[CANCER RESEARCH 64, 5535–5538, August 15, 2004] Advances in Brief Dendritic Cell Subsets Differentially Regulate Angiogenesis in Human Ovarian Cancer Tyler J. Curiel, 1 Pui Cheng, 1 Peter Mottram, 1 Xavier Alvarez, 1 Lieve Moons, 2 Melina Evdemon-Hogan, 1 Shuang Wei, 1 Linhua Zou, 1 Ilona Kryczek, 1 Gary Hoyle, 1 Andrew Lackner, 1 Peter Carmeliet, 2 and Weiping Zou 1 1 Tulane University Health Science Center, New Orleans, Louisiana, and 2 Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium Abstract Angiogenesis is essential for both primary and metastatic tumor growth. Tumor blood vessel formation is complex and regulated by many factors. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovas- cularization. To examine tumor angiogenesis in the tumor microenviron- ment, we studied malignant ascites of patients with untreated ovarian carcinoma. We observed high numbers of plasmacytoid dendritic cells (PDCs) and significant stromal-derived factor (CXCL-12/SDF)-1 in their malignant ascites, attracting PDCs into the tumor environment. We now show that tumor-associated PDCs induced angiogenesis in vivo through production of tumor necrosis factor  and interleukin 8. By contrast, myeloid dendritic cells (MDCs) were absent from malignant ascites. MDCs derived in vitro suppressed angiogenesis in vivo through production of interleukin 12. Thus, the tumor may attract PDCs to augment angio- genesis while excluding MDCs to prevent angiogenesis inhibition, demon- strating a novel mechanism for modulating tumor neovascularization. Because dendritic cells (DCs) have long been known to affect tumor immunity, our data also implicate DCs in regulation of tumor neoangio- genesis, suggesting a novel role of DCs in tumor pathology. Introduction Angiogenesis is essential for both primary and metastatic tumor growth. Work to date suggests that vascular endothelial growth factor (VEGF) plays a central role in tumor angiogenesis. How- ever, blood vessel formation is complex and regulated by many factors. For example,  3 and  5 integrins were thought to support angiogenesis based on in vitro work. However, recent in vivo experiments failed to show support for these molecules in angio- genesis in vivo (1, 2). Furthermore, the proangiogenic molecule basic fibroblast growth factor was found to be positively related to the prolonged survival of cancer patients (3). Importantly, early human clinical cancer treatment trials with antiangiogenic mole- cules have only demonstrated modest benefits (4 – 6). More strik- ingly, recent reports (7, 8) suggest that angiogenesis inhibitors (or antagonists) alone, by depriving tumors of oxygen, could have an unintended effect: promotion of tumor metastasis. These results reflect our growing understanding of the complexity of the tumor angiogenic process and metastasis process. Dendritic cells (DCs) prime naı ¨ve T cells and thereby activate antigen-specific immu- nity. The two principal human DC subtypes are MDCs (DC1) and plasmacytoid dendritic cells [PDCs (DC2; Ref. 9)]. MDCs express- ing tumor antigens have been used in human clinical trials to induce significant clinical responses against some tumors (10). Although much work has focused on the relevance of DCs to tumor immunity, there are no reports regarding how DCs influence tumor angiogenesis. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompa- nied by intense neovascularization. Because immune factors are known to modulate blood vessel formation in some settings (11), we hypothesized that specific DC subsets might differentially affect tumor neovascularization. Materials and Methods Human Subjects. We studied patients with International Federation of Gynecology and Obstetrics stage III or IV ovarian epithelial carcinomas. All patients gave written, informed consent. The study was approved by the local institutional review board. No patients received prior specific cancer treat- ments. Plasmacytoid Dendritic Cells. We collected peripheral blood mononu- clear cells and ovarian tumor ascites aseptically, harvested cells by centrifu- gation over a Ficoll-Hypaque density gradient (Amersham), and cryopreserved them at -86°C until use. CD3-, CD14-, CD16-, CD19-, and CD56-expressing cells were depleted using paramagnetic beads (Miltenyi, Auburn, CA), and blood PDCs or tumor ascites PDCs were sorted by flow cytometry gating on CD4 + CD123 + CD11c - cells. Cell populations were 99% pure by flow cytometry. Myeloid Dendritic Cells. MDCs were differentiated from CD14 + tumor ascites cells with granulocyte macrophage colony-stimulating factor plus in- terleukin (IL)-4 as described previously (12, 13). Activation of Dendritic Cells and Detection of Dendritic Cell-Derived Cytokines. Tumor-associated PDCs or MDCs were activated for 24 h with CD40 ligand (CD40L) stimulation (200 ng/ml; Immunex) or without stimula- tion. In some cases, the culture plates were precoated with growth factor- reduced Matrigel (BD Bioscience, Bedford, MA). Dendritic cells were col- lected for in vivo Matrigel assay. Culture supernatants were collected for detecting cytokines with commercial enzyme-linked immunosorbent assay kits (all from R&D Systems, Minneapolis, MN). In vivo Matrigel Assay. NOD.SCID mice (6 – 8 weeks old; The Jackson Laboratory, Bar Harbor, ME) were inoculated with growth factor-reduced Matrigel Matrix (BD Bioscience) bearing fresh or CD40L-activated tumor- associated PDCs or MDCs and/or the indicated cytokines and/or mouse anti- human antibody. Recombinant human VEGF, fibroblast growth factor (FGF), tumor necrosis factor (TNF)-, IL-8 (all at 10 ng/ml) were from R&D Systems. Mouse antihuman TNF- (clone 1825; IgG1), mouse antihuman-IL-8 antibody (clone 6217; IgG1), and mouse antihuman IL-12 antibody [clone 24910; IgG1 (500 ng/ml each)] were from R&D Systems. After 12 days, we isolated the Matrigel plugs. Matrigel plugs were subjected to immunohisto- chemistry with anti-von Willebrand factor antibody (polyclonal antibody; 1:10 dilution; DAKO, Carpinteria, CA). Microvessel density was analyzed (14) and quantified with ImagePro Plus software (Media Cybernetics, Silver Spring, MD) and expressed as a mean percentage of microvessel surface area by confocal Leica microscope. Hemoglobin (Hb) content in Matrigel plugs was detected with a commercial kit (Sigma, St. Louis, MO). Received 4/9/04; revised 6/12/04; accepted 7/7/04. Grant support: National Cancer Institute Grants CA092562 and CA100227, Depart- ment of Defense Grant OC020173, Louisiana Board of Regents Grant 126A, the Concern Foundation (W. Zou), and Grant RR00164 (X. Alvarez and A. Lackner). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Weiping Zou, Tulane University Health Science Center, 1430 Tulane Avenue, New Orleans, LA 70112. 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