CXC Chemokine Ligand 12 (Stromal Cell-Derived Factor 1) and CXCR4-Dependent Migration of CTLs toward Melanoma Cells in Organotypic Culture 1 Tianqian Zhang,* Rajasekharan Somasundaram,* Klara Berencsi,* Laura Caputo,* Pyapalli Rani,* DuPont Guerry, † Emma Furth, ‡ Barrett J. Rollins, ¶ Mary Putt, § Phyllis Gimotty, § Rolf Swoboda,* Meenhard Herlyn,* and Dorothee Herlyn 2 * Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor- positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a melanoma patient’s CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag. The Journal of Immunology, 2005, 174: 5856 –5863. C hemokines produced by tumor cells have been demon- strated to attract chemokine receptor-positive T lympho- cytes into the tumor area, potentially leading to tumor growth inhibition in vitro and in vivo (1–3). These studies have been largely confined to mouse tumor systems, and the role of human chemokines in human T lymphocyte trafficking toward hu- man tumor cells is relatively unexplored. In mouse systems, ex vivo transduction of chemokines into tumor cells has provided potent tumor vaccines inducing tumor rejection, which was medi- ated by infiltrating T cells at the vaccine site. Furthermore, in vivo transduction of mouse tumor cells with chemokines leads to T cell infiltration into the tumor area and rejection of both transduced and nontransduced, subsequent challenge tumor cells (1–3). In both the ex vivo and in vivo chemokine transduction studies, effective che- mokines were chemokine C motif ligand-1 (lymphotactin), CCL1 (I-309), CCL2 (MCP-1), CCL3 (MIP-1), CCL5 (RANTES), CCL7 (MCP-3), CCL16 (hemofiltrate CC chemokine-4/liver-ex- pressed chemokine), CCL19 (EBV-induced receptor ligand che- mokine), CCL20 (MIP-3), CCL21 (chemokine with six cys- teines/secondary lymphoid tissue chemokine), and CXCL10 (IFN- -inducible protein-10) (1, 3). CD4 + T cell subsets have been implicated in tumor rejection induced by vaccination of mice with CCL19-transduced tumor cells (4), and CD8 + CTL were instru- mental in tumor growth rejection in mice following intratumoral delivery of CCL20 via an adenovirus vector (5) or injection of CCL16-expressing tumor cells (6). Both CD4 + and CD8 + T cells were required for tumor growth inhibition to occur in mice injected intratumorally with CCL21 (7). In addition to the approaches transducing whole tumor cells with chemokines, chemokines may be fused to tumor Ag-specific Ab to attract adoptively transferred or endogenous T cells to the tumor site, or incorporated into Ag vaccines to attract T cells to the vaccine site, which has been shown to lead to tumor destruction in mouse models (8, 9). To develop immunotherapeutic strategies for cancer patients based on chemokines and their receptors, similar to the approaches already successfully used in mice (see above), we must identify chemokines and their receptors involved in T cell migration to- ward tumor cells in patients. Such information is scarce in human tumor systems. In related studies, T cell infiltrates in human tu- mors were correlated to the expression of chemokines by tumor cells and chemokine receptors by T cells. Ohshima et al. (10) have investigated chemokine expression in Hodgkin’s lymphoma in re- lation to chemokine receptor expression on tumor-infiltrating lym- phocytes. They found a positive correlation between CXCL9 (monokine induced by IFN-), CXCL10, and CCL17 (thymus- and activation-regulated chemokine) expression by tumor cells and presence of Th2-type, CXCR3- or CCR4-positive (CXCL9, *The Wistar Institute, † Pigmented Lesion Clinic, Hospital of the University of Penn- sylvania, ‡ Department of Pathology and Laboratory Medicine, Hospital of the Uni- versity of Pennsylvania, and § Department of Biostatistics and Epidemiology, Univer- sity of Pennsylvania, Philadelphia, PA 19104; and ¶ Dana-Farber Cancer Institute, Boston, MA 02115 Received for publication October 5, 2004. Accepted for publication February 23, 2005. 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. 1 This work was supported by Specialized Program of Research Excellence Grant P50 CA93372 and Grant CA10815 from the National Institutes of Health and by the Commonwealth Universal Research Enhancement Program, Pennsylvania Depart- ment of Health. 2 Address correspondence and reprint requests to Dr. Dorothee Herlyn, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: dherlyn@wistar.upenn.edu The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00