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DCs are a diverse population of cells with remarkable plasticity1,and are therefore viewed as crucial regulators of adaptive immuneresponses2,3. They can break tolerance to tumor antigens and inducetumor regression4–6. Vegf-A, a critical factor promoting tumorangiogenesis7, inhibits DC differentiation and maturation8,9, medi-ating tumor immune evasion10,11. Vegf-A overexpression has beenrecently associated with the absence of T cells or mature DCs inovarian cancer12.

β-defensins are antimicrobial inflammatory peptides that recruitimmature DCs through CCR6, linking innate and adaptive immu-nity13. Although the role of inflammation in promoting tumor angiogenesis has been established14,15, whether β-defensins or theirtargets, DCs, are involved in these mechanisms remains unknown. Wedescribe here a new pathogenic mechanism supporting vasculogene-sis in tumors. Our data indicate that β-defensins recruit DC precur-sors through CCR6 into the tumor, where Vegf-A transforms theminto endothelial-like cells that engage in vasculogenesis and functionas promoters of tumor progression.

Defb29 cooperates with Vegf-A to promote tumor angiogenesisTo analyze the effect of β-defensins on tumors, we retrovirally trans-duced the complete open reading frame (ORF) of the newly clonedDefb29, encoding β-defensin-29 (Defb29), into Defb29–/– ID-8 mouseovarian cancer cells16 or ID8 cells engineered to overexpress mouse

Vegf-A164 (ID8-Vegf)17. Expression of Defb29 did not alter the growthof ID8 or ID8-Vegf cells in vitro. ID8 tumors with or without Defb29,and with high or low expression of Vegfa, were transplanted subcuta-neously into C57BL/6 mice. As previously reported17, levels of Vegf-Ain ID8 or ID8-Vegf tumors are within the range observed in humanovarian carcinoma. Expression of Defb29 had no significant effect onthe growth of ID8 tumors expressing low amounts of Vegfa (n = 15;Supplementary Fig. 1 online), but markedly accelerated the growth oftumors overexpressing Vegfa (ID8-Vegf Defb29, 1045.7 ± 191.1 mm3

compared with ID8-Vegf, 110.1 ± 26.7 mm3 after two months;P = 0.03; Wilcoxon test; Fig. 1a,b). Intraperitoneal ID8-Vegf Defb29tumors resulted in significantly shorter survival than control tumors(ID8-Vegf Defb29: 33.4 ± 9.8 d compared with ID8-Vegf: 96 ± 32.0 d;P < 0.01; Mann-Whitney test) (Fig. 1a).

CD31+ and CD34+ vessel density was more pronounced in ID8-Vegf Defb29 than control ID8-Vegf tumors (Fig. 1c,d); furthermore,levels of CD31 and von Willebrand factor (VWF) mRNA were 29 and5 times higher, respectively, in ID8-Vegf Defb29 than ID8-Vegftumors (Supplementary Fig. 1 online). Thus, β-defensin interactedwith Vegf-A to increase tumor vascularization.

DC precursors undergo endothelial-like specializationBecause β-defensins chemoattract DCs13,18, we examined the local-ization of CD11c+ cells in ID8-Vegf Defb29 tumors. CD11c+ cells

1Center for Research in Reproduction and Women’s Health, 2Abramson Family Cancer Research Institute and 3Department of Cell and Developmental Biology,University of Pennsylvania Medical Center, BRBII/III, 421 Curie Blvd, Philadelphia, Pennsylvania 19104, USA. 4Department of Obstetrics and Gynecology, Universityof Turin, 10126 Turin, Italy. 5These authors contributed equally to this work. Correspondence should be addressed to G.C. (gcks@mail.med.upenn.edu).

Published online 29 August 2004; doi:10.1038/nm1097

Tumor-infiltrating dendritic cell precursors recruited by aβ-defensin contribute to vasculogenesis under theinfluence of Vegf-AJose R Conejo-Garcia1,5, Fabian Benencia1,5, Maria-Cecilia Courreges1, Eugene Kang1, Alisha Mohamed-Hadley1,Ronald J Buckanovich1, David O Holtz1, Ann Jenkins1, Hana Na1, Lin Zhang1,2, Daniel S Wagner3,Dionyssios Katsaros4, Richard Caroll2 & George Coukos1,2

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumorvasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of β-defensins and vascular endothelialgrowth factor-A (Vegf-A). Expression of mouse β-defensin-29 recruited DC precursors to tumors and enhanced tumorvascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC andendothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and β-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow–derived DCsunderwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing highlevels of Vegf-A. We show that β-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks:β-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specializationand migration to vessels, which is mediated by Vegf receptor-2.

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localized mainly to capillary-like structures (Fig. 1e), where CD31and CD11c colocalized in >40% of vessels (Fig. 1f), suggestingendothelial-like specialization of CD11c+ cells.

