tumor lymphangiogenesis promotes t cell infiltration and … · antibodies against vegf...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

CANCER

1Institute of Bioengineering, Swiss Federal Institute of Technology Lausanne(EPFL), Lausanne, Switzerland. 2Institute for Molecular Engineering, University ofChicago, Chicago, IL 60637, USA. 3Department of Oncology and Ludwig Cancer Re-search, University of Lausanne, Lausanne, Switzerland. 4Department of Cell, Develop-mental and Cancer Biology and Knight Cancer Institute, Oregon Health and ScienceUniversity, Portland, OR 97239, USA. 5The Bioinformatics and Biostatistics Core Facil-ity, EPFL, Lausanne, Switzerland. 6Swiss Institute for Experimental Cancer Research,School of Life Sciences, EPFL, Lausanne, Switzerland. 7The Ben May Departmentfor Cancer Research, University of Chicago, Chicago, IL 60637, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected]

Fankhauser et al., Sci. Transl. Med. 9, eaal4712 (2017) 13 September 2017

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Tumor lymphangiogenesis promotes T cell infiltrationand potentiates immunotherapy in melanomaManuel Fankhauser,1* Maria A. S. Broggi,1,2* Lambert Potin,1,2 Natacha Bordry,3 Laura Jeanbart,1

Amanda W. Lund,1,4 Elodie Da Costa,1 Sylvie Hauert,1,2 Marcela Rincon-Restrepo,1

Christopher Tremblay,1 Elena Cabello,5 Krisztian Homicsko,3,6 Olivier Michielin,3

Douglas Hanahan,6 Daniel E. Speiser,3 Melody A. Swartz1,2,6,7†

In melanoma, vascular endothelial growth factor–C (VEGF-C) expression and consequent lymphangiogenesiscorrelate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggestingthat lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressedthis concept in mouse melanoma models with VEGF receptor–3 (VEGFR-3)–blocking antibodies and unexpectedlyfound that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further foundthat this effect was mediated by VEGF-C–induced CCL21 and tumor infiltration of naïve T cells before immuno-therapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, geneexpression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrationsassociated with both T cell activation and expansion after peptide vaccination and clinical response to checkpointblockade. We propose that VEGF-C potentiates immunotherapy by attracting naïve T cells, which are locally acti-vated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarkerfor immunotherapy response.

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INTRODUCTIONMany tumors including melanoma are considered lymphangiogenicbecause they associate with lymphatic vessels (LVs) and can inducelymphatic expansion and activation via vascular endothelial growthfactor–C (VEGF-C), and both VEGF-C expression and LV density cor-relate with poor prognosis in melanoma patients (1–3). Apart fromoffering physical routes for metastatic spread (4, 5), recent evidence isemerging that tumor-associated LVs are involved in shaping antitumorimmunity (6, 7). On a physical level, they passively drain antigens, cy-tokines, and danger signals from the tumor to sentinel lymph nodes(LNs), which are essential for the generation of a T cell–inflamedmicro-environment inmousemelanoma (8).Moreover, lymphatic endothelialcells (LECs) actively affect immune cell function by releasing immuno-modulatory cytokines and by presenting endogenous and exogenousantigens on major histocompatibility complex (MHC) class I and IImolecules (9–16). In this context, it was previously shown that tumor-associated LECs in lymphangiogenic tumors can directly suppressantitumor T cell responses by tolerogenic cross-presentation of tumorantigens (11). Finally, VEGF-C induces LECs to up-regulate CCL21, achemokine expressed by LN stromal cells that, together with CCL19,guides immune cell subsets into the LN paracortex for education(17–19). We have previously shown that tumor-associated CCL21recruits CCR7+ immune cells into primary mouse melanomas andinduces the formation of a lymphoid-like stroma with hallmarks ofan immunosuppressive tumor microenvironment (20). Together, these

observations have promoted the idea that tumor-associated LECs areone of themany cell types that promote a suppressivemicroenvironmentand help the tumor escape host immunity. A better understanding of themechanisms that govern immunoregulation by tumor-associated lym-phatics should thus enable the rational development of immuno-therapeutic strategies.

We asked whether inhibiting tumor-associated lymphangiogenesis,thereby reducing its suppressive effects, would enhance the efficacy ofimmunotherapy, but we observed the opposite. Instead, we identify amechanismwherebyCCL21-dependent recruitment of naïveT cells intolymphangiogenic melanomas renders the tumor microenvironment moreresponsive to systemic immunotherapy.Wesuggest thatonce in the tumor,naïve T cells are locally primed and activated after immunotherapy-induced tumor cell death, leading to epitope spreading and long-lasting antitumor immunity. These results reveal an unappreciated roleof tumor-associated lymphangiogenesis in shaping the tumor immunemicroenvironment.

RESULTSVEGF receptor–3 inhibition decreases suppressive featuresof VEGF-C–expressing B16 melanomaLymphangiogenesis was not seen in ovalbumin (OVA)–expressingB16-F10 tumors (B16-OVA), so we modified the cells to expressVEGF-C (B16-OVA/VC) (Fig. 1A).We then assessedwhether blockingantibodies against VEGF receptor–3 (VEGFR-3) (aR3) altered LECdensity after implantation. As expected, VEGF-C increased the densityof intratumoral Lyve-1+ LVs, whereas aR3 starting the day of inocula-tion prevented this lymphangiogenesis (Fig. 1B). After tumor digestionoptimized for stromal and immune cell retrieval (21), we confirmed byflow cytometry that LECs (gp38+CD31+), but not blood endothelial cells(BECs; gp38−CD31+) ormacrophages (Macs; F4/80+), were enriched inlymphangiogenic tumors (Fig. 1, C and D). B16-OVA tumor growthwas unaffected by VEGFR-3 blockade, whereas B16-OVA/VC tumorsgrew somewhat larger than the B16-OVA tumors (Fig. 1E); this increase

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was eliminated with aR3.No differences were seenamong any of the groupsin overall survival (Fig. 1E).

