IL17A-Mediated Endothelial Breach Promotes Metastasis Formation Paulina Kulig1, Sara Burkhard1, Joanna Mikita-Geoffroy1, Andrew L

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

Priority Brief Cancer Immunology Research IL17A-Mediated Endothelial Breach Promotes Metastasis Formation Paulina Kulig1, Sara Burkhard1, Joanna Mikita-Geoffroy1, Andrew L. Croxford1, Nadine Hovelmeyer€ 2, Gabor Gyulv€ eszi 1, Christian Gorzelanny3, Ari Waisman2, Lubor Borsig4, and Burkhard Becher1

Abstract

The role of the IL23/IL17A axis in tumor–immune interactions is consistently develop fewer pulmonary tumor colonies. IL17A a matter of controversy. Although some suggest that IL17A-pro- speciﬁcally increased blood vessel permeability and the expres- ducing T cells (TH17) can suppress tumor growth, others report that sion of E-selectin and VCAM-1 by lung endothelial cells in vivo. IL17A and IL23 accelerate tumor growth. Here, we systematically In transgenic mice, speciﬁc targeting of IL17A to the endothe- assessed the impact of IL17A-secreting lymphocytes in several lium increased the number of tumor foci. Moreover, the direct murine models of tumor lung metastasis. Genetic fate mapping impact of IL17A on lung endothelial cells resulted in impaired revealed that IL17A was secreted within lung metastases predom- endothelial barrier integrity, showing that IL17A promotes inantly by gd T cells, whereas TH17 cells were virtually absent. the formation of lung metastases through tumor-endothelial / Using different tumor models, we found Il17a mice to transmigration. Cancer Immunol Res; 4(1); 26–32. 2015 AACR.

Introduction (10, 11). The receptors required to transduce IL17A signals, the IL17RA and IL17RC complex, are expressed predominantly by There is clear evidence that the immune system controls the epithelial and endothelial cells (12, 13). Using transplantable development of malignancies, a process termed tumor immune and spontaneous mouse models of tumor metastasis, we surveillance (1). Nonetheless, cancers usually subvert antitu- found IL17A to promote the formation of lung metastasis by mor immune responses, resulting in progressive tumor growth regulating endothelial barrier function and extravasation of (2). Given the current limitations of conventional cancer treat- tumor cells from the circulation into the lung parenchyma. ment, reinforcement of antitumor immune responses remains an attractive therapeutic strategy (3). Studies on the role of the IL23/IL17A axis and TH17 cells in cancer have yielded contro- Materials and Methods versial data (4, 5), because both pro- and antitumorigenic Mice / properties have been proposed for IL17A (6, 7). Nonetheless, C57BL/6 mice were purchased from Janvier. Il17a mice were ﬁ the in ltration of IL17A-producing lymphocytes into human provided by Y. Iwakura (University of Tokyo, Tokyo, Japan), tumors suggests a potential function of IL17A in human malig- IL17Aind and IL17F-Cre were described before (14, 15), nancies (8). ROSA26-EYFP mice were provided by A. Diefenbach (University The metastasis of tumor cells is the main cause of cancer- of Freiburg, Freiburg, Germany), VE-cadherin-Cre mice were related deaths. Metastasis formation requires the invasion of provided by C. Halin-Winter (University of Zurich, Zurich, Swit- / / cancer cells into the lymphatic system or the bloodstream. To zerland), and Tcrbd were bred in-house from Tcrb and / disseminate, tumor cells need to clasp hold in the vascular or Tcrd mice obtained from The Jackson Laboratory. Mice were lymphatic endothelium, extravasate, and colonize target kept in house under speciﬁc pathogen-free conditions. organs (9), a process involving both integrins and selectins

