[CANCER RESEARCH 62, 1832–1837, March 15, 2002] Use of the Stromal Cell-derived Factor-1/CXCR4 Pathway in Prostate Cancer Metastasis to Bone 1 Russell S. Taichman, 2 Carlton Cooper, Evan T. Keller, Kenneth J. Pienta, Norton S. Taichman, and Laurie K. McCauley Departments of Periodontics, Prevention, and Geriatrics, and the Center for Biorestoration of Oral Health, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109-1078 [R. S. T., L. K. M.]; Division of Hematology and Medical Oncology, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan 48109 [C. C., K. J. P.]; Department of Pathology and Unit for Laboratory Animal Medicine, School of Medicine, University of Michigan, Ann Arbor, Michigan [E. T. K.]; and Department of Pathology, University of Pennsylvania Dental School, Philadelphia, Pennsylvania 19104 [N. S. T.] ABSTRACT Neoplasms have a striking tendency to metastasize or “home” to bone. Hematopoietic cells also home to bone during embryonic development, where evidence points to the chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12; expressed by osteoblasts and endothelial cells) and its receptor (CXCR4) as key elements in these processes. We hypothesized that metastatic prostate carcinomas also use the SDF-1/CXCR4 pathway to localize to the bone. To test this, levels of CXCR4 expression were determined for several human prostate cancer cell lines by reverse tran- scription-PCR and Western blotting. Positive results were obtained for cell lines derived from malignancies that had spread to bone and marrow. Prostate cancer cells were also observed migrating across bone marrow endothelial cell monolayers in response to SDF-1. In in vitro adhesion assays, pretreatment of the prostate cancer cells with SDF-1 significantly increased their adhesion to osteosarcomas and endothelial cell lines in a dose-dependent manner. Invasion of the cancer cell lines through base- ment membranes was also supported by SDF-1 and inhibited by antibody to CXCR4. Collectively, these results suggest that prostate cancers and perhaps other neoplasms may use the SDF-1/CXCR4 pathway to spread to bone. INTRODUCTION Prostate neoplasms have a striking tendency to metastasize to bone. For metastases to occur, the malignant cells must escape the primary tumor, penetrate and circulate through the bloodstream, and subse- quently arrest and proliferate in target tissues. The mechanisms that account for bone homing behavior have not yet been elucidated but may include a “direct” vascular pathway, highly permeable marrow sinusoids, chemotactic factors produced by marrow stromal cells, and the synthesis of growth factors by resident cells within the bone and marrow that support the survival, growth, and proliferation of “seed- ed” cancer cells (1, 2). It is well known that hematopoietic stem cells also “home” to bone during fetal life and during marrow transplantation (3). In this context, a CXC chemokine SDF-1 3 (or CXCL12) and its receptor, CXCR4 appear to be critical molecular determinants for these events (4, 5). For instance, although normal fetal liver hematopoiesis still occurs in SDF-1 or CXCR4 gene knockouts, marrow engraftment by these hematopoietic cells is not observed (5, 6). In addition, the levels of CXCR4 expression correlate with the ability of human progenitors to engraft into the marrow of nude mice (7). Finally, osteoblasts and marrow endothelial cells express SDF-1 protein that functions as a chemoattractant for human hematopoietic progenitor cells (8 –10). Thus, it appears that SDF-1 and CXCR4 represent at least one of the critical determinants for bone marrow homing by hematopoietic cells. On the basis of the hematopoietic model, we hypothesize that metastatic prostate carcinomas may use a similar pathway to localize to the bone marrow. In the present investigation, we demonstrate that several human prostate cancer cell lines express functional CXCR4 receptors, and that SDF-1 alters the adherence, migration, and inva- sion of human prostate cancer cell lines. These data are consistent with a role of SDF-1/CXCR4 in metastatic cascades of prostatic carcinomas and suggest novel targets for therapeutic intervention. MATERIALS AND METHODS Primary HOB Cells, Osteosarcoma, Endothelial and Prostate Cancer Cell Lines. Enriched HOB cultures were established as detailed previously (11). MG-63 (CRL1424) and SaOS-2 (ATCC 85-HTB) osteosarcoma cell lines were purchased from the ATCC (Rockville, MD). Bone marrow endothelial cells (HBME) were isolated from a normal Caucasian male and immortalized with SV40 large T-antigen (12). PC-3 and DU145 prostate cancer cells originally isolated from vertebral and brain metastases from prostate cancer patients were obtained from ATCC. LNCaP cells were isolated from a lymph node of a patient with disseminated bony and lymph node involvement (UroCor, Inc., Oklahoma City, OK). The rat MatLyLu cell line were obtained from Dr. John Isaacs (John Hopkins University, Baltimore, MD). MCF-7 cells were established from a patient with metastatic breast cancer (ATCC). Prostate cancer cell lines were passaged and allowed to grow to confluence over 5 days. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine. RT-PCR. RT-PCR was performed as described previously (9, 11). Sense and antisense primers were prepared to cross intron/exon boundaries including: SDF-1, 5'-CGT CAG CCG CAT TGC CCG CT and 3'-GGT CTA GCG GAA AGT CCT (380 bp); CXCR4, 5'-GGC AGC AGG TAG CAA AGT GA and 3'-TGA TGA CAA AGA GGA GGT CGG (341 bp); glyceraldehyde-3- phosphate dehydrogenase, 5'-GAC AAC AGC CTC AAG ATC ATC AGC and 3'-AAG TCA GAG GAG ACC ACC TGG TGC; and -actin, 5'-TCC TGT GGC ATC CAT GAA ACT ACA TTC AAT TCC, 3'-GTG AAA ACG CAG CTC AGT AAC AGT CCG CCT AG (347 bp). The samples underwent thermal cycling at 94°C for 1 min and 60°C for 1 min and 72°C for 1 min for 35 cycles for SDF-1, followed by a 10-min extension at 72°C (Perkin-Elmer, Foster City, CA). PCR for CXCR4 was performed at 94°C, 55°C, and 72°C. False positives and DNA contamination were controlled by omitting reverse transcriptase in control reactions. Immunohistochemistry. Indirect immunohistochemistry was performed for CXCR4 on cells grown in eight-well tissue slides (Costar Corp). Cells fixed in 2% paraformaldehyde at 25°C for 30 min with Triton X-100 were incubated with either 10 g/ml of a murine antihuman CXCR4 monoclonal antibody (BD PharMingen, San Diego, CA) in PBS with 10% normal goat serum or an isotype-matched control at 25°C (Sigma Chemical Co., St. Louis, MO), followed by a goat antimurine FITC-conjugated serum at a 1:50 dilution (Sigma Chemical Co.). SDF-1 ELISA. For determination of SDF-1 levels in conditioned medium, primary human osteoblasts and osteosarcoma cell lines were plated to an initial density of 2.0  10 5 cells/cm 2 in Ham’s F-12/DMEM (1:1, v/v) medium Received 8/20/01; accepted 1/18/02. 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 from a grant from the Specialized Program of Research Excellence in Prostate Cancer at the University of Michigan and NIH Award AR46024. 2 To whom requests for reprints should be addressed, at Department of Periodontics, Prevention, and Geriatrics, University of Michigan School of Dentistry, 1011 North University Avenue, Ann Arbor, MI 48109-1078. Phone: (734) 764-9952; Fax: (734) 763- 5503; E-mail: rtaich@umich.edu. 3 The abbreviations used are: SDF-1, stromal cell-derived factor 1; HOB, human osteoblast; ATCC, American Type Culture Collection; RT-PCR, reverse transcription- PCR; ERK, extracellular signal-regulated kinase. 1832 Research. on December 20, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from