We speculated that theincreased growth rate inlymphangiogenic tumors[that is, B16-OVA/VC tu-

mors treated with isotype control (Iso) antibody] was due to increasedinflammatory cell infiltration seen previously (11). We found VEGFR-3–dependent increases in immune cell infiltration (Fig. 1F), most nota-bly in regulatory T (Treg) cells (Fig. 1G), immunosuppressive myeloidsubsets, such as immature myeloid cells and myeloid-derived suppres-


sor cells (MDSCs) (Fig. 1,H and I), and antigen-presenting cells, includ-ing conventional, cross-presenting (CD8+), and myeloid dendritic cell(DC) subsets (Fig. 1, J and K). We did not detect any significant effectsof aR3 on immune cell infiltration in non-lymphangiogenic B16-OVAtumors (Fig. 1, D and F to K). These data demonstrate that VEGF-C

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Fig. 1. Blocking VEGFR-3signaling in lymphangiogenicmelanomas decreases Treg cellinfiltration and delays primarytumor growth. B16-OVA andB16-OVA/VC tumor–bearingmicewere treated with control (Iso) oraR3 starting on the day of inoc-ulation, and tumors were charac-terized on day 9. (A) IntratumoralVEGF-C concentration (n = 5). N.D.,not detected. (B) Immunostainedwhole-tumor sections [scale bars,500 mm and 200 mm (in zoomedimages)] showing overall lymphaticdensity [green, Lyve-1; gray, 4′,6-diamidino-2-phenylindole (DAPI)].(C) Representative flow cytometryplots of tumor cell suspensions forLECs (CD45−gp38+CD31+), BECs(CD45−gp38−CD31+), and Macs(CD45+F4/80+). (D) Quantificationof LECs, BECs, and Macs in tumors(n = 5). (E) Growth and survivalcurves (n ≥ 5). (F) Total infiltratingleukocytes (CD45+). (G) Treg cells(CD4+FoxP3+) and effector CD8+

T (Teff) cells (CD62L−CD44+).

(H) Gating strategy for myeloidsubsets. (I) Quantification ofmyeloid subsets (mature, m-Myeloid, CD11c−CD11b+MHCII+

and immature, imm-Myeloid,CD11c−CD11b+MHCII−) andMDSCs (granulocytic, G-MDSCs,CD11c−CD11b+MHCII−Ly6G+Ly6Clow,and monocytic, Mo-MDSCs,CD11c−CD11b+MHCII−Ly6G+Ly6Clow).(J) Gating strategy for DC subsets.(K) Quantification of DC subsets: con-ventional (cDCs; CD11c+CD11b−),cross-presenting (CD8+ DCs;CD11c+CD11b−CD8+), and mye-loid (Myel. DCs; CD11c+ CD11b+)(n = 5). All data represent twoindependent experiments. *P <0.05, **P < 0.01 by two-tailed Stu-dent’s t test.

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expression promotes features of an immunosuppressive tumor micro-environment, whereas inhibiting VEGFR-3 signaling prevents tumorlymphangiogenesis and decreases suppressive cell infiltrates such as Tregcells and MDSCs in B16 melanomas.

Lymphangiogenic melanomas are highly sensitiveto immunotherapyHaving confirmed that inhibiting VEGFR-3 signaling and preventinglymphangiogenesis decreases cellular hallmarks of immunosuppressionin B16-OVA/VC tumors, we hypothesized that VEGFR-3 blockadewould enhance the efficacy of immunotherapy in this model. To testthis, we adoptively transferred ex vivo activated OVA-specific CD8+

OT-I cells into tumor-bearing mice on day 9 after tumor inoculation.At early times after adoptive T cell therapy (ATT), aR3-treated tumorsresponded earlier, withmore rapid declines in tumor volume on day 12(Fig. 2A). However, these tumors reversed course and began progressingagain shortly thereafter, whereas lymphangiogenic (Iso-treated) tumorsshowed a more profound and long-lasting response to ATT. This trans-lated into significantly decreased tumor volume in the progression phase(day 23, P < 0.0001; day 27, P = 0.002) and increased survival of controlIso-treated B16-OVA/VC tumor–bearing mice. In contrast, aR3 treat-ment had no effect on the efficacy of ATT in B16-OVA tumors, indicat-ing that VEGF-C was necessary to potentiate immunotherapy (fig. S1A).Furthermore, we performed the same experiment inmice lacking dermalLVs (K14-VEGFR-3-Ig mice) (22) and found no difference betweenB16-OVA and B16-OVA/VC tumor growth or host survival (Fig. 2B),confirming that the VEGF-C–mediated potentiation of ATT was de-pendent on host lymphangiogenesis.

We next asked whether the lymphangiogenic status of B16 mela-nomas modulates the efficacy toward an immunotherapy approachthat relies on raising an endogenous antitumor response. We used atherapeutic DC vaccination (DC vax), in which ex vivo activated,SIINFEKL peptide–pulsed, bone marrow–derived DCs were intra-venously injected into tumor-bearing mice on days 4 and 10 aftertumor inoculation. As with ATT, we found that aR3-treated tumorsbegan to regress earlier following immunotherapy but then reversedcourse, whereas lymphangiogenic tumors underwent profound andlong-lasting regression (Fig. 2C). Accordingly, the median survivalincreased from 21 days for aR3-treated mice to more than 2 monthsfor Iso-treated mice bearing B16-OVA/VC tumors.

