Effectiveness of insulin-like growth factor I receptor antisense strategy against Ewing’s sarcoma cells Katia Scotlandi, 1 Cecilia Maini, 1 Maria Cristina Manara, 1 Stefania Benini, 1 Massimo Serra, 1 Vanessa Cerisano, 1 Rosaria Strammiello, 1 Nicola Baldini, 1 Pier-Luigi Lollini, 2 Patrizia Nanni, 2 Giordano Nicoletti, 3 and Piero Picci 1 1 Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy; 2 Istituto di Cancerologia, Universita ` degli Studi di Bologna, Bologna, Italy; and 3 Istituto Nazionale per la Ricerca sul Cancro-Genova, Sezione di Biotecnologie di Bologna, Bologna, Italy. The insulin - like growth factor I receptor ( IGF - IR ) plays an essential role in the establishment and maintenance of transformed phenotype of Ewing’s sarcoma ( ES ) cells, and interference with the IGF - IR pathways by a neutralizing antibody causes reversal of the malignant potential of this neoplasm. In this paper, we stably transfected an IGF - IR antisense mRNA expression plasmid in an ES cell line to determine the effectiveness of antisense strategies against the in vitro and in vivo growth of ES cells. Doxorubicin sensitivity of TC - 71 cells expressing antisense targeted to IGF - IR mRNA was also examined. Cells carrying antisense IGF - IR had a reduced expression of the receptor, a modest decrease in cell proliferation, a significant increase in anoikis - induced apoptosis, and a severely reduced ability to form colonies in soft agar. Moreover, TC / AS cells showed a marked reduction in their motility. In vivo, when cells carrying antisense IGF - IR were injected subcutaneously in nude mice, tumor formation was delayed and survival increased. Metastatic ability of ES cells was also significantly reduced. Furthermore, TC / AS clones showed a significantly higher sensitivity to doxorubicin — a major drug in the treatment of ES. These results indicate that inhibiting IGF - IR by antisense strategies may be relevant to the clinical treatment of ES patients by reducing the malignant potential of these cells and enhancing the effectiveness of chemotherapy. Cancer Gene Therapy (2002) 9, 296 – 307 DOI: 10.1038 / sj / cgt / 7700442 Keywords: Ewing’s sarcoma; insulin - like growth factor I; antisense; malignancy; nude mice E wing’s sarcoma (ES) is a malignant small round cell tumor of bone and soft tissues, preferentially arising in children and young adults. ES is characterized by the presence of specific chromosomal translocations, which produce EWS/ets gene rearrangements [ in more than 95% of cases, the gene fusion is EWS/FLI-1 due to the t(11;22) (q24;q12), or EWS/ERG due to the t(21;22) (q22;q12)], 1 as well as the expression, at extremely high levels, of an antigen encoded by the MIC2 gene (also known as CD99 or p30/32 MIC2 ). 2,3 ES shows an extremely aggressive clinical course. At diagnosis, approximately 25% of patients has detectable metastases in the lungs and/or bones and bone marrow, but nearly all patients have micrometastases, as evidenced by a less than 10% cure rate with local therapy alone. 4 The adoption of multimodal treatments with very aggressive chemotherapeutic regimens associated with surgery or radiotherapy for local control has significantly improved the chance of survival of ES nonmetastatic patients, shifting the 5-year survival rate to around 50– 60%. 5,6 However, despite advances in therapy, one third of patients with nonmetastatic disease, and the great majority of patients with metastases at diagnosis, do not survive, regardless of therapy. 7 Furthermore, recent clinical studies have indicated that the survival rate of ES patients has reached a plateau phase and, possibly, the highest levels achievable by conventional multimodal therapy. 8,9 Success- ful treatment of therapy-resistant disease therefore requires new strategies. Recent findings have indicated the insulin-like growth factor I receptor I (IGF-IR) as a promising target for tailored biological therapies of ES. IGF-IR is implicated in the autocrine and paracrine control of ES growth and appears to be particularly important in the pathogenesis of this tumor. In particular, IGF-IR–mediated loop is constantly present in ES and is a major autocrine circuit of this neoplasm. 10,11 In addition, Toretsky et al 12 reported IGF-IR expression to be necessary for EWS/FLI-1– mediated transformation of primary fibroblasts. Thus, the IGF-IR pathway may be considered a good site for therapeutic intervention in EWS-FLI-1–mediated tumor proliferation. We confirmed this finding by demonstrating that impairment of IGF-IR by neutralizing antibody and suramin reduces growth and increases apoptosis of ES cells both in vitro and in vivo, and significantly decreases migration, invasion, and metastatic spread to the lungs and Received December 13, 2001. Address correspondence and reprint requests to: Dr Katia Scotlandi, Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Via di Barbiano, 1 /10, Bologna 40136, Italy. E- mail: katia.scotlandi@ior.it Cancer Gene Therapy (2002) 9, 296 – 307 D 2002 Nature Publishing Group All rights reserved 0929-1903 / 02 $25.00 www.nature.com/ cgt