Cell lines 1Institute of Experimental Immunology, University of Zurich, Zurich, Mouse B16F10 melanoma was purchased from the ATCC 2 Switzerland. Institute for Molecular Medicine, University Medical (CRL-6475), B16F10 cells expressing luciferase (B16F10-luc) Center of the Johannes Gutenberg, University of Mainz, Mainz, Germany. 3Department of Dermatology, Experimental Dermatology, were purchased from Caliper LifeScience, and Lewis lung carci- Medical Faculty Mannheim, Heidelberg University, Mannheim, noma (LLC) was kindly provided by R. Schwendener (University 4 Germany. Institute of Physiology, University of Zurich, Zurich, of Zurich, Zurich, Switzerland). All cell lines were tested to be Switzerland. mycoplasma free. No genomic reauthentication was performed. Note: Supplementary data for this article are available at Cancer Immunology Tumors were established from cryopreserved stocks that have Research Online (http://cancerimmunolres.aacrjournals.org/). been split less then four times. B16F10 and LLC cells were cultured Corresponding Author: Burkhard Becher, University of Zurich, Winterthurer- in DMEM medium supplemented with 10% FCS, 1% penicillin, strasse 190, 8057 Zurich, Switzerland. Phone: 41-44-635-3701; Fax: 41-44-635- 1% streptomycin, and 1% glutamine. B16F10-luc cells were 6883; E-mail: [email protected] selected with a DMEM medium supplemented with 0.2 mg/mL doi: 10.1158/2326-6066.CIR-15-0154 zeocin (Invitrogen), 10% FCS, 1% penicillin, 1% streptomycin, 2015 American Association for Cancer Research. and 1% glutamine.

26 Cancer Immunol Res; 4(1) January 2016

Role of IL17A in Tumor Control

Experimental and spontaneous tumor metastasis model lungs, suggesting a tumor-promoting function of IL17A in the Six- to 10-week-old mice were used for all the experiments. lung microenvironment (Fig. 1C). For subcutaneous tumor challenge, 5 105 LLC cells were s.c. These findings contrast with those from a report in which the injected into the lateral flank of the mouse. The tumor area was same B16F10 cell line was shown to be more metastatic in IL17A- þ calculated from two perpendicular diameters of the tumor deficient animals (6). In that study, TH17 cells promoted CD8 T- measured with a digital caliper. Once the tumor size reached cell activation, which was necessary for lung tumor eradication. 80 to 100 mm2 (days 10–14 after injection), mice were anes- Although we have no obvious explanation for this discrepancy, we thetized and the tumor was surgically removed. Animals were found that, similar to B16F10 cells, LLC tumor colonies also were / kept for an additional 4 weeks before being sacrificed for tumor reduced in Il17a mice (Fig. 1D). colony counts in the lung. For the induction of lung metastasis, 2 105 B16F10, 3 105 gd T cells are the main source of IL17A in tumor-bearing lungs B16F10-luc, or 2 105 LLC were injected i.v. After 6, 12, or 24 In previous reports, both ab T cells and gd T cells (4, 16) were hours or at days 14 or 16 after tumor inoculation, mice were found to produce IL17A in tumor tissues. To delineate the source sacrificed and perfused, followed by lung extraction for tumor of IL17A in the metastatic lung tissue, on day 16 after tumor colony counts, flow cytometry analysis, or RNA isolation. challenge, lung-invading leukocytes were isolated, stimulated for 4 hours with phorbol myristate acetate (PMA) and ionomycin, In vivo bioluminescence imaging and next analyzed by intracellular cytokine staining. Of the IL17A- Tumor-bearing mice were injected with D-Luciferin (50 mg/kg producing cell types, approximately 50% could be attributed to þ þ þ body weight; Caliper Lifesciences). Luminescence signal was CD3 T cells. Among CD3 IL17A T cells, only small fractions of measured using the Xenogen IVIS 100 (Caliper Lifesciences) þ þ þ them were CD4 TH17 cells. IL17A gd TCR T cells were signif- imaging system. Recorded data were analyzed using Living Image icantly more abundant (Fig. 2A and B). The CD3 lymphocytes, 2.5 software (Caliper Lifesciences). A region of interest was but not natural killer cells, accounted