To test whether the lymphangiogenic status of B16 tumors alsomodulates non–antigen-specific immunotherapy, we used an adjuvant-only treatment with the TLR9 (Toll-like receptor 9) ligand CpG. Intra-dermal CpG injection into the hind footpads on days 4, 7, and 10 aftertumor inoculation was more effective at controlling tumor growth andenhancing survival in Iso-treated versus aR3-treated mice (Fig. 2D).When CpG was combined with OVA protein, vaccine efficacy wasnearly complete (Fig. 2E); in all but two mice, tumors regressed com-pletely. The VEGF-C potentiating effects on immunotherapy were notlimited to vaccinating againstOVAbecause a vaccine composed ofCpGand nanoparticle-bound endogenous melanoma peptide Trp2 (NP-Trp2) (23) showed similar trends (Fig. 2F). Furthermore, to rule outthe possibility that lymphangiogenic potentiation was only effectiveagainst tumors expressing the highly immunogenic antigen OVA, weperformed a set of experiments with wild-type B16 (B16-WT) andVEGF-C–overexpressing (B16/VC) tumors lacking OVA. As before,VEGFR-3 blockade alone did not affect the growth of B16-WT orB16/VC tumors (fig. S1B), but only VEGF-C–expressing (Iso-treated)tumors showed enhanced response to immunotherapy, either to CpG


adjuvant-only therapy (fig. S1C) or to the NP-Trp2 + CpG vaccine(Fig. 2G).

To extend our results beyond transplantable tumor models, weperformed immunotherapy in a more clinically relevant, geneticallyengineered mouse model of melanoma driven by mutated BrafV600E

and biallelic deletion of Pten (BrafV600E/Pten−/−) (24). These tumors,induced via topical application of 4-OH-tamoxifen, were naturallylymphangiogenic, and blocking VEGFR-3 led to decreased intratu-moral lymphatics (fig. S2, A and B). To raise a potent antitumor immuneresponse, BrafV600E/Pten−/− mice received a combinatorial immuno-therapy consisting of a peptide vaccine (CpG + gp100 peptide) combinedwith anti–PD-1 treatment starting 8 days after tumors were visible. Asobserved in the B16 model, anti–VEGFR-3–treated BrafV600E/Pten−/−

mice responded less well to immunotherapy intervention, whereasimmunoglobulin G (IgG)–treated mice showed delayed tumor out-growth and increased survival (Fig. 2H). Together, these data demon-strate that VEGF-C–mediated lymphangiogenesis potentiates theeffects of immunotherapy in several different mouse models of mela-noma, despite promoting an immunosuppressive microenvironment.

CCL21 is increased in lymphangiogenic melanomasand drives recruitment of naïve T cells intoVEGF-C–overexpressing B16 tumorsWe next asked why the more immunosuppressed, invasive tumorswould be more responsive to immunotherapy. When examiningthe immune cell infiltrates before immunotherapy, we found a sig-nificant increase (~2.5-fold, P = 0.03) in CD4+FoxP3− T cell densityas well as increased CD8+ T cell density in Iso-treated B16-OVA/VCtumors versus aR3-treated tumors (Fig. 3A). The infiltrating subsetsresponsible for these increases were mainly naïve (CD62L+CD44−)T cells, particularly in the CD4+ T cell compartment (Fig. 3B), shiftingthe ratio between naïve and effector (CD62L−CD44+) T cells in lym-phangiogenic tumors (Fig. 3C).

Because naïve, but not effector, T cells express the chemokinereceptor CCR7, we assessed tumor expression of the CCR7 ligandCCL21, which is normally expressed by LN stromal cells to guide naïveand memory T cells as well as mature DCs into the LN parenchyma(17, 25). Because CCL21 is expressed by LECs and up-regulated in re-sponse to VEGF-C/VEGFR-3 signaling (17, 19, 26–28), we were notsurprised to find that CCL21 protein was substantially increased inlymphangiogenic B16 (Fig. 3D) and BrafV600E/Pten−/− (fig. S2B) tu-mors. Furthermore, CCL21 was detected within and around lymphaticendothelium in both tumor models (Fig. 3E and fig. S2C). This effectwas restricted to the local tumor microenvironment because no changein CCL21 concentration could be detected in the tumor-draining LNs(dLNs) or non-draining LNs (ndLNs) (Fig. 3D). Accordingly, increasednumbers of conventional T cells, but not Treg cells, expressing theCCL21 receptor CCR7 were observed within B16-OVA/VC as com-pared to B16-OVA tumors (Fig. 3F).

To test whether CCL21/CCR7 signaling was responsible for theincreased recruitment of naïve T cells into lymphangiogenic tumors,B16-OVA/VC tumor–bearing mice were treated with CCR7-blockingantibodies (Fig. 3, G to J). CCR7 blockade mainly reduced the infil-tration of naïve T cells (Fig. 3, G and H), with a very large reduction inthe ratio of naïve versus effector phenotype for both tumor-infiltratingCD4+ and CD8+ T cells (Fig. 3I). We also observed this more directlyby adoptive transfer of allogeneic (CD45.2) naïve OT-I cells into micebearing B16-OVA/VC tumors; after 24 hours, nearly 10-fold fewer OT-Icells were found in aCCR7-treated tumors compared with control-treated

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tumors (Fig. 3J). Together, these data demonstrate that the CCL21/CCR7 axis drives not only regulatory CD4+ but also naïve T cells intolymphangiogenic B16 melanomas.