CD11c+ cells from ID8-Vegf Defb29 tumors or ascites were sortedand characterized by flow cytometry. More than 90% ofCD11c+CD45+ cells were MHC-II+, DEC-205+ and CD8α+, high-lighting their DC lineage, but only 4% expressed CD80, suggestingan immature phenotype. Most CD11c+CD45+DEC-205+CD8α+ cellsexpressed CD34 and CD31, whereas 40% expressed highly specificendothelial markers VE-cadherin and P1H12 (ref. 19; Fig. 2a). Afraction of tumor CD11c+ cells expressed Vegf receptors(Supplementary Fig. 1 online). Immunopurified CD11c+CD31+ cellsexhibited diploid content of X chromosomes by fluorescence in situhybridization (FISH; Supplementary Fig. 1 online), ruling out cellfusion as a cause for such a hybrid phenotype. Thus, a new popula-tion of CD11c+ leukocytes exhibiting both endothelial and DC fea-tures was identified.

To further test whether tumor-associated CD11c+ cells migratetowards tumor vessels, we immunomagnetically procured a >98%pure population of CD11c+ cells from ascites of ID8-Vegf Defb29

tumors. Cells were cultured in ID8-Vegf Defb29 tumor-conditioned media for 24 h, stained with 7-amino-4-chloromethyl-coumarin (CMAC) and injected subcutaneously around flank ID8-Vegf Defb29 tumors (106 cells/tumor). Transplanted cells infiltratedtumors, migrating toward functional dextran-perfusable bloodvessels at a luminal location (Fig. 2b). Cells were still CMACbright

10 d after injection, suggesting that they had undergone no substantial proliferation.

The frequency of tumor-associated CD11c+ cells increased withprogressive tumor burden in ID8-Vegf Defb29 tumors(Supplementary Fig. 1 online). To test whether tumor-infiltratingCD11c+ cells could account for enhanced tumor growth, flanktumors were generated with an equal number of ID8-Vegf cellstransplanted alone (7 × 106 cells) or admixed (20:1 ratio,ID8-Vegf:CD11c+) with 99% pure CD11c+ cells immunopurified asabove from ascites. The presence of CD11c+ cells markedly acceler-ated tumor growth, giving rise to congested tumors filled with vascu-lar lakes (Supplementary Fig. 2 online). Thus, tumor-infiltratingCD11c+ cells exhibited endothelial features and promoted tumorvascular development and growth.

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Figure 1 Defb29 and Vegfa cooperatively promote tumor growth. (a) ID8-Vegf Defb29 tumors (Defb29 Vegf) exhibit accelerated growth (left) insubcutaneous locations and result in reduced survival (right) when inoculated intraperitoneally compared with ID8-Vegf tumors (Vegf). (b) IntraperitonealID8-Vegf Defb29 tumors cause rapid accumulation of ascites (right mouse) compared with ID8-Vegf tumors (left mouse). Subcutaneous ID8-Vegf Defb29tumors (left) seem more congested and hemmorhagic than ID8-Vegf tumors (right). (c) CD31+ microvasculature is more pronounced in ID8-Vegf Defb29tumors (Defb29 Vegf) compared with ID8-Vegf tumors (Vegf). (d) CD31 microvascular density (percentage of the vessel area per microscopic computerizedfield) is significantly higher in ID8-Vegf Defb29 tumors (Defb29 Vegf) compared with ID8-Vegf tumors (Vegf). Error bar, s.e.m. of the microvascular densityof ten different tumors. (e) CD11c+ cells localize to capillaries in Defb29 Vegfa-expressing tumors. (f) Colocalization expression of CD31 and CD11c invascular areas of an ID8-Vegf Defb29 tumor.

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CD11c+ cells assemble in vascular structuresWe surmised that CD11c+ cells promoted the growth of ID8-VegfDefb29 tumors through vasculogenesis. To test the ability of CD11c+

cells to form vessels, a >98% pure population of tumor-derivedCD11c+ cells was procured immunomagnetically, fluorescentlylabeled ex vivo with carboxyfluorescein diacetate (CFSE) orchloromethyl-benzoyl-amino tetramethylrhodamine (CMTMR),suspended in Matrigel containing ID8-Vegf conditioned medium andinjected subcutaneously into C57BL/6 mice. Cells were CD31+

(>90%) or CD34+ (>80%) at isolation in different experiments.Within two weeks, cells assembled into mature capillaries anastomos-ing with the host’s vascular tree (Fig. 3a–d). Most transplanted cellsassembled in blood vessel formation and were CFSEbright, suggestingthat no considerable cell proliferation had occurred. This ruled outthe possibility that vessels were formed by overgrowth of contaminat-ing stem cells. Furthermore, cells assembling into these capillariesstrongly expressed CD11c, CD45, DEC-205 and CD8α, indicatingtheir DC lineage. To further test the ability of tumor-derived CD11c+

cells to expand, single CD11c+ cells were sorted by FACS from dis-persed ID8-Vegf Defb29 tumors or ascites and individually culturedin 96-well plates (n = 576) in ID8-Vegf Defb29 conditioned medium.We were unable to expand any of these cells ex vivo, ruling out blastproperties.

To dismiss the idea that such neovessels were formed by endothelialprecursors recruited from the host, we transplanted fluorescentlylabeled CD11c+ cells (isolated as above) in CD31 knockout C57BL/6mice20. Two weeks later, resected Matrigel plugs exhibited perfusable,intact capillaries formed by fluorescently labeled cells expressingCD31, indicating their exogenous origin. Cells were also MHC-II+CD34+ (Fig. 3e); thus, tumor-associated CD11c+ leukocytes areindependently capable of forming neovasculature while preservingexpression of DC gene products.