for the remaining IL17A- fi fl þ de ned around the tumor site and photon ux from that area expressing cell types. We found that the majority of CD3 IL17A þ was read out. cells were CD5 positive (Fig. 2C), showing that this population consists of T cells that downregulate the CD3/TCR complex after Electric cell-substrate impedance sensing PMA activation. Impact of 1,000 ng/mL of IL17A (PeproTech) on transendothe- To unambiguously identify the source of IL17A in the tumor lial electrical resistance was measured using electric cell-substrate microenvironment, we used the IL17F-CreEYFP fate mapping impedance sensing (ECIS). Endothelial cells were seeded into strain, where IL17-expressing cells are irreversibly labeled with gelatine-coated 8-well ECIS slides (8W1E PET; ibidi GmbH) at a enhanced yellow fluorescent protein (EYFP) after IL17F promoter 5 concentration of 1 10 cells/well. Impedance at a frequency of activity (15). IL17A and IL17F are juxtaposed in the genome, and 4,000 Hz was measured every 48 seconds (ECIS-zeta system; both genes are usually coexpressed (17–19). Thus, IL17F-CreEYFP Applied BioPhysics Inc.) while cells were continuously main- mice can be used to faithfully track IL17A-producing lymphocytes fi tained in a humidi ed atmosphere at 37 C and 5% CO2. (20). On day 16 after tumor challenge, tumor-infiltrating leukocytes were isolated and directly analyzed. A clear majority of þ þ Evans Blue vascular permeability test EYFP cells in the lung tissue were CD3 positive, among which þ Mice were injected i.v. with 2 105 tumor cells and 24 hours 80% were gdTCR (Fig. 2D), confirming that gdT cells and not ab later, 2 mg of Evans Blue (Sigma-Aldrich) was i.v. applied. After T cells are the major IL17-producing tumor-infiltrating lympho- 30 minutes, animals were euthanized and lungs were perfused cyte subset in tumor-bearing lungs. with PBS and homogenized in 1 mL of 50% trichloroacetic acid In line with a recent study in which IL17A-secreting gd T cells (Sigma-Aldrich). Evans Blue fluorescence was measured at an were shown to promote tumor cell dissemination in a mouse excitation of 620 nm and an emission wavelength of 680 nm. model of spontaneous breast cancer metastasis (21), we confirmed the tumor-promoting role of IL17-producing lympho- / Study approval cytes, using Tcrbd mice, which lack both ab and gd T cells. Animal experiments were approved by the Swiss Cantonal These mice formed significantly fewer tumor lung colonies (Sup- Veterinary Office (16–2009 and 147/2012; Zurich, Switzerland). plementary Fig. S1). See the Supplementary Material and Methods for additional details. IL17A promotes the formation of lung metastasis Tumor cell arrest in the lung vasculature and subsequent tissue Results and Discussion extravasation requires interaction between cell surface receptors IL17A promotes the formation of lung tumor foci and their ligands on tumor cells, leukocytes, and endothelial cells To determine the role of IL17A in lung tumor development, we (22). Selectins, which belong to the family of vascular adhesion /– injected B16F10 cells i.v. into Il17a and wild-type C57Bl/6 molecules, mediate tumor cell interactions with the endothelium (WT) control mice. Mice lacking IL17A showed a significant (23). Because epithelial and endothelial cells are the main targets decrease in tumor burden compared with control mice (Fig. of IL17A, we tested the impact of IL17A on E-selectin expression / 1A). Injection of luciferase-expressing B16F10 cells (B16F10-luc) by injecting B16F10 cells i.v. into Il17a and WT mice. First, we permitted in vivo monitoring of tumor growth (Fig. 1B). Tumor- confirmed the source of IL17A in tumor-bearing lungs 24 hours free mice (control) were used to set up background detection after tumor injection. Again, gd T cells were the main producers signal of luminescence. Again, tumor cells transplanted into of IL17A (Supplementary Fig. S2A and S2B). Next, after 6, 12, and / Il17a mice showed a significant delay in accumulation in the 24 hours, lungs were isolated and E-selectin expression was

