TheUniversity research priority project "URPP-translationalcancer research" (B. Becher), The European Union FP7 project ATECT(B. Becher), EU-FP7-ITN_NeuroKine (A. Waisman and B. Becher), DFG grantHO 4440/1-1 (N. H€ovelmeyer), and EMBO long-term fellowship ALTF 508-2011 (A.L. Croxford).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 23, 2015; revised September 1, 2015; accepted September 16,2015; published OnlineFirst November 19, 2015.

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Kulig et al.




2016;4:26-32. Published OnlineFirst November 19, 2015.Cancer Immunol Res Paulina Kulig, Sara Burkhard, Joanna Mikita-Geoffroy, et al. FormationIL17A-Mediated Endothelial Breach Promotes Metastasis

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il17a-mediated endothelial breach promotes metastasis ... · b16f10-luc, or 2 510 llc were injected...

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