To test whether tumor-infiltrating CD31+CD11c+ cells are able toacquire a DC phenotype, we pulsed one half of CD11c+ cells isolatedfrom tumors or ascites with ovalbumin (OVA), or cultured themwith control medium. Cells were thentreated with tumor necrosis factor-α(TNF-α), CpG and antibody to interleukin-10 receptor to reverse tumor-induced DCparalysis21. OVA-pulsed cells, but not con-trol CD11c+ cells, induced proliferation ofnaive CD4+ T lymphocytes purified fromαβT cell receptor (TCR) OT-II transgenicmice22 after 8 d of coculture (Fig. 3f).Furthermore, OVA-pulsed CD31+CD11c+

cells increased day-7 production of IL-2 bynaive T-cells 15 times, compared with non-pulsed controls (Fig. 3g). No substantialproliferation or production of IL-2 wasinduced by CD31+CD45− (bona fideendothelial) cells extracted from the sametumors. Moreover, tumor-derived CD11c+

cells (activated as above) developed den-drites, became CD80+ (>80%) andexpressed high levels of CD86 (>95%) andMHC-II (>98%; Fig. 3h).

Freshly isolated CD11c+ cells from greenfluorescent protein (GFP)+ tumors orascites expressed GFP, suggesting previousengulfment of tumor antigen in vivo.Without ex vivo pulsing, immunomagneti-

cally purified tumor CD11c+ cells induced expansion and type-1polarization of tumor-reactive splenic T cell precursors fromtumor-bearing mice (Supplementary Fig. 1 online). Similar resultswere obtained with the tumor-derived, sorted CD11c+ CD45low

DEC205+ VE-cadherin+ cell fraction. Taken together, these data indicate that tumor-associated CD11c+ cells acquire antigen and function as endothelial-like cells or DCs, dependingon the milieu.

CD11c+ cells can undergo endothelial-like specializationWe reasoned that CD31+CD11c+ cells found in ID8-Vegf Defb29tumors derive from circulating DC precursors. We thus askedwhether bona fide DCs can undergo endothelial-like specializa-tion. We procured a >99% pure population of CD34−CD11c+ bonemarrow–derived DCs through GM-CSF treatment23, followed byimmunomagnetic depletion of CD34+ and positive selection ofCD11c+ cells. CD34+ cells accounted for less than 1% of the totalin multiple experiments (Fig. 4a). Purified cells exhibited a CD34−CD8α− MHC-IIlowCD11chighCD11bhigh myeloid DC phe-notype, engulfed tumor antigen and efficiently activated type-1tumor-reactive T cells in vitro (Supplementary Fig. 2 online).We tested the ability of Vegf-A to induce functional VEGF receptor-2 (VEGFR-2) in these cells by assessing receptorautophosphorylation after incubation in ID8-Vegf cell-condi-tioned medium, or RPMI medium with or without recombinantmouse Vegf-A (100 ng ml–1). We immunoprecipitated cell lysateswith antibodies to phosphorylated tyrosine (anti-Tyr(P)) orVEGFR-2, and immunoblotted immunoprecipitates with antibod-ies to VEGFR2 or to phosphorylated VEGFR-2, respectively. Wedetected a specific signal for tyrosine-phosphorylated VEGFR-2only in CD11c+CD34− cells exposed to ID8–Vegf medium or torecombinant Vegf-A, but not in cells maintained in controlmedium (Fig. 4b).

Cells treated with conditioned medium showed no prolifera-tion, as documented by [3H]thymidine uptake, intensity of CFSE

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Figure 2 Tumor-infiltrating DC precursors undergo endothelial-like specialization. (a) CD11c+ cellssorted by FACS from ascites of 8-week intraperitoneal ID8-Vegf Defb29 tumors coexpress specific DCand endothelial markers. Most gated CD11c+MHC-II+DEC205+CD45+ cells are CD8α+, but alsoCD31+ and CD34+. A fraction also expresses the endothelial markers P1H12, VE-cadherin andVEGFR-2/KDR. (b) CD11c+ cells immunopurified from ID8-Vegf Defb29 tumors labeled with CMACand injected subcutaneously around ID8-Vegf Defb29 tumors migrate towards dextran-perfusiblevessels at an endothelial location in the tumor.

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staining and abundance of Ki-67 mRNA (Fig. 4c), or apoptosis(data not shown); this excluded the expansion or elimination ofcell subpopulations. A large fraction of CD11c+ cells acquiredCD31 and CD34, whereas a portion downregulated CD45 (Fig. 4a). Cells assumed a spindle-like shape within 3 d, upregu-lated CD31 and VWF protein (Fig. 4d and Supplementary Fig. 3online) and progressively aligned, forming strings. These pro-gressed within two weeks into cord-like structures, where >85% ofcells clustered (Fig. 4e). This was due to recruitment of cells, asdocumented by time-lapse video microscopy (Supplementary Fig. 2 online). A marked upregulation of Defb29 mRNA was observed in DCs under these circumstances (Fig. 4f).Lumen structures and pathognomonic Weibel-Palade bodies24

were detected by transmission electronmicroscopy (Fig. 4g). Similar results wereobtained when DCs were incubated in ID8-Vegf medium or, albeit at a slowerrate, in RPMI medium enriched withrecombinant Vegf-A.