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Kulig et al.

100 A 250 B16F10 D LLC ** * 200 B16F10 80

150 WT Il17a-/- 60 oci per lung 100 40 Number Number of tumor foci per lung of tumor foci of tumor f 50 20

0 0 WT Il17a-/- WT Il17a-/-

B C 3,000,000 B16F10-luc 16 d 2,500,000

2,000,000

1,500,000

1,000,000 Control B16F10-luc ** 600,000 12 d OI flux (p/s)

R 400,000 8 d 2 h 24 h 48 h

200,000

0 /------/- -/- -/ -/ -/ WT WT WT WT WT WT Il17a Il17a Il17a Il17a Il17a Il17a

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, 14 days after B16F10 inoculation, lungs of the mice were harvested and photographed and tumor colonies were counted. Pooled data from more than three / independent experiments (n ¼ 23 WT, n ¼ 28 Il17a , average SEM). B, representative picture of luminescence imaging. C, kinetics of B16F10-luc tumor growth in the lungs. The gray dotted line represents luminescence detection threshold. Pooled data from two independent experiments (n ¼ 8 WT, n ¼ 7 / Il17a , 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 11 / Il17a , 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).

quantified by qPCR. Six hours after cell transfer, we found that WT less permeable compared with those from WT mice (Fig. 3E). To mice displayed significantly higher levels of the E-selectin tran- functionally confirm the impact of IL17A on vascular integrity, we / scripts compared with lungs of Il17a mice (Fig. 3A), suggesting performed electrical resistance measurements of primary lung endo- that IL17A induces the expression of adhesion molecules by the thelial monolayers. IL17A caused decrease of the relative electrical endothelium when challenged with tumor cells. As with E-selec- resistance already at 1 hour after treatment (Fig. 3F). Lastly, IL17A tin, expression of integrin ligands, such as vascular cell adhesion engagement of primary lung endothelial cells increased B16F10 / molecule 1 (VCAM-1), was also reduced in Il17a mice (Fig. melanoma cell transmigration in vitro (Fig. 3G). 3B). When we concomitantly injected mice with recombinant IL17A and B16F10 cells, we found that IL17A alone slightly IL17A regulates tumor dissemination in a spontaneous lung induced E-selectin expression, which was further augmented by metastasis model the presence of tumor cells (Fig. 3C). This was apparently not due To confirm that IL17A promotes vessel activation and perme- to upregulation of the IL17R expression in lung tissues after ability and thereby contributes to tumor dissemination, we chose encountering B16 melanoma cells and/or IL17A (Fig. 3D). a different model in which lung metastases arise from a primary / Because IL17A regulated adhesion molecule expression, we de- tumor mass located at a distant site. WT and Il17a mice were termined the impact of IL17A on vessel permeability by measuring injected s.c. with LLC cells, and the tumor mass was resected after / Evans Blue dye uptake. Lungs of Il17a mice were significantly reaching a size of 80 to 100 mm2. On the day of resection, no

28 Cancer Immunol Res; 4(1) January 2016 Cancer Immunology Research

Role of IL17A in Tumor Control

5 5 5 10 0 1.61 10 2.02 0.321 10 66.4 0.828

4 10 4 4 10 10

3 10 3 10 3 10

2 10 2 10 0 0 0 98.4 58.9 38.8 0 18.4 14.4

IL17A NK1.1 γδ TCR 3 4 5 2 3 4 5 3 4 5 01010 10 010 10 10 10 01010 10

CD45 CD3 CD4 B CD11b− CD11b− CD11b− CD45+IL-17A+ cells CD45+IL-17A+CD3+ cells CD45+IL-17A+CD3− cells 100 100 100

80 80 80

60 60 60 cells (%) cells (%) cells (%) + + + 40 40 40 Composition of IL17A Composition of Composition of IL17A IL17A 20 20 20

0 0 0 + − − CD3 CD3 + + +

TCR CD4 Other γδ NK1.1 - NK1.1 C CD3

105 Gate 1 105 Gate 2 105 Gate 3

104 104 104 64.9 93.7 47.5 103 103 103

102 102 102 0 0 0

CD3

CD3 CD3

0102 103 104 105 0102 103 104 105 0102 103 104 105

CD11b CD11c B220 Gated on gate 3

5 10 0 1.86 105 3.36 1.29 105 81.1 0

4 10 104 104

3 10 103 103

2 10 102 102

0 0 0 0 98.1 61.5 33.6 19.3 0

CD5 IL17A

NK1.1 2 3 4 5 010 10 10 10 0102 103 104 105 0102 103 104 105

CD45 CD3 CD3 D

− CD45+CD11b CD45+CD11b−CD3+EYFP+ 5 105 0.0197 0.191 10 11.1 1.83 100 100

4 104 10 80 80

3 103 10 60 60 cells (%) cells (%)

+ + 102 0 40 40 0 Composition of 43 56.8 8.13 78.9 Composition of IL17F IL17F

IL17F-EYFP CD4 0102 103 104 105 0103 104 105 20 20

CD3 γδ TCR 0 0 + − CD3 CD3 + +

CD4 Other γδ TCR

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 for ﬂow 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 among CD45þ lineage negative (CD11bCD11cB220) 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.

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Kulig et al.

