Cancer Research Annual Meeting 2010 Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carlo-Stella, C.
Right arrow Articles by Gianni, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carlo-Stella, C.
Right arrow Articles by Gianni, M. A.
[Cancer Research 62, 6152-6157, November 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

Defibrotide in Combination with Granulocyte Colony-stimulating Factor Significantly Enhances the Mobilization of Primitive and Committed Peripheral Blood Progenitor Cells in Mice1

Carmelo Carlo-Stella2, 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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment 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 µg/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 administration 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 significant increase (P <= 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 105 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 homing 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 synergizes 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
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The availability of adequate amounts of HSCs/HPCs3 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 substantial 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 mechanism(s) regulating HSC trafficking (5 , 6) .

The localization of hematopoietic cells to the BM involves developmentally 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 extracellular 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 characterized by severe leukocytosis, expanded splenic hematopoiesis, elevated hematopoietic cytokine levels, and increased levels of circulating HPCs (11) . The {alpha}4ß1 integrin also plays a key role in homing of HSCs/HPCs to marrow stroma, and administration of function-blocking antibodies against {alpha}4ß1 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 leukotriene B4, inhibits monocyte superoxide anion generation, and stimulates expression of thrombomodulin in vascular endothelium (15 , 16 , 17) . Defibrotide is avidly bound to vascular endothelium and significantly 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 preliminary insights into the mechanism(s) of action of Defibrotide.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 Committee 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, clone M/K-2) was purchased from Southern Biotechnology Associates Inc. (Birmingham, AL).

Mobilization Protocols.
The standard mobilization protocol included treatment of BALB/c with rhG-CSF (5 µg/mouse, i.p.) once daily for 5 days. Defibrotide (1–15 mg/mouse, i.p.) was administered once daily for 5 days either as a single agent or in combination with rhG-CSF. Controls were injected either with PBS/MSA or with isotype control IgG.

Cell Harvesting and Separation.
PB, BM, and spleen were collected before and 3, 5, and 8 days after the onset of treatment. PB was harvested from the orbital plexus into heparin-containing tubes. After WBC counting, PB was diluted (1:4, v/v) with PBS, and MNCs were separated by centrifugation (280 x g, 30 min, room temperature) on a Ficoll discontinuous density gradient. Cells were then washed twice in Iscove’s modified Dulbecco’s medium (Seromed, Berlin, Germany) supplemented with 10% FBS (Stem Cell Technologies, Vancouver, British Columbia, Canada), 2 mM L-glutamine, and antibiotics. BM cells were harvested by repeatedly flushing femura of each animal in PBS by using a 26-gauge needle. Spleen cell suspensions were prepared by mincing the tissue with surgical scissors, grinding tissue fragments using the plunger of a 2-ml syringe, and finally passing cell suspension through a 21-gauge needle to obtain a single cell suspension free of fragments of the splenic capsule. In selected experiments, Sca-1+lin- cells were used. Briefly, blood MNCs were labeled with Sca-1 multisort microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and FITC-conjugated antibodies anti-CD4 (L3T4), anti-CD8a (Ly-2), anti-CD45R (B220), anti-CD11b (Mac-1), anti-Gr-1 (all purchased from PharMingen, San Diego, CA), and anti-Ter-119 (MBL, Milan, Italy). Sca-1+ cells were isolated by double-positive selection using MiniMACS separation columns (Miltenyi Biotec) according to manufacturer instructions. After releasing the magnetic beads with the multisort release reagent, Sca-1+ cells were labeled with anti-FITC microbeads (Miltenyi Biotec), and depletion of lineage+ cells from preselected Sca-1+ cells was achieved using MiniMACS separation columns. The mean (±SD) purity of Sca-1+lin- cells as evaluated with a phycoerythrin-conjugated anti-Sca-1 antibody was 73% ± 6%.