Highly purified fluorescent-labeledCD34−CD11c+ cells treated with ID8-VegfDefb29 media for 7 d, assembled three-dimensional tubes in Matrigel in vitro(Fig. 4g) and migrated towards tumor ves-sels in vivo when injected around flankID8-Vegf Defb29 tumors (data not shown). Single fluorescent CD34−CD11c+

cells migrating towards tumors isolatedthrough laser capture microdissection25

(Supplementary Fig. 2 online) exhibitedupregulated CD31 (60-fold), VE-cadherin(22-fold) and VEGFR-2 (20-fold) mRNA 7 d after injection (Fig. 4h).

Defb29 recruits CD11c+ cells; Vegf induces endothelializationTo explain the accelerated growth of ID8-Vegf Defb29 tumors in vivo,we surmised that β-defensin mainly recruits CD11c+ cells, whereasVegf-A mainly transforms them into endothelial-like cells. To testwhether tumor enrichment with CD11c+ cells could reproduce theeffects of Defb29, we subcutaneously inoculated an equal number ofID8-Vegf or ID8 cells alone, or admixed with bone marrow–derivedCD11c+CD34− cells (20:1 ratio; tumor cells, DCs). Addition of DCsresulted in highly vascularized tumors (Supplementary Fig. 2 online)and increased tenfold the growth of Vegfa-overexpressing tumors (P < 0.05; n = 6 per group), but not tumors expressing normal levelsof Vegfa (ID8/DC, 28 ± 4 mm3 compared with ID8, 35 ± 2 mm3 at twomonths; P = 0.19; n = 10 per group; data not shown). Thus, addition

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Figure 3 DC precursors can promotevasculogenesis or induce T cell responses. (a) CFSE-labeled CD11c+ cells (1) assemble aneovessel perfusable by rhodamine-dextran (2)and retain expression of CD11c in vivo (4 and5; 5, ×400 detail). (b) CFSE-labeled CD11c+

cells (1) localize to a neovessel perfused withrhodamine-dextran (2) and express CD45 (4).(3, combined image; 5, phase image). (c,d) CMTMR or CFSE-labeled CD11c+ cellsform functional vessels and retain expressionof DEC-205 and CD8α. (e) Neovessels areformed by wild-type CMTMR-labeled CD11c+

cells in a CD31 knock-out mouse (KO):fluorescently labeled cells assembleperfusable neovessels expressing CD31, MHC-II and CD34. (b–e) 5, phase image. (f–h) If pulsed with OVA and stimulated withTNF-α, monoclonal antibody to IL-10R andCpG, tumor-derived CD11c+ cells induceproliferation of OVA-specific OT-II naive CD4+

lymphocytes (f), which secrete abundant IL-2.(g) CD11c− cells represent CD31+CD45−

CD11c− endothelial cells immunopurified fromthe same specimens. Error bars, s.e.m. of atleast three observations. (h) Tumor-derivedactivated CD11c+ cells undergo maturation,expressing CD80, CD86 and MHC-II (dots,isotype control).

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of DCs reproduces the effects of Defb29, promoting the growth onlyof tumors overexpressing Vegfa.

Next, we tested whether Defb29 chemoattracts CD11c+ cells13.Highly purified bone marrow–derived immature DCs were chemoat-tracted similarly by ID8-Defb29 and ID8-Vegf Defb29 cell super-natants, which mimicked recombinant mouse MIP-3α (Fig. 5a),whereas ID8 cell supernatants did not chemoattract DCs. DirectedDC migration, rather than chemokinesis, was confirmed by simpli-fied checkerboard analysis (data not shown). Intraperitoneal inocula-tion in healthy mice of cationic liposomes carrying the entire Defb29ORF, but not of control liposomes, resulted in increased exudation ofperitoneal CD11c+ cells expressing DEC-205, a marker of peripheralDCs26 (Fig. 5a).

MIP3-α (but not other chemokines) and Defb29 largely attenu-ated each other’s chemotactic gradient for immature DCs (Fig. 5a),suggestive of CCR6-mediated chemotaxis. A CCR6-specific antibodymarkedly reduced the chemotactic migration of DCs towards undi-luted ID8-Vegf Defb29 conditioned medium (Fig. 5b). A weaker,dose-dependent inhibition by a VEGFR1-specific antibody was alsodetected (Fig. 5b). In vivo, CMTMR-labeled CD11c+ cells adoptivelytransferred intraperitoneally to mice bearing a subcutaneous ID8-Vegf and a contralateral ID8-Vegf Defb29 tumor migrated at signifi-cantly higher frequency to tumors expressing Defb29 than tocontralateral tumors lacking Defb29 (4.1 ± 0.6 compared with 0.52 ± 0.2 cells per high power field; P < 0.01; Mann-Whitney test).Preincubation of CD11c+ cells with a CCR6-specific antibody, but notwith an isotype control, substantially impaired their migration toID8-Vegf Defb29 tumors (SupplementaryFig. 4 online). Additionally, the frequency ofCD11c+ cells by immunostaining was 5 timeshigher in ID8-Vegf Defb29 compared withID8-Vegf tumors, but was not substantially

different between ID8 and ID8-Vegf tumors (data not shown). Thus,tumors recruited CD11c+ cells mainly through interaction ofβ-defensin with CCR6.