In human melanoma, VEGFC expression correlates withCCL21, CCR7, and a T cell signatureWe next asked whether the VEGF-C/CCL21 axis was relevant forshaping the immune microenvironment in human melanoma. Wefirst performed immunofluorescence analysis of the lymphaticmarker podoplanin in sections from 14 untreated primary humanmelanomas (Fig. 4A) and found that roughly half were lymphan-giogenic, that is, showing substantially higher lymphatic density inthe tumor than in the adjacent skin (Fig. 4B). Furthermore, thesetumors stained positively for VEGF-C expression (Fig. 4C) as wellas CCL21 expression in intratumoral LECs (Fig. 4D).


Several recent reports have described genetic signatures that stratifypatient response to immunotherapy according to T cell infiltration,where high-expression levels are seen in responders (24, 29–33). Wethus analyzed primary tumors from 469 metastatic melanoma patientsfrom The Cancer Genome Atlas (TCGA) database and found strongcorrelations between VEGFC, but not VEGFA or FIGF (VEGFD), andgenes correlating with immunotherapy response (Fig. 4E, left). In ad-dition, several genes previously correlated with immunotherapy resistance,including CTNNB1 andMYC (24), were inversely correlated with VEGFC(Fig. 4E, right). In line with our findings in mice, gene expression ofVEGFC correlated with that of CCL21 and CCR7 in human primarymelanoma, whereas expression of the other main VEGFR-3 ligand,FIGF (VEGFD), showed no correlation, and VEGFA showed an inversecorrelation (Fig. 4F, top two rows). Similarly, expression of CD8, CD4,CD11c, FoxP3, and CD127 (expressed by naïve T cells), but not CD44

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Fig. 2. VEGF-C/VEGFR-3 signaling increases responsiveness of melanoma to immunotherapy. Tumor growth and survival of three different melanoma modelstreated with control (Iso) or aR3-blocking antibodies receiving different immunotherapies (arrows indicate times of administration). (A and B) B16-OVA/VC tumorstreated with ATT in (A) WT (n ≥ 15) and (B) K14-VEGFR-3-Ig mice that lack dermal lymphatics (n = 4). ns, not significant. (C to F) B16-OVA/VC tumors in WT mice treated with (C)ex vivo activated DCs (DC vax; n = 6), (D) 50 mg of CpG (n = 6), (E) 10 mg of OVA + 50 mg of CpG (n ≥ 8), and (F) 2 mg of Trp2 peptide–conjugated nanoparticles (NP-Trp2) +50 mg of CpG (n = 7). (G) B16/VC tumors treated with NP-Trp2 + 50 mg of CpG (n = 6). (H) Tamoxifen-induced tumors in BrafV600E/Pten−/− mice treated with CpG + gp100peptide (days 8 and 12) and anti–PD-1 antibody (day 12 and every 4 days thereafter). Each panel shows data from one (B to D, F, and G), two (E), or three (A) independentexperiments. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test for growth curves and log-rank (Mantel-Cox) test for comparing survival curves.

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Fig. 3. VEGFR-3 signaling increases infiltration of naïve T cells in a CCR7-dependent manner. (A to D) B16-OVA and B16-OVA/VC tumor–bearing mice weretreated with control (Iso) or VEGFR-3–blocking antibodies (aR3) starting on the day of inoculation, and tumors were characterized on day 9 by flow cytometry (datarepresent two independent experiments, n = 5 each). (A) Quantification of tumor-infiltrating conventional CD4+ T cells (conv CD4+; FoxP3−) and CD8+ T cells. (B) Activationstatus of CD4+ (top) and CD8+ (bottom) T cells. Naïve, CD44−CD62L+; effector/effector memory (EM), CD44+CD62L−; central memory (CM), CD44+CD62L+. (C) Ratio of naïve/EMin infiltrating CD4+ and CD8+ T cells. (D) CCL21 concentration as assessed by enzyme-linked immunosorbent assay (ELISA) in the tumor, dLNs, and ndLNs. (E) Representativeimage of a lymphangiogenic B16/VC tumor section immunostained for CD4+ T cells (red), CCL21 (green), LECs (Lyve-1, white), and DAPI (blue). Scale bar, 100 mm. (F) Quan-tification of CCR7+ T cell subsets in untreated B16-OVA and B16-OVA/VC tumors after 14 days (n ≥ 4). (G to J) B16-OVA/VC tumor–bearing mice were treated with Iso or anti-CCR7 (aCCR7)–blocking antibodies on days 0, 3, and 6, and (G to I) tumors were characterized on day 9 by flow cytometry or (J) mice were given adoptive transfer of 106 naïveOT-I CD8+ T cells (n ≥ 6). (G) Representative flow cytometry plots of T cell activation status. (H) Quantification of naïve and CM fractions of conventional CD4+ (left) and CD8+

(right) T cells. (I) Ratio of naïve/EM subsets. (J) Quantification of intratumoral OT-I cells as percentage of overall CD8+ T cells on day 10 after inoculation. *P < 0.05, **P < 0.01,***P < 0.001 performed with two-tailed Student’s t test or one-way analysis of variance (ANOVA).

