[CANCER RESEARCH 62, 3453–3458, June 15, 2002] Interferon Stimulated Gene 15 Constitutively Produced by Melanoma Cells Induces E-Cadherin Expression on Human Dendritic Cells1 Elisabetta Padovan,2 Luigi Terracciano, Ulrich Certa, Barbara Jacobs, Anca Reschner, Martin Bolli, Giulio C. Spagnoli, Ernest C. Borden, and Michael Heberer Department of Surgical Research [E. P., A. R., M. B., G. C. S., M. H.] and Institute of Pathology [L. T.], University of Basel, 4031 Basel, Switzerland; Roche Genetics, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland [U. C.]; and The Cleveland Clinic Foundation, Cleveland, Ohio 44195 [B. J., E. C. B.] ABSTRACT promise the antigen-presenting cell functions of infiltrating DCs, thereby favoring tumor immune escape. In a consistent number of The immunobiology of tumor-infiltrating dendritic cells (DCs) can be reports, discrete tumor-derived factors have been shown to prevent strongly influenced by the cytokine environment present in the malignant DC differentiation and maturation and hamper the induction of tissue. We have previously identified discrete melanoma lines, inducing E-cadherin expression on monocyte-derived DCs in vitro. We demonstrate antitumor immunity (14–17). Along with these observations, we here that this effect, independent of cell contact, is not inducible in the have previously identified discrete melanoma cell lines inducing presence of tumor lysates and requires the constitutive expression of IFN E-cadherin expression on monocyte-derived DCs in vitro, potentially stimulated gene 15 (ISG15) by malignant cells. impairing their migratory behavior (9). High-density oligonucleotide arrays were used to investigate the expres- In the present study, melanoma cell clones endowed with different sion pattern of 7000 genes in RNA from two melanoma cell clones com- functional capacities were subjected to gene profiling to identify petent for E-cadherin induction and two clones devoid of DC-modulating differentially expressed genes. We provide evidence that defined capacity. A total of 13 genes encoding soluble proteins were expressed at soluble factors are responsible for the melanoma-induced modulation higher magnitude in melanomas able to induce E-cadherin expression on of DC phenotype. DCs. Combining those data with quantitative protein assays, we could narrow our investigation down to three factors: the chemokine CCL5 and the cytokines ISG15 and type I IFNs. Strikingly, >7 ng/ml of ISG15 could MATERIALS AND METHODS be detected in the corresponding melanoma-conditioned medium and induction of E-cadherin on DCs failed in the presence of antibodies Media and Reagents for Cell Culture. Cells were cultured in RPMI 1640 neutralizing ISG15 protein. Most importantly, strong cytoplasmic expres- supplemented with 1% Ciproxin (Bayer, Zu¨rich, CH), 2 mML-glutamine, 1% Ϫ sion of ISG15 was detected by immunohistochemistry in the original nonessential amino acids, 1% sodium pyruvate, 5 ϫ 10 5 M 2-mercaptoethanol tumor specimen from which the melanoma cell lines under investigation and 10% FCS (Life Technologies, Inc., Paisley, Great Britain), thereafter were derived. referred to as complete medium. LPS content in serum was tested by Limulus These data describe a novel property of ISG15 targeting induction of LAL assay, and only LPS-free batches were used. Human recombinant IL-4 E-cadherin on DCs and possibly influencing their migratory behavior. was produced in our laboratory. Granulocyte/macrophage-colony stimulating factor was provided by Novartis (Basel, CH). INTRODUCTION Cell Cultures. The original cell line Me67 was generated in our laboratory from a metastatic melanoma. Lines Me67.3, Me67.5, Me67.9, and Me67.10 3 DCs are potent inducers of immunity, the properties of which can were derived by culturing the parental cell line in limiting dilution at 0.3 be strongly influenced by the nature of the microenvironment they cells/well in 96-well plates. All cell lines were grown in complete medium and infiltrate (1). Experimental data indicate that the migratory behavior were free from Mycoplasma infection, as monitored by specific reverse tran- of mature DCs can be profoundly different, even when their apparent scription-PCR. Cell lysates were prepared by three cycles of freeze and antigen-presenting capacity is similar (2). In particular, homing of thawing. mature DCs to secondary lymphoid organs, following the induction of Generation of DCs from Peripheral Blood Monocytes. Immature DCs were generated from human peripheral blood mononuclear cells according to specific chemokine receptors (3, 4), allows priming of naı¨ve T cells. published methods (18). Briefly, monocytes were purified by positive sorting On the other hand, induction of chemokines