To confirm the role of tumor-infiltrating CCR6+ cells in tumorgrowth, we transplanted ID8-Vegf Defb29 tumors combined withCCR6-specific or control rat IgG antibodies, plus a secondary anti-body to rat conjugated to ribosome-inactivating saporin. Treatmentwith CCR6-specific/immunotoxin complex resulted in a 95%decrease in the frequency of tumor-infiltrating CD11c+ cells (data notshown) and reduced tumor growth compared with controls treatedwith rat IgG/immunotoxin (226.0 ± 39.1 mm3 compared with 584.2 ±54.2 mm3 at day 14; P < 0.001; Wilcoxon test; Fig. 5c,d). Therefore,targeting of CCR6+ cells resulted in delayed tumor growth.

Next, we tested the contribution of Vegfa or Defb29 to the processof endothelialization of CD11c+ cells in vitro. Vegf-A inhibition with aneutralizing antibody against VEGFR-2 prevented downregulation ofCD45 and upregulation of CD34 in CD11c+CD34− cells cultured inID8-Vegf Defb29 conditioned medium, whereas neutralizing anti-bodies against VEGFR-3 or CCR6 had no effect (Fig. 5e). Blockade ofVEGFR-1 mimicked VEGFR-2 blockade. Thus, Vegf-A, but notDefb29, induced DC endothelialization.

To further dissect the effects of Defb29 and Vegfa on endothelializa-tion of CD11c+ cells, fluorescently labeled CD11c+CD34− cells wereincubated in ID8-Vegf Defb29 conditioned medium containing neu-tralizing antibodies, suspended in Matrigel and injected subcuta-neously into healthy mice (Fig. 5f). DCs cultured without antibodiesor in the presence of antibodies to VEGFR-3 or CCR6- assembled

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Figure 4 Endothelial-like differentiation of DCs in vitro. (a) Input cells are CD11c+CD45+ (1),CD31low and CD34− (2, shaded). Cells cultured insupernatants from ID8-Vegf Defb29 cellsupregulate CD31 and acquire CD34 (2, white and3), and a fraction loses CD45 (3). (b) Lysates of DCsexposed to (ID8 + Vegf) supernatants, medium enriched with Vegf-A (Vegf) or controlmedium were immunoprecipatated with antibodiesto VEGFR-2 (top) or Tyr(P) (bottom) and thenimmunoblotted with antibodies to total VEGFR-2 orphosphospecific VEGFR-2. (c) DCs treated withID8-Vegf Defb29 supernatants for 6 d do notproliferate, on the basis of [3H]thymidine uptake(left, controls: proliferating CD4+ lymphocytes andDCs incubated with mitomycin-C); FACS analysis ofCFSE staining (center, proliferation index = 1.3)and Ki-67 mRNA expression (right). (d–g) Treatment with ID8-Vegf Defb29 supernatantsinduces expression of CD31 (d, left; magnification,×200) and VWF (d, right; magnification, ×200);formation of strings and cord-like aggregates (e); a4,000-fold upregulation of endogenous Defb29mRNA (f); and acquisition of endothelial features(g, left; L, lumen; solid arrows, cell junctions;arrowheads, endocytotic vesicles; ×10,000) anddevelopment of Weibel-Palade bodies (g, middleand right; ×50,000). PKH26-labeled DCs organizecapillary-like structures on Matrigel (g, right). (h) Microdissected PKH26-labeled DCs upregulateCD31, VEGFR-2/KDR and VE-cadherin, but noteNOS 7 d after peritoneal injection.

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perfusable capillary networks within 2 weeks. Less dense capillarynetworks were formed by CD11c+ cells treated with VEGFR1-specificantibody. In contrast, CD11c+ cells cultured in the presence ofVEGFR2-specific antibody, or in RPMI medium alone, failed to formcapillaries and were absent from the injection sites after 2 weeks (datanot shown). Thus, Vegf-A (through VEGFR-2), but not Defb29,induced the assembly of vascular-like structures by DC precursors.

We next examined the effects of VEGFR-2 blockade in vivo.Treatment of mice bearing ID8-Vegf Defb29 tumors with the tyrosine kinase inhibitor SU5416 (ref. 27) arrested the growth ofID8-Vegf Defb29 tumors (Fig. 5g). Although no change in the intensity of CD11c+ cell infiltration was observed, SU5416 abolished the vascular migration of CD11c+ cells (Fig. 5h),confirming the importance of tyrosine kinase signaling in DCendothelialization, but not recruitment.