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2 4 6 82468

1012r = 0.29 P = 0.003

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Fig. 4. Primary humanmetastatic melanomascontain CCL21-expressingLECs, and expression ofVEGFCpositivelycorrelateswithhallmarksof tumor in-flammation. (A) Represent-ative image of a humanprimarymelanoma immuno-stained for LVs (green, podo-planin; blue,DAPI). Scalebars,500 mm (left) and 200 mm(right). (B) Quantification ofLV density in tumor (n = 14)and, when present, neigh-boring skin (n = 7) of primarymelanoma tumor sectionsstratifying patients with ele-vated intratumor LV density(closed circles), indicatingtumor lymphangiogenesis,from those without (opencircles). (C) Representativeimage of a lymphangio-genic melanoma immuno-stained for VEGF-C (brown).Scale bar, 100 mm. (D) Rep-resentative image of an in-tratumoral LV (podoplanin,green) expressing CCL21(red). Blue, nuclei (DAPI). Scalebar, 10 mm. (E and F) Corre-lations of gene expressiondata of human primary cu-taneous metastatic mela-noma patients from TCGA.(E) Heat map showing cor-relation between the expres-sionof 30genes indicative ofT cell inflammation versusVEGFC, FIGF (VEGFD), andVEGFA. Colors indicate min-imumandmaximum rvaluesusing nonparametric Spear-man’s test. (F) Dot plots ofgenes of interest (n = 103)shown with linear regres-sion correlations using non-parametric Spearman’s test.

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(expressed by activated T cells), correlated with that of VEGFC (Fig. 4F,bottom three rows). The same trends were observed in metastatic(secondary) tumors from the same database (fig. S3). Together, thesedata show that VEGFC and CCL21 expression are strongly correlated inhuman melanoma and are consistent with the notion that VEGF-C/CCL21 up-regulation shifts the immune microenvironment to driveT cell infiltration.

VEGF-C correlates with response to immunotherapy inhuman metastatic melanoma patientsWenext sought to determinewhetherVEGF-C correlatedwith responseto immunotherapy in human melanoma patients. Using sera storedfrom an earlier clinical study of 20 patients who underwent Melan-Aanalog vaccination (34), we measured VEGF-C concentrations andfound direct correlations with T cell response to immunotherapy, interms of both numbers of circulating Melan-A–specific CD8+ T cells(Fig. 5A) and their expression of the effector cytokine interferon-g(IFN-g) (Fig. 5B). Furthermore, Melan-A–specific T cells in patientswith high VEGF-C concentration displayed superior polyfunctionality(Fig. 5C).

We next measured serum VEGF-C concentrations in a larger clin-ical trial of humanmetastatic melanoma patients who underwent com-bined anti–CTLA-4 (ipilimumab) and anti–PD-1 (nivolumab) therapyto determine correlationswith progression-free survival (PFS). Strikingly,PFS correlated with high levels of serum VEGF-C but not VEGF-A orVEGF-D, each stratified into mid, low, and high according to means ±0.4 SD (Fig. 5D). Together, these data demonstrate that serumVEGF-Cconcentration before immunotherapy not only predicts the magnitudeand quality of immune responses raised by a cancer vaccine but also


stratifies long-term patient responses to combined checkpoint blockadeand further strengthens the case for investigating the use of serumVEGF-C as a predictive biomarker for immunotherapy candidates.

Lymphangiogenic potentiation of immunotherapy isdependent on CCR7 signaling and local activationof naïve T cellsNaïve T cells can be recruited and primed within primary tumors(35–37), and we asked whether the increased accumulation of naïvetumor-infiltrating lymphocytes (TILs) within lymphangiogenic B16tumors was responsible for the increased susceptibility to immuno-therapy. We hypothesized that naïve CCR7+ T cells within lymphan-giogenic tumors could be locally activated after immunotherapy-inducedrelease of tumor antigen and innate immune activation, therebyincreasing antigen spreading as well. Accordingly, we found that, 3 daysafter ATT, Iso-treated B16-OVA/VC tumors contained higher num-bers of activated as well as naïve endogenous CD8+ T cells togetherwith more transferred OT-I cells as compared to aR3-treated tumors(Fig. 6A). This was not the case in the dLNs, where the observed im-mune cell subsets accumulated to a similar extent, independent ofVEGFR-3 signaling (Fig. 6B). To test whether the lymphangiogenic po-tentiation of ATT was dependent on VEGF-C–induced recruitment ofnaïve T cells into the tumor, CCR7-blocking antibodies wereadministered during tumor development until a few days before im-munotherapy to avoid confounding effects. We found that after theinitial response to ATT, B16-OVA/VC tumors of aCCR7-treated miceresponded similarly as aR3-treated mice, namely, with a faster initialresponse to ATT but later relapse and rapid progression compared toIso-treated mice (Fig. 6C). This suggested that the recruitment of

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Fig. 5. Serum VEGF-C correlates with antitumor immune responses and PFS after immunotherapy in human metastatic melanoma patients. (A to C) Correla-tions of magnitude and quality of T cell responses with serum VEGF-C concentrations (n = 20) in patients enrolled in a phase 1 clinical study (NCT00112229) evaluatingan antitumor Melan-A/MART-1 peptide vaccine. T cell responses reflect peak values across four weekly blood samples in Melan-A tetramer+ CD8+ T cells, and serumVEGF-C was measured before therapy. (A) Antigen-specific T cells as % of circulating CD8+ T cells versus serum VEGF-C. Left: Absolute values for each patient (dottedline indicates mean VEGF-C). Right: Comparison of T cell numbers in patients with low (mean) VEGF-C. (B) IFN-a expression and (C) poly-functionality in terms of IFN-a, TNF-a (tumor necrosis factor–a), IL-2 (interleukin-2), and CD107 expression in tetramer+ CD8+ T cells. (D) PFS of human melanomapatients (n = 76) enrolled in a phase 2 clinical study (NCT01927419) receiving combined aPD-1 and aCTLA-4 checkpoint blockade. Patients were stratified into threegroups (high, mid, low) according to serum VEGF-C, VEGF-D, and VEGF-A concentrations measured before immunotherapy. Groups were compared using a non-parametric Spearman’s test for correlations, two-tailed Student’s t test for dot plots (*P < 0.05), and log-rank (Mantel-Cox) test for survival curves.

