[CANCER RESEARCH 62, 6152– 6157, November 1, 2002] Defibrotide in Combination with Granulocyte Colony-stimulating Factor Significantly Enhances the Mobilization of Primitive and Committed Peripheral Blood Progenitor Cells in Mice 1 Carmelo Carlo-Stella, 2 Massimo Di Nicola, Michele Magni, Paolo Longoni, Marco Milanesi, Claudio Stucchi, Loredana Cleris, Franca Formelli, and Massimo A. Gianni “Cristina Gandini” Bone Marrow Transplantation Unit [C. C-S., M. D. N., M. Ma., P. L., M. Mi., M. A. G.], Health Physics Service [C. S.], and Department of Experimental Oncology [L. C., F. F.], Istituto Nazionale Tumori, and Chair of Oncology, University of Milano [C. C-S., M. A. G.], Milan, Italy 20133 ABSTRACT Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize pe- ripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treat- ment with Defibrotide alone (1–15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony- forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 g/mouse/day) for 5 days significantly (P < 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined ad- ministration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a signifi- cant increase (P < 0.001) of the frequency of circulating long-term cul- ture-initiating cells. In addition, transplantation of 2  10 5 rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow hom- ing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide syner- gizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans. INTRODUCTION The availability of adequate amounts of HSCs/HPCs 3 represents an essential prerequisite for the feasibility of high-dose therapy programs (1). Optimal mobilization of autologous PBPCs in cancer patients requires both chemotherapy and growth factors, whereas allogeneic PBPCs are mobilized by short courses of G-CSF. Either because of prior extensive chemoradiotherapy or disease-related factors, a sub- stantial proportion of cancer patients fail to mobilize optimal amounts of CD34 + cells (2). Failure to mobilize the required target cell dose of CD34 + cells may also occur in healthy donors (3). PBPC mobilization might be enhanced either by using early acting cytokines capable of expanding marrow progenitors (4) or by interfering with the mecha- nism(s) regulating HSC trafficking (5, 6). The localization of hematopoietic cells to the BM involves devel- opmentally regulated adhesive interactions between hematopoietic cells and stromal cells (7). A wide variety of CAMs participate in the adhesion of HSCs/HPCs to stromal cells and their associated extra- cellular matrix components (8). P-, E- and L-selectins are involved in leukocyte rolling on vascular endothelium (9, 10). Mice lacking endothelial selectins display abnormalities in hematopoiesis charac- terized by severe leukocytosis, expanded splenic hematopoiesis, ele- vated hematopoietic cytokine levels, and increased levels of circulat- ing HPCs (11). The 41 integrin also plays a key role in homing of HSCs/HPCs to marrow stroma, and administration of function-block- ing antibodies against 41 integrin or its cellular receptor, VCAM-1, inhibits homing and induces PBPC mobilization (12, 13). Defibrotide, a single-stranded polydeoxyribonucleotide, is derived from porcine mucosa by controlled depolymerization and has been found to have antithrombotic, anti-ischemic, anti-inflammatory, and thrombolytic properties without significant systemic anticoagulant effects (14). Defibrotide is an adenosine receptor agonist, which increases levels of endogenous prostaglandins, reduces levels of leuk- otriene B4, inhibits monocyte superoxide anion generation, and stim- ulates expression of thrombomodulin in vascular endothelium (15, 16, 17). Defibrotide is avidly bound to vascular endothelium and signif- icantly decreases expression of CAMs, such as P-selectin (18) and intercellular adhesion molecule-1 (19), on endothelial cells. Therefore, we hypothesized a role for Defibrotide in inducing PBPC mobilization by interfering with HSC/HPC trafficking. To test this hypothesis, we investigated the capability of Defibrotide alone or in combination with rhG-CSF to mobilize PBPCs in a mouse model. In addition, homing experiments were performed to provide prelim- inary insights into the mechanism(s) of action of Defibrotide. MATERIALS AND METHODS Animals. Six-to-8-week-old female BALB/c mice, with body weight of 20 –25 g, were purchased from Charles River (Milan, Italy). Experimental procedures performed on animals were approved by the Ethical Committee for Animal Experimentation of the Istituto Nazionale Tumori and were carried out in accordance with the guidelines of the United Kingdom Coordinating Com- mittee on Cancer Research (20). The mice were injected daily i.p. with rhG-CSF and reagents in 0.2 ml low-endotoxin PBS containing 0.1% MSA as a carrier. Each experiment was performed on at least three separate occasions, and 3– 4 mice per group per time point were used. Unless otherwise stated, all of the animal groups were sacrificed 2–3 h after the last treatment. Cytokines, Drug, and Reagents. rhG-CSF, Neupogen, was from Roche (Milan, Italy). Defibrotide was kindly provided by Crinos s.p.a. (Como, Italy). Low endotoxin, azide-free monoclonal antibodies against VCAM-1 (CD106, Received 12/17/01; accepted 8/26/02. 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 Supported in part by grants from “Ministero dell’Universita ` e della Ricerca Scien- tifica e Tecnologica” (MURST, Rome, Italy) and “Associazione Italiana per la Ricerca sul Cancro” (AIRC, Milan, Italy). 2 To whom requests for reprints should be addressed, at “Cristina Gandini” Bone Marrow Transplantation Unit, Istituto Nazionale Tumori, Via Venezian, 1, 20133 Milan, Italy. Phone: 39-02-2390-2717; Fax: 39-02-2390-2678; E-mail: carmelo.carlostella@unimi.it. 3 The abbreviations used are: HSC, hematopoietic stem cell; HPC, hematopoietic progenitor cell; G-CSF, granulocyte colony-stimulating factor; rh, recombinant human; PBPC, peripheral blood progenitor cell; CFC, colony-forming cell; LTC-IC, long-term culture-initiating cell; CAM, cell adhesion molecule; VCAM, vascular cell adhesion molecule; MSA, mouse serum albumin; PB, peripheral blood; BM, bone marrow; MNC, mononuclear cell; FBS, fetal bovine serum; CFU-GM, granulocyte-macrophage colony- forming unit; BFU-E, erythroid burst-forming unit; CFU-GEMM, multilineage colony- forming unit; rm, recombinant mouse; IL, interleukin; CFDASE, carboxyfluorescein diacetate succinimidyl ester. 6152 Research. on September 27, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from