Endothelial-like CD11c+ cells in human ovarian carcinomaTo test whether β-defensins also function in human ovarian carcinogenesis, we assessed the expression of human β-defensin-4(DEFB4) and β-defensin-103A (DEFB103A), both of which recruitDCs through CCR6 (refs. 13,28), in human epithelial ovarian neo-plasms and normal postmenopausal ovaries. We detected DEFB4peptide in most ovarian cancers (36 of 45) with the strongestexpression located in tumor islets, although stromal leukocytesoccasionally exhibited DEFB4 staining (Fig. 6a). DEFB103A pep-tide was found in all cancer specimens analyzed (45 of 45), withpatchy staining within tumor islets. DEFB4 and DEFB103A mRNAlevels in human ovarian cancer resembled those of Defb29 in trans-duced mouse tumors. DEFB4 mRNA levels were 7 times higher instage-III than in stage-I or borderline tumors by TaqMan PCR (P = 0.02; Mann-Whitney test). DEFB4 mRNA was undetectable in

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Figure 5 Defb29 and Vegf carry out different tasks. (a) Defb29 chemoattracts DCs. Left, Supernatants from ID8-Defb29 (Defb29) or ID8-Vegf Defb29cells (Vegf Defb29) and MIP-3α chemoattract mouse bone marrow–derived DCs. Middle, increased intraperitoneal exudation of CD45+CD11c+DEC205+

cells upon inoculation of Defb29+ (bottom) compared to empty plasmid (top). Right, MIP-3α and ID8-Defb29 media counteract each other’s chemotacticgradient for DCs (Ctrl, control media). (b) CCR6-specific, but not VEGFR1-specific, antibody induces a marked decrease in DC chemotaxis towardsundiluted Vegf Defb29 supernatants. (c,d) Impairment of ID8-Vegf Defb29 tumor growth by a CCR6-immunotoxin complex, compared to irrelevantantibody plus immunotoxin. (e) Upregulation of CD34 and downregulation of CD45 in bone marrow–derived DCs (open peaks) is partly blocked byantibodies against VEGFR-2 or VEGFR-1, but not against VEGFR-3 or CCR6 (shaded).(f) Formation of capillary networks in Matrigel plugs containing theindicated antibodies by DCs treated with conditioned medium. (g) Tumor growth induced by Defb29 and Vegf cooperation is abrogated by SU5416. (h)SU5416 (bottom; magnification, ×400) abolishes vascular homing of CD11c+ cells (top; magnification, ×400).

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benign tumors or normal postmenopausal ovaries (SupplementaryFig. 4 online).

We tested whether CD11c+ cells exhibit endothelial markers inhuman ovarian cancer coexpressing increased VEGFA and β-defensins. A large fraction of CD11c+ cells isolated from freshly dis-persed human ovarian carcinomas coexpressed CD45 (>65%) andthe endothelial markers CD34 (>50%) and CD31 (>85%; Fig. 6b). Aproportion (40%) of CD11c+CD45+ cells expressed CCR6, implicat-ing β-defensin-mediated chemoattraction. In randomly analyzedascites specimens, we identified CD11c+CD45+CCR6+ cells coex-pressing VE-cadherin, P1H12, CD31, CD8α and CD86 at variablepercentages. Although the absolute frequency of this cell subset waslow in unselected specimens, they confirmed the existence of this celltype. Finally, ovarian carcinomas coexpressing increased VEGFAand DEFB4 harbored capillaries exhibiting CD11c+CD123+,CD45+P1H12+, CD45RO+, CD45+CD31+ or CD45+CD34+ leukocytesat a typical endothelial location (Fig. 6c–g). Thus, a population ofCD11c+ leukocytes expressing endothelial and DC markers was alsoidentified in human tumors coexpressing β-defensins and VEGFA.

DISCUSSIONThis work has uncovered a new mechanism of tumor vasculogenesisinvolving the cooperation of Vegf-A with β-defensins, mediated by anew population of leukocyte precursors exhibiting properties of DCsand endothelial-like cells. Although previous reports have shown thatmonocytes can acquire endothelial features in vitro29–34, this is thefirst evidence that leukocytes may have an important role in tumorvasculogenesis in vivo.

We found that Defb29 exhibited bona fide β-defensin properties,including defense against viral infection (F.B., J.R.C.-G. and G.C.,unpublished observations) and chemoattraction of DCs throughCCR6. Coexpression of Defb29 and Vegfa in mouse tumors at levelsresembling DEFB4 and VEGFA in human ovarian carcinomas, pro-moted tumor growth through the accumulation of CD11c+ DCs withvascular competence and expressing endothelial markers. CD11c+

cells did not expand ex vivo, but promoted tumor growth when trans-planted with tumor cells, and engaged in vasculogenesis in vivo whentransplanted to Matrigel plugs. Although endothelial cells mayexpress CD11c (ref. 35), negative selection of CD34+ cells virtually

excluded all endothelial progenitors36 inthese experiments. These cells largelymigrated to capillaries. Despite inclusion ofhigh-resolution immunohistochemical pho-tographs, we cannot ensure that CD11c+ cellsare incorporated only luminally, becomingtrue endothelial-like cells, or rather incorpo-rate perivascularly. It is possible that CD11c+

cells contribute to tumor angiogenesis bothin a reconstitutive as well as an instructivemanner. Clearly, their contribution to tumorgrowth is important, as indicated by target-ing experiments through CCR6. Thus, tumorvasculogenesis may occur not only throughthe recruitment of bone-marrow endothelialprecursor cells37, but also through therecruitment of differentiated CD11c+CCR6+

myeloid DC precursors, which emerge as atherapeutic target.