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naïve T cells to the tumor before immunotherapy, which dependson CCR7 signaling, was required for the VEGF-C enhancement ofimmunotherapy.

To assess whether these naïve T cells were activated withinthe tumor, we next performed ATT while blocking lymphocyteegress from LNs using the inhibitor FTY720. The enhanced efficacyof ATT in B16-OVA/VEGF-C tumors was unaffected by FTY720(Fig. 6D), although it severely depleted circulating numbers of lym-phocytes (Fig. 6, E and F), indicating that lymphangiogenic po-tentiation was independent of T cells activated in the LN afterimmunotherapy. Because VEGF-C–mediated potentiation of immu-notherapy in B16 melanomas was dependent on CCR7-mediated at-traction of naïve T cells to the tumor and independent of effectorT cell recruitment after immunotherapy, it is likely that the localactivation and expansion of recruited naïve T cells within the tumormicroenvironment is the key mechanism underlying lymphangiogenicpotentiation.


Mice that reject lymphangiogenic B16 melanomas afterimmunotherapy show epitope spreading and protectionto rechallengeIf lymphangiogenic tumors attract more naïve T cells that can becomeactivated locally upon tumor cell killing initiated by immunotherapy,then one would expect the antigenic repertoire of tumor-reactive T cellsto broaden beyond the targeted antigen (that is, OVA in these experi-ments) asmore of these recruited cells expand—a process called antigenor epitope spreading (38–40). Accordingly, we found that, 2 weeks afterOVA vaccination (day 23 after tumor inoculation), mice bearing lym-phangiogenic tumors (Iso) had increased numbers of endogenous effec-tor CD4+ and CD8+ T cells as well as the OVA-specific CD8+ T cells inblood as compared to either aR3-treated or non–tumor-bearing micevaccinated with OVA + CpG (Fig. 7A).

Because the tumorshad completely regressed inmost of the Iso-treatedmice that had been therapeutically vaccinated with OVA-CpG (Fig. 2E),we rechallenged thosemice at least 14 days after complete regression, with

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Fig. 6. Increased efficacy of immunotherapy in lymphangiogenic B16 melanomas depends on CCR7 signaling before therapy and local activation and ex-pansion of TILs after therapy. (A and B) B16-OVA/VC tumor–bearing mice treated with control IgG (Iso) or anti–VEGFR-3 (aR3)–blocking antibodies were euthanized 3 daysafter ATT, and tumor single-cell suspensions were analyzed by flow cytometry (n = 5). Quantification of overall naïve CD8+ (CD45+CD8+CD44−CD62L+), effector CD8+

(CD45+CD8+CD44+CD62L−), and OT-I (CD45+CD8+CD45.1+) T cells (A) in the tumor and (B) in the dLNs. (C) Tumor growth and survival curves of B16-OVA/VC tumor–bearing mice treated with anti-CCR7 (aCCR7), control IgG (Iso), or aR3 antibodies combined with ATT on day 9. CCR7 blockade was performed only before ATT (days 0, 3,and 6) (data pooled from two or more independent experiments, n ≥ 15 total). (D) Tumor growth curves of B16-OVA/VC tumor–bearing mice treated with control IgG (Iso)or aR3 antibodies received daily injections of the small molecular S1P inhibitor FTY720 starting on the same day as ATT was performed (day 9) (n ≥ 5). Statistics showdifferences between Iso + FTY720 and aR3 + FTY720 by one-way ANOVA. (E) Representative flow cytometry plots and (F) quantification of circulating CD4+ and CD8+ T cells(after B220 exclusion) in blood 26 days after tumor inoculation. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test or one-way ANOVA and log-rank (Mantel-Cox)test for survival curves.

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an intravenous injectionof eitherB16-OVA/VCorB16-WTcells. Becausethe tumor-bearing mice that had been aR3-treated did not survive, weused non–tumor-bearing mice vaccinated with OVA + CpG and naïvemice as positive and negative controls, respectively. In naïve mice, theB16-OVA/VC cells drove more extensive lung metastasis than the B16-WT (Fig. 7, B and C), consistent with our expectations. Also as expected,theOVA-vaccinatedmicewere partially protected againstmetastasis fromB16-OVA/VC tumors but not from B16-WT tumors. However, the micethat had previously rejected B16-OVA/VC tumors were almost com-pletely protected against colonization of both B16-WT and B16-OVA/VC, despite having been vaccinated originally only against OVA(Fig. 7, B and C). Moreover, higher numbers of circulating OVA-specific CD8+ T cells were detected in these mice compared to the OVA-vaccinated only (Fig. 7D). Together, these data suggest that antigen-specificimmunotherapy in lymphangiogenic B16 tumors not only potentiatesongoing antitumor immune responses but also induces secondary immuneresponses against a variety of endogenous tumor antigens, conferring long-term memory and protection against pulmonary metastasis.