specifically attracting using anti-CD14 conjugated microbeads (Milteny I., Bergisch-Gladbach, D). The antigen-specific T cells (5, 6) will enhance effector functions in the sorted cells were cultured for 6–7 days in complete medium supplemented with 50 periphery and recruitment of memory lymphocytes (7). Derangements ng/ml granulocyte/macrophage-colony stimulating factor and 1000 units/ml IL-4. of these migratory patterns could crucially influence the outcome of Induction of DC Phenotypic Modulation. Immature DCs were cocultured immune responses, including those that are tumor specific (8, 9). with either tumor cells at a 5:1 ratio, in the presence of tumor cell culture The presence of DCs in malignant tissues has been extensively supernatant or their lysates (1:2 dilution). In some experiments, purified rabbit IgG studied, with conflicting results. For some types of cancers, such as anti-ISG15 (19), goat anti-IFNARI, mouse anti-CCR5 IgG1 antibodies, or their oral (10), ovarian (11), colorectal cancers (12) and renal carcinoma isotype matched controls (R&D Systems, Oxon, United Kingdom and BD Bio- ␮ (13), DC infiltration has been associated with prolonged patients sciences, Heidelberg, Germany), were added to the cultures at a 50 g/ml final concentration. Cell cultures were analyzed, after 24-h incubation time, by FACS survival and reduced metastatic disease, specially if associated with staining. T-cell infiltration (10, 12). FACS Analysis. The DC phenotype was monitored by cell surface staining On the other hand, factors secreted by neoplastic cells can com- using FITC-conjugated mouse antibodies from BD Biosciences (Heidelberg, Germany) to human CD86 (clone IT2.2) and CD15 (clone MMA). The mouse Received 1/28/02; accepted 4/18/02. antihuman E-cadherin (clone SHE78–7; R&D Systems) antibody was used in The costs of publication of this article were defrayed in part by the payment of page combination with a goat antimouse IgG2a FITC-conjugated (Southern Biotech- charges. This article must therefore be hereby marked advertisement in accordance with nology Associates, Birmingham, AL) antibody. Samples were analyzed on a 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Basel Regional Cancer League Grant 6/00 (to E. P.) and National FACSCalibur (Becton Dickinson, Mountain View, CA) using propidium io- Cancer Institute CA90914 (to E. C. B.). dide to exclude dead cells. 2 To whom requests for reprints should be addressed, at Department of Surgery, Oligonucleotide Array Analysis. Cultured melanoma cells were harvested Research Division, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland. by scraping, and total cellular RNA was extracted (20). Ten ␮g from each sample Phone: 41-61-2652376; Fax: 41-61-2653990; E-mail: [email protected]. 3 The abbreviations used are: DC, dendritic cell; IL, interleukin; FACS, fluorescence- were reverse transcribed, labeled, and processed by using a commercial kit activated cell sorter; ISG, interferon stimulated gene. (Affymetrix, Santa Clara, CA) according to the supplier’s instructions. Upon 3453 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. ISG15 AND DC PHENOTYPE alkaline heat fragmentation, cDNA were hybridized to the arrays following stand- ard procedures as supplied with the microchips (Affymetrix). Raw data were collected with a confocal laser scanner (Hewlett Packard, Palo Alto, CA), and pixel levels were analyzed using a commercial software (GeneChip v3.1; Af- fymetrix). Three repeats for each array were performed. Expression levels for each gene were calculated as normalized average difference of fluorescence intensity as compared with hybridization to mismatched oligonucleotides, expressed in arbi- trary units. On average, Ͼ25% of the genes under investigation were positive in the cell lines tested. A threshold of 20 normalized average difference units was assigned to any gene with a calculated expression level Ͻ20, because mRNA levels in this low range could not be reliably assessed. Chemokines and Cytokines Detection. CXCL1, CCL5, IL-1␤, IL-6, and IGF-II production was determined by quantitative ELISA assays (sensitivity, Ն10 pg/ml) using cell supernatants of confluent cultures. Antibody pairs and standards were provided by BD Biosciences or R&D Systems. ISG15 was detected by ELISA assay (19). Release of type I IFNs was quantified on HeLa cells as described elsewhere (21). Assays were performed on coded samples. Immunohistochemistry. The original tumor specimen from which Me67 melanoma lines were derived, conserved as paraffin-embedded material, was retrieved and analyzed as follows. Serial sections were incubated overnight at 4°C with anti-ISG15 or isotype-matched control antibodies, followed by avidin-biotin-peroxidase