The present findings unveil a previouslyunsuspected role of β-defensins in tumor

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Figure 6 β-defensins and vascular CD11c+ cellsin human ovarian carcinoma. (a) Expression ofDEFB4 was localized to tumor islets (75% ofstage-III tumors; magnification, ×200) andstromal inflammatory cells (additional 5% oftumors; magnification, ×100). Patchy expressionof DEFB103A within tumor islets was found inall tumor specimens analyzed (magnification,×200). (b) A fraction of tumor-derived CD11c+

cells (gated) expresses CD34 and CD31, whereas a fraction expresses no CD45 (top).CD11c+CD45+CCR6+ cells concomitantlyexpressing typical DC and endothelial markers CD31, P1H12 and VE-cadherin (bottom) are detected in human ovarian cancer.(c–f) Capillaries in human tumors harborCD11c+CD123+ cells and CD45+ cellscoexpressing endothelial markers P1H12, CD31or CD34. CD45RO expression, a feature ofleukocytes (including DCs) but not endothelialcells, is also noted at a typical vascular location.c–g: magnification, ×200.

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biology, and indicate that β-defensins and Vegf-A can cooperate topromote vasculogenesis by carrying out different functions. Defb29promoted recruitment of CD11c+ leukocytes through CCR6, whereasVegf-A was largely responsible for inducing their endothelial-like spe-cialization and migration to vessels as vascular constituents — mech-anisms which mainly depended on VEGFR-2. It is unclear whetherthe effect of tumor-derived Vegf is endocrine or paracrine, andwhether such CD11c+ precursors already exist in the periphery as adistinct population with vascular features or, rather, acquire these fea-tures in the tumor microenvironment. Although not mutually exclu-sive, this mechanism is different from previous models ofangiogenesis38–40, which depend on the paracrine action of mono-cyte-derived angiogenic factors and proteases14, and can be abrogatedby blockade of VEGFR-1, but not VEGFR-2 (ref. 39).

The present findings extend previous evidence of phenotypic over-lap between monocyte-derived DCs and microvascular endothe-lium41,42 and have important implications for tumor angiogenesisand immunology. Further investigation is warranted to assess whetherthe described mechanism is model-specific and pertains only to ovar-ian carcinoma or rather applies to solid tumors generally. As many as27 new candidate β-defensins have been recently identified43, war-ranting investigation in the context of physiologic or pathologicangiogenesis.

METHODSMice. Mice were purchased from Charles River Laboratories. Animal experi-ments were approved by the University of Pennsylvania Institutional AnimalCare and Use Committee. Human experiments were approved by theUniversity of Pennsylvania Institutional Review Board.

Cloning of Defb29. Applying a hidden Markov model profile with the maturepeptide sequences of known β-defensins to the RIKEN full-length enrichedlibrary, we cloned Defb29 from kidney. Defb29 contains a 406-bp ORF encod-ing a 78-amino-acid pre-propeptide that exhibits 18–23% homology toknown β-defensins (see Supplementary Note online).

Cells and tissues. We transduced ID8 cells using retroviral vectors pLXSN44

(Defb29) or MIGR1 (mouse Vegfa164), as previously described17. Cell-con-ditioned media were collected every 48 h. We generated mouse DCs by cul-turing flushed bone marrow precursors in RPMI medium supplementedwith GM-CSF (20 ng ml–1; Peprotech) for 8 d23. We separated CD34+ andCD11c+ cells immunomagnetically using RAM34 and HL3 monoclonalantibodies (BD Pharmingen), respectively. Matrigel (BD Biosciences)diluted with an equal volume of cell-conditioned medium was seeded ontosix-well plastic dishes. In some experiments, cells were incubated withrecombinant Vegf-A (Peprotech). For neutralization experiments, we usedgoat polyclonal antibodies against mouse VEGFR-2, VEGFR-1 or VEGFR-3;and a monoclonal CCR6-specific antibody (140706; R&D Systems). Weanalyzed DEFB4 and DEFB103A in 33 stage-III, 3 stage-II and 9 stage-Iovarian carcinomas; 4 borderline and 4 benign tumors; and 2 normal post-menopausal ovaries. All patients gave oral informed consent before enroll-ment in the study12.