A B C

0.5 0.4 *** 2.5 * *** *** *** 2.0 0.4 0.3 1.5 n.s. 0.3 0.2 1.0 0.2 *** n.s. 0.1 0.5 0.1

0.0 0.0 0.0 to the control gene Polr2a to the control gene Polr2a to the control gene Polr2a e h e h ïv v ïve 6 6 h normalized Eselectin expression 6 h 6 h normalized Vcam1 expression 12 h 24 h 12 h 24 h 12 h 24 h 12 24 h F10 expression normalized Eselectin expression Naïve Naïve Na Naï Na IL17A16 B WT Il17a-/- WT Il17a-/- 10+IL17A 16F B

D E 50,000 F

* ) 1.5 1.5 Ω 1.4 40,000 ( Medium n.s. n.s. IL17A * 1.0 1.0 30,000 (4000 Hz) 1.2

20,000 per lung 1.0 0.5 0.5 10,000 0.8 Fluorescence Intensity 0.4 0.0 0.0 0 0.0 to the control gene Polr2a Normalized TEER

to the control gene Polr2a - − − expression normalized Il17rc expression e -/ -/- 4 2024 expression normalized expression Il17ra Naïv WT WT NaïveIL17A IL17A l17a Time (h) B16F10 B16F10 I Il17a Naïve B16F10 B16F10+IL17A B16F10+IL17A

100 * Medium IL17A 80

20 cells per field view 0 Number of transmigrated tumor Number of transmigrated IL17A Medium

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, lungs were resected 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 performed on the whole lung tissue. Pooled data from three independent experiments (n ¼ 9 per group, average SEM). E, spectrophotometric quantification of EB extracted 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 stimulated with 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 of IL17A for 10 hours. Next, media were changed and cells were incubated with red fluorescent–labeled B16F10 tumor cells for the next 16 hours. Results show the number of transmigrated tumor cells per field view. Pooled data from two independent experiments (n ¼ 6 per medium group, n ¼ 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; , P < 0.001 (one-way ANOVA with Bonferroni posttest for A–D or unpaired two tailed t-test for E–G). n.s., not statistically significant.

signiﬁcant difference in primary tumor size was observed compared with WT controls (Fig. 4B). Of note, primary tumor between the two mouse strains (Fig. 4A). Four weeks after surgery, size at the time of resection did not correlate with the number / Il17a mice showed a reduced number of lung metastases of metastases (not shown).

30 Cancer Immunol Res; 4(1) January 2016 Cancer Immunology Research

Role of IL17A in Tumor Control

A B D E F n.s. 15 * 200 2,000 Serum 200 n.s. 60 * ) )

2 ** 2 150 1,500 50 10 150 30

100 1,000 100 20 Number

5 Number

50 IL17A (pg/mL) 500 50 10 of resection (mm of resection (mm of tumor foci per lung of tumor foci of tumor foci per lung of tumor foci Tumor size on the day size Tumor 0 0 0 on the day size Tumor 0 0 -/- -/- + WT Il17a WT Il17a /+ /+ ind/+ ind ind/ ind ind/+ ind/+

IL17A + IL17A IL17A + IL17A IL17A + IL17A

-Cre

VE-cadherin-Cre VE-cadherin VE-cadherin-Cre C IL17Aind/+ IL17Aind/+ IL17Aind/+ 20K 250K 38.9 105

200K 15K 104

150K 63 10K 103 100K

5000 102 3.69 50K 0

SSC 0 CD34 0 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 0103 104 105

CD45 CD31 EGFP

VE-cadherin-Cre+IL17Aind/+ VE-cadherin-Cre+IL17Aind/+ VE-cadherin-Cre+IL17Aind/+ 250K 35.2 105 8000 200K 104 46.6 6000 150K

103 4000 100K

2 75.1 50K 10 2000 0

SSC 0 CD34 0 0102 103 104 105 0102 103 104 105 0103 104 105

CD45 CD31 EGFP

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 tumor reached average size of 80 to 100 mm2, tumors were removed, and 4 weeks later, lungs were resected and spontaneously arising tumor colonies were / þ þ counted. Pooled data from three independent experiments (n ¼ 13 WT, n ¼ 10 Il17a , average SEM). C, na€ve lungs from VE-cadherin-Cre IL17Aind/ and þ IL17Aind/ control littermates were resected and processed for ﬂow cytometry analysis. Cells were stained for CD45, CD31, and CD34 markers. EGFP expression is displayed as a histogram plot after indicated gating. The frequency of gated cells is shown. Depicted results are from a representative experiment performed independently two times. D, IL17A expression in a serum isolated from na€ve VE-cadherin-CreþIL17Aind/þ and IL17Aind/þ littermate controls. Pooled data from two independent 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 control littermates were challenged with LLC cells as described above. Pooled data from three independent experiments (n ¼ 11 VE-cadherin-CreþIL17Aind/þ, þ n ¼ 10 IL17Aind/ , 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.