DISCUSSIONCollectively, these data reveal a new and unexpected role for tumor-associated lymphangiogenesis in enhancing the efficacy of systemicimmunotherapy. Although lymphangiogenic tumors are characterizedby hallmarks of immunosuppression before immunotherapy, theywere far more sensitive to systemic immunotherapy as compared to thosewhere VEGFR-3 signaling was blocked. Our data suggest that lymphan-giogenic potentiation of immunotherapy depends on the recruitment


and local activation of CCR7+ cells, particularly naïve T cells and DCs, inmelanoma tumors. We therefore hypothesize that upon immunotherapy-induced cytotoxicity, the release of antigens and danger signals promoteslocal T cell activation, thereby leading to antigen spreading and long-lasting memory. Furthermore, because T cell infiltration has been shownto correlate with patient response to immunotherapy (24, 29–33, 41),our findings have translational implications in suggesting that serumVEGF-C may serve as a predictive biomarker for responsiveness toimmunotherapy. We demonstrated two independent clinical immu-notherapy trials in metastatic melanoma patients, one using peptidevaccination and another using combined PD-1 and CTLA-4 check-point blockade that prospectively measured serum VEGF-C corre-lates with the magnitude and quality of antitumor immune responsesand PFS, respectively. This makes the case for VEGF-C– or tumor-associated lymphangiogenesis to be a key determinant of a patient’s“cancer immunogram” (42).

Our findings are surprising on the one hand because VEGF-C ex-pression in human tumors is strongly correlated with LN metastasesandpoor prognosis (1–3, 43).However, we have also previously demon-strated (i) that local lymphatics are important for initiating and estab-lishing the inflammatory tumormicroenvironment via communicationwith the dLN (8), (ii) that VEGF-C up-regulates CCL21 as well as sup-pressive factors in the tumor microenvironment (further supported inthe current work with human genomic data) (11, 18), and (iii) thatCCL21 expression in mouse melanoma drives LN-like changes in thetumor stroma that support the recruitment and education of immunecells (20). In the absence of immunotherapy, locally recruited T cellseither remained naïve or were rendered anergic or tolerant within the

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Fig. 7. Mice rejectingprimary lymphangiogenicB16melanomas in response to immunotherapy showepitope spreadingand long-termprotection. (A to D) B16-OVA/VC tumor–bearing mice that rejected the primary tumor [primary intradermal (1° i.d.) challenge] after therapeutic vaccination received a metastatic rechallenge withintravenous injections of 2 × 105 B16-WT or B16-OVA/VC cells [secondary intravenous (2° i.v.) challenge] at least 10 days after complete regression. Mice that receivedeither no treatment (naïve) or vaccination only (Vax only) served as controls. (A) Flow cytometry analysis of circulating effector CD4+ (CD45+B220−CD4+CD44+CD62L−),effector CD8+ (CD45+B220−CD8+CD44+CD62L−), and tumor antigen–specific CD8+ (CD45+B220−CD8+SIINFEKL-pentamer+) T cells 23 days after 1° i.d. challenge butbefore 2° i.v. rechallenge. (B) Representative images of lung metastases. (C) Quantification of metastatic nodules per lung of mice. (D) Circulating tumor antigen–specificCD8+ T cell responses 9 days after the 2° i.v. challenge (data pooled from two independent experiments, n ≥ 5 total). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.

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highly suppressive microenvironment (11, 20). Our current observa-tions demonstrate that these cells can be activated or reactivated uponimmunotherapy (or initiation of tumor cell killing) to formamore pow-erful defense against the tumor than if they had not been present at all.They also confirm relevance in human melanoma patients, in terms ofVEGF-C correlatingwith bothT cell infiltration and response to immu-notherapy, despite the well-established correlation of VEGF-C withworse prognosis in the absence of immunotherapy.

Limitations of this study include uncertainty in the following: (i) thepotential roles of other immune cell subsets that may be altered inVEGF-C–expressing tumors, including natural killer T cells, gd T cells,and especially various DC subsets that we found to be enriched; (ii) theextent to which numerous other cytokines that are altered in lymphan-giogenic tumors contribute to immunotherapy potentiation; (iii) cellsources of VEGF-C in human tumors; and (iv) indirect effects of naïveT cell recruitment on the immune microenvironment [for example,competition with Treg cells for nutrients (44) or homeostatic cytokinessuch as IL-7 (45)]. These will require further investigation.

Although tumor lymphangiogenesis has always been correlatedto increasedmetastasis and poor patient prognosis, this work reveals itsflip side, bringing into focus a more comprehensive understanding ofhow it shapes the immune microenvironment. We now appreciate thenumerous mechanisms of immunosuppression that a T cell–inflamedtumor develops to survive, including lymphangiogenesis. On the otherhand, lymphangiogenesis promotes immune recognition and supportsthe recruitment and local priming of naïve T cells. In an untreated de-veloping tumor, the suppressive environment prevents the functionalactivation of these cells, but when the scales are tipped toward activatingfactors dominating over suppressive ones, as is the case with immuno-therapy or possibly other means that drive immunogenic cell death,these T cells become robust participants in antitumor immunity. In thislight, tumor-associated lymphatics can play on both teams: that of thetumor and that of host immunity. Figuring out howwe can harness thelatter is an exciting challenge for immunotherapy.

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MATERIALS AND METHODSStudy designExperiments were designed to correlate infiltrating immune cells,chemokine levels, and response to various immunotherapy approacheswith lymphangiogenesis and VEGFR-3 signaling in several mousemodels. They included randomization across different cages and re-searcher blinding during calipermeasurements. To determine relevanceof findings to humanmelanoma patients, we first analyzed TCGA datasets for VEGFC, VEGFD, and VEGFA compared to gene signatures ofimmune infiltration. In two different clinical trials for immunotherapyof melanoma patients, serumVEGF-C was measured and correlated toimmune status and patient survival. Details of the clinical trial designsare included below. Sample numbers and numbers of replicates per-formed for each experiment are included in the figure legends. Allprimary data, used to compile all figures, are given in table S1.