CFC Assay.
Total CFCs, i.e., CFU-GM, BFU-E, and CFU-GEMM were assessed in standard methylcellulose cultures. Briefly, 1-ml aliquots of marrow (5 x 103 to 1 x 104 nucleated cells), blood (5 x 104 to 2 x 105 MNCs), or spleen cells (1 x 105 to 3 x 105 nucleated cells) were plated in 35-mm Petri dishes in methylcellulose-based medium (HCC-3434; Stem Cell Technologies) supplemented with rm stem cell factor (50 ng/ml), mouse rmIL-3 (10 ng/ml), rhIL-6 (10 ng/ml), and rh erythropoietin (3 units/ml). Colonies were scored according to standard criteria after 12–14 days of incubation at 37°C in a humidified atmosphere of 5% CO2 in air (21) .

Long-Term Culture Initiating Cell Assay.
The frequency of LTC-IC was assayed under limiting dilution conditions according to Lemieux et al. (22) with slight modifications. Briefly, serial dilutions of test cells (3 x 104 to 0.9 x 103) were resuspended in 150 µL complete medium in 96-well flat-bottomed plates. For each test cell dose, 16–22 replicates were plated. Complete medium consisted of {alpha}-medium (Life Technologies, Inc., Grand Island, NY) supplemented with FBS (12.5%), horse serum (12.5%; Stem Cell Technologies), L-glutamine (2 mM), 2-ME (10-4 M), inositol (0.2 mM), folic acid (20 µM), and freshly dissolved hydrocortisone (10 µM). Test cells were seeded into plates containing a feeder layer of irradiated (2000 cGy) murine AFT024 cells (kindly provided by Dr. Kateri Moore, Princeton University, Princeton, NJ; Ref. 23 ). Cultures were fed weekly by replacement of half of the growth medium with fresh complete medium. After 4 weeks in culture, nonadherent and adherent cells from individual wells were harvested by trypsinization, washed, and assayed together for CFCs. Cultures were scored as positive (>=1 colony) or negative (no colony), and the LTC-IC frequencies were calculated with the L-Calc software program (Stem Cell Technologies) that uses Poisson statistics and the method of maximum likelihood.

Assay for Radioprotective Cells.
Radioprotective cells were studied by comparing the survival of lethally irradiated (1050 cGy) recipient mice transplanted with MNCs obtained from donor mice that had been treated for 5 days with PBS/MSA, rhG-CSF (5 µg/d), or rhG-CSF plus Defibrotide (15 mg/d). Fifteen recipient mice were injected with control MNCs from PBS/MSA-treated animals, 25 mice were injected with 2 x 105 rhG-CSF- or Defibrotide/rhG-CSF-mobilized cells, and 15 mice were injected with 5 x 105 rhG-CSF- or Defibrotide/rhG-CSF-mobilized cells. Control irradiated mice were included in all of the experiments. A dose of irradiation (1050 cGy) that in preliminary experiments resulted in 100% mortality by day 17 was chosen. Recipient mice were placed in a polymethylmetaacetate box and given total body irradiation by a Roentgen tube (Stabilipan Siemens AG, Berlin, Germany), supplied with a voltage of 300 kV and a current of 12 mA, at a dose rate of 20 cGy/min. Recipient mice were injected via the tail vein 2–3 h after irradiation (24) .

Assays for Hematopoietic Progenitor Homing.
To investigate the effect of Defibrotide on CFC trafficking, untreated or Defibrotide-treated (15 mg/mouse, i.p., 48, 24, and 2 h before reinfusion) recipient mice were lethally irradiated (1050 cGy) and injected, immediately after irradiation, with freshly prepared BM cells (1 x 107 cells, i.v.). Aliquots of injected cells were assayed for CFCs to determine the numbers of injected progenitors (input CFCs). Transplanted cells were allowed to circulate and home for 14 h. For each experiment, hematopoietic organs of 3 irradiated but noninjected mice served as controls for background colony counts. After the homing period, blood, spleen, and femora were harvested. MNCs from 500 µl of blood and cell suspensions corresponding to 10–15% of the BM and spleen volumes were plated, and CFCs were determined. The proportion of homed CFCs was calculated by using the following formula: homed CFCs = (number of CFCs per organ in transplanted mice - number of CFCs per organ in background controls)/input CFCs. To represent the whole BM, the number of homed CFCs per femur was multiplied by 16.9 because one femur represents ~5.9% of the total murine BM (25) . In control experiments using anti-VCAM-1, the antibody or the isotype control (300 µg/mouse, i.p.) were injected 3 h before irradiation of recipient mice.