þ þ To validate the protumorigenic function of IL17A, we took lungs of VE-cadherin-Cre IL17Aind/ mice compared with lit- advantage of a mouse strain in which IL17A expression can be termate controls (Fig. 4F). conditionally regulated (IL17Aind;ref.14).Crossingthe Whereas we present here one tangible mechanism by which IL17Aind mouse with VE-cadherin-Cre–expressing mice led to IL17A promotes the formation of lung metastasis, namely though expression of IL17A (and EGFP) by endothelial cells (Fig. 4C), altering vessel permeability, it is likely that IL17A additionally allowing us to directly target IL17A to the endothelium, in affects tumor dissemination through other means, such as the which IL17A acts in an auto/paracrine manner. Serum from induction of survival or angiogenic genes (24). Several lines of þ þ na€ve VE-cadherin-Cre IL17Aind/ micehadelevatedlevelsof evidence suggest that IL17A may play a protective role in tumor þ þ IL17A (Fig. 4D). VE-cadherin-Cre IL17Aind/ mice and litter- surveillance in the context of T-cell responses (25). In vitro– mate controls were challengedwithLLCcellsasdescribed generated IL17A-secreting TH17 cells for instance were able to above. Whereas transgenic expression of IL17A again did not eradicate established tumors in an IFNg-dependent manner upon have a signiﬁcant impact on the primary tumor size (Fig. 4E), a transfer into tumor-bearing mice (5). Conversely, a growing signiﬁcantly higher number of metastases were found in the body of data support the protumorigenic function of IL17A,

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Kulig et al.

which is associated with its impact on the nonhematopoietic Writing, review, and/or revision of the manuscript: P. Kulig, A.L. Croxford, € compartment (16, 24, 26). Here, we found gd T cells to be the N. Hovelmeyer, B. Becher main source of IL17A in the context of lung metastasis using Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Burkhard several preclinical models. IL17A modulates endothelial integrity, Study supervision: B. Becher leading to enhanced tumor cell extravasation into the lung parenchyma. By combining an array of IL17-transgenic mice with Acknowledgments several models of tumor metastasis, we propose a function of The authors thank S. Hasler and J. Jaberg for their excellent technical IL17A in promoting tumor cell–endothelial interactions. assistance.

Disclosure of Potential Conﬂicts of Interest Grant Support This study was supported by The Swiss National Science Foundation No potential conﬂicts of interest were disclosed. 310030_146130 (B. Becher) and 316030_150768 (B. Becher), Swiss Cancer League (B. Becher), The University research priority project "URPP-translational Authors' Contributions cancer research" (B. Becher), The European Union FP7 project ATECT Conception and design: P. Kulig, J. Mikita-Geoffroy, A.L. Croxford, B. Becher (B. Becher), EU-FP7-ITN_NeuroKine (A. Waisman and B. Becher), DFG grant € Development of methodology: P. Kulig, S. Burkhard, J. Mikita-Geoffroy, HO 4440/1-1 (N. Hovelmeyer), and EMBO long-term fellowship ALTF 508- A.L. Croxford, G. Gyulv€ eszi, A. Waisman, L. Borsig 2011 (A.L. Croxford). Acquisition of data (provided animals, acquired and managed patients, pro- The costs of publication of this article were defrayed in part by the payment advertisement vided facilities, etc.): P. Kulig, S. Burkhard, J. Mikita-Geoffroy, N. Hovelmeyer,€ of page charges. This article must therefore be hereby marked C. Gorzelanny, L. Borsig in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. Kulig, S. Burkhard, J. Mikita-Geoffroy, Received June 23, 2015; revised September 1, 2015; accepted September 16, C. Gorzelanny 2015; published OnlineFirst November 19, 2015.

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32 Cancer Immunol Res; 4(1) January 2016 Cancer Immunology Research

IL17A-Mediated Endothelial Breach Promotes Metastasis Formation

Paulina Kulig, Sara Burkhard, Joanna Mikita-Geoffroy, et al.

Cancer Immunol Res 2016;4:26-32. Published OnlineFirst November 19, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-15-0154

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