Vaccination trial in metastatic melanoma patientsSerum VEGF-C concentrations from stage III and IV melanoma pa-tients that had been enrolled in a prospective phase 1 study evaluatingan antitumor peptide vaccine (ClinicalTrials.gov, NCT00112229) wereanalyzed (34). Patients were enrolled upon written informed consent.Briefly, patients had received monthly subcutaneous injections of avaccine composed of CpG 7909 (PF-3512676) oligonucleotides and


Melan-A/MART-1 peptide, emulsified inMontanide ISA-51.Melan-A–specific CD8+ T cell frequency and function weremeasured in bloodby flow cytometry at the time point of peak response (after an average ofeight injections). Serum VEGF-C was assessed using a commercialELISA kit (DVEC00; R&D Systems).

Checkpoint blockade trial in metastatic melanoma patientsSerum was collected from treatment-naïve patients with unresectable,stage III/IVmetastaticmelanoma in a randomized, double-blind, placebo-controlled, multicenter, two-arm, phase 2 trial, BMS (Bristol-MyersSquibb) CheckMate-069 (CA209-069, NCT01927419). All patientsprovided written informed consent for the use of biological materialsincluding serum analysis. The study compared ipilimumab (n = 47),given 3 mg/kg every 3 weeks for four doses, to combined (n = 89),ipilimumab (3 mg/kg) + nivolumab (1 mg/kg), given every 3 weeksfor four cycles, followed by nivolumab alone (3mg/kg) every 2 weeksuntil disease progression or unacceptable toxicity (46). Only patientsin the ipilimumab + nivolumab arm were included here (n = 76),excluding 13 patients who had either very short follow-up (



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4. S. A. Stacker, S. P. Williams, T. Karnezis, R. Shayan, S. B. Fox, M. G. Achen,Lymphangiogenesis and lymphatic vessel remodelling in cancer. Nat. Rev. Cancer 14,159–172 (2014).

5. S. Karaman, M. Detmar, Mechanisms of lymphatic metastasis. J. Clin. Invest. 124, 922–928(2014).

6. A. W. Lund, T. R. Medler, S. A. Leachman, L. M. Coussens, Lymphatic vessels, inflammation,and immunity in skin cancer. Cancer Discov. 6, 22–35 (2016).

7. M. A. Swartz, Immunomodulatory roles of lymphatic vessels in cancer progression.Cancer Immunol. Res. 2, 701–707 (2014).

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Acknowledgments: We thank B. Pytowski and Eli Lilly for the mF4-31C1 antibody, the BMS forsharing clinical data, P. Corthésy-Henrioud and Y. B. Saida for technical assistance, T. Gajewskifor the BrafV600E/Pten−/−mice, K. Alitalo for the K14-VEGFR-3-Igmice, and the Swiss Federal Instituteof Technology Lausanne (EPFL) Flow Cytometry and Bioimaging Core Facilities. Funding: Thisstudy was supported by the Swiss National Science Foundation (CR23I2_143754 and31003A_153471), the European Research Council (AdG-323053), SwissTransMed (35/2013), andFonds Pierre-François Vittone. Author contributions:M.F., M.A.S.B., L.P., and M.A.S. designed andanalyzed experiments. M.F., M.A.S.B., L.P., L.J., A.W.L., S.H., M.R.-R., E.D.C., and C.T. performedexperiments. M.A.S.B., N.B., D.E.S., K.H., O.M., and D.H. collected, characterized, and analyzed humanmelanoma specimens. E.C. performed bioinformatics analysis. M.F., M.A.S.B., and M.A.S. wrote the


manuscript with input and revisions from L.P., L.J., A.W.L., D.E.S., K.H., and D.H. Competing interests:Anti–VEGFR-3–blocking antibodies (mF4-31C1) were from Eli Lilly under a material transferagreement with the EPFL and the University of Chicago. M.A.S., M.F., and M.A.S.B. are inventorson a patent application (62/329,133) submitted by the University of Chicago that covers thepotential diagnostic and therapeutic uses of VEGF-C for cancer immunotherapy. The authors declarethat they have no other competing interests.

Submitted 24 November 2016Resubmitted 30 May 2017Accepted 11 July 2017Published 13 September 201710.1126/scitranslmed.aal4712

Citation: M. Fankhauser, M. A. S. Broggi, L. Potin, N. Bordry, L. Jeanbart, A. W. Lund, E. Da Costa,S. Hauert, M. Rincon-Restrepo, C. Tremblay, E. Cabello, K. Homicsko, O. Michielin, D. Hanahan,D. E. Speiser, M. A. Swartz, Tumor lymphangiogenesis promotes T cell infiltration andpotentiates immunotherapy in melanoma. Sci. Transl. Med. 9, eaal4712 (2017).

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melanomaTumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in

Michielin, Douglas Hanahan, Daniel E. Speiser and Melody A. SwartzCosta, Sylvie Hauert, Marcela Rincon-Restrepo, Christopher Tremblay, Elena Cabello, Krisztian Homicsko, Olivier Manuel Fankhauser, Maria A. S. Broggi, Lambert Potin, Natacha Bordry, Laura Jeanbart, Amanda W. Lund, Elodie Da

DOI: 10.1126/scitranslmed.aal4712, eaal4712.9Sci Transl Med

findings have important implications for the use and predictions of response to immunotherapy.clinical trials confirmed that indicators of lymphangiogenesis were associated with robust T cell responses. Theseactually disrupted recruitment of naïve T cells and subsequent antitumor immunity. Data from patients enrolled in

. used mouse models of melanoma to show that blocking lymphangiogenesiset alcancer treatment. Fankhauser Metastatic spread depends on lymphangiogenesis, and mediators of this pathway are targeted clinically for

Unintentional immunotherapy inhibition

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