Generation of tumors and expression of Defb29 in vivo. Tumors were gener-ated as previously described17. For flank injections, 7 × 106 ID8-Defb29 cellsand ID8 cells were injected subcutaneously into contralateral flanks ofC57BL/6 mice (n = 15). Similarly, 7 × 106 ID8-Vegf Defb29 cells and ID8-Vegf cells were injected subcutaneously into contralateral flanks of C57BL/6mice (n = 15). An equal number of mice was injected subcutaneously nearthe hips. Tumor volumes were calculated by the formula V = 0.5 (L × W2),where L is length and W is width. For survival analysis, we injected 1 × 107

ID8-Vegf Defb29 cells (n = 15), or ID8-Vegf cells (n = 15) intraperitoneally.For in vivo neutralization of CCR6+ cells, we injected ID8-Vegf Defb29 cellsadmixed with 20 µg ml–1 rat antibody to mouse CCR6 or an isotype control(R&D Systems), plus 20 µg ml–1 rat-Zap (Advanced Targeting Systems). We

treated mice with a subcutaneous injection of 100 µl of CCR6-specific orisotype control antibodies plus rat-Zap (in PBS; 20 µg ml–1) around thetumor site after 7 or 14 d. To test the recruitment of DCs in vivo, 12 µg ofpCEFL-KZ-HA–Defb29 expression vector, or plasmid alone admixed withDOSPER transfection agent (Roche Diagnostics), were injected intraperi-toneally into C57BL/6 mice twice, 1 d apart. Peritoneal washings wereobtained 2 d later.

Angiogenesis in Matrigel plugs. Flank ID8 tumors (n = 5) were resected,minced and digested with collagenase-A (Roche). CD11c+ cells were immuno-magnetically separated from freshly digested flank tumors or peritoneal ascitesof tumor-bearing mice using an HL3 monoclonal antibody. CD11c+ cells wereincubated overnight with cell-conditioned medium and labeled with celltracker fluorochrome CFSE (green), CMTMR (orange) or CMAC (blue;Molecular Probes). Suspensions of 1 × 106 labeled cells in 250 µl were mixedwith an equal volume of cold Matrigel and injected subcutaneously into wild-type (n = 4) or CD31−/− C57BL/6 mice (n = 3). After two weeks, the vasculartree was perfused live with 3 ml of intravenous FITC-dextran or TRITC-dextran solution (50 mg ml–1, relative molecular mass ∼ 150,000; Sigma). Micewere killed 20 min later, and Matrigel plugs were removed for histologicalexamination.

Histology, flow cytometry and immunoblotting. Cryosections (6-µm thick)were immunostained as described12. Mouse-specific antibodies used were:CD31-specific (MEC13.3), CD45-specific (30-F11), CD11c-specific (HL3;all from BD Pharmingen), and VWF-specific (DAKO). We carried out cellsorting on a MoFlo cell sorter (Cytomation), using antibodies against CD8α(53-6.7), CD11b (M1/70), CD11c (HL3), CD14 (rmC5-3), CD16 (2.4G2),CD31 (MEC13.3), CD34 (RAM34), CD45 (30-F11), CD80 (16-10A1), CD86(GL1), GR1 (RB6-8C5), MHC-II (KH74), NK1.1 (PK136; BD Pharmingen);DEC-205 (NLDC-145 Serotec); VE-cadherin (R&D Systems); and VEGFR-2(Avas 12α1; Research Diagnostics). For analysis of human cells, we used thefollowing antibodies: CD11c (SHCL-3), CD45 (HI30), CCR6 (11A9),CD8-α (RPA-T8), CD45RO (UCHL1), CD34 (581; BD Pharmingen),CD31 (JC70A; Dako), P1H12 (Chemicon), VE-Cadherin (MedsystemsDiagnostics), DEFB103A (Orbigen) and DEFB4 (Santa CruzBiotechnology). For immunoblotting, we incubated 3 × 106 DCs in ID8-Vegfconditioned medium for 5 d or in serum-free RPMI medium containing 1 mM Na3VO4 for 1 h and treated with Vegfa (100 ng ml–1; Peprotech) for 3 h at 4 °C. We carried out immunoprecipitation with antibody to Tyr(P)(Py20; Transduction Laboratories) or goat VEGFR-2-specific antibody(R&D). We used VEGFR-2-specific or rabbit antibody to phosphorylatedVEGFR-2 (ab5472; Abcam) for immunoblotting. We carried out FISH with aprobe for mouse X chromosome labeled with Cy3 (Cambio), following therecommended protocol.

GenBank accession number. Defb29, AF515626.

ACKNOWLEDGMENTSWe thank C. H. June and C. B. Thompson for critical review of the manuscript andhelpful discussions; S. M. Albelda for the CD31 knock-out mice; P. Terranova forthe mouse ID8 cell line; A. D. Miller for the retroviral vector pLXSN; P. D’Amorefor the Vegf164 cDNA; and J. M. Palacios for the pCEFL-KZ-HA vector. This workwas supported by National Cancer Institute ovarian SPORE P01-CA83638;National Institute of Health R01 CA098951; and grants from the Sidney KimmelFoundation and the Ovarian Cancer Research Fund, as well as institutionalfunding by the Abramson Family Cancer Research Institute and the Department ofObstetrics and Gynecology at the University of Pennsylvania. The LCM facility wassupported by a generous grant by the Fannie Rippel Foundation. F.B. and M.C.C.were supported by National Institutes of Health Research Grant #D43 TW00671funded by the Fogarty International Center. F.B. is a member of the ConsejoNacional de Investigaciones Cientificas Argentinas. D.K. was supported byAssociazione Italiana per la Ricerca sul Cancro.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 27 May; accepted 3 August 2004Published online at http://www.nature.com/naturemedicine/

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