Homing experiments were also performed in nonirradiated mice using pooled blood cells mobilized with rhG-CSF or Defibrotide/rhG-CSF. For these experiments, Sca-1+lin- cells enriched as above were fluorescently labeled with CFDASE (Molecular Probes, Inc., Eugene, OR). Briefly, 1 x 106 Sca-1+lin- cells that have been washed extensively in PBS were incubated with a 20 µM CFDASE solution for 10 min at room temperature while slowly agitating. The labeling reaction was stopped by the addition of a 10-fold volume excess of cold Iscove’s modified Dulbecco’s medium with 10% FBS, followed by three washes in cold medium. After CFDASE staining, Sca-1+lin- cells were transplanted (1–2 x 105/mouse, i.v.) in nonirradiated recipients that had been treated with PBS/MSA or Defibrotide (15 mg/mouse, i.p., 48, 24, and 2 h before reinfusion). At 14 h after transplantation, mice were killed and single-cell suspensions were prepared from the BM, spleen, and blood. After transfer of the cell suspensions to flat bottomed multiwell tissue plates, the number of CFDASE+ cells in 2–4 x 106 cells per sample was detected visually using a Leica DMIRB (Milan, Italy) inverted microscope with an external fluorescent light source. Phase contrast visualization was used to confirm the viable nature of the cells counted. Nontransplanted CFDASE-labeled cells provided a staining control. After quantification of total cell numbers per sample, results were normalized and reported as CFDASE+ cells per 107 total cells.

In a third group of homing experiments, we analyzed homing properties of PBPCs mobilized by Defibrotide/rhG-CSF. BALB/c mice were transplanted shortly after lethal irradiation with syngeneic blood MNCs (5 x 106, i.v.) mobilized with either rhG-CSF alone or Defibrotide/rhG-CSF. Aliquots of injected cells were assayed for CFCs to determine the numbers of injected progenitors (input CFCs). For each experiment, hematopoietic organs of 3 irradiated but noninjected mice served as controls for background colony counts. At 14 h after transplantation, BM and spleen of transplanted mice were harvested, and CFCs were determined to calculate the proportion of CFCs migrating into marrow and spleen.

Statistical Analysis.
Two plates were scored for each data point per experiment, and the results were expressed as the mean ±1 SE. Statistical analysis was performed with the statistical package Prism3 (GraphPad Software, Inc., San Diego, CA) run on a Macintosh G3 personal computer (Apple Computer Inc., Cupertino, CA). The Student t test for unpaired data (two-tail) was used to test the probability of significant differences between groups. The LTC-IC frequencies were calculated with the L-Calc software program (Stem Cell Technologies).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Defibrotide and rhG-CSF on Circulating WBCs.
As compared with PBS/MSA-treated mice (data not shown), treatment with Defibrotide alone (1–15 mg/mouse/day) had no effect on the number of WBCs, whereas administration of rhG-CSF (5 µg/mouse/day) for 5 days induced an ~2-fold increase in the mean (±SE) number of WBCs (3088 ± 309 versus 6,790 ± 721, P <= 0.0001; Fig. 1Citation ). As compared with mock-injected mice, addition of Defibrotide to rhG-CSF induced a statistically significant (P <= 0.0001) and dose-dependent increase of WBC counts (Fig. 1)Citation . WBC counts achieved with Defibrotide/rhG-CSF treatment were significantly increased over those detected using rhG-CSF alone (P <= 0.001). Three days after cessation of therapy WBC counts had returned to pretreatment levels.



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Effects of Defibrotide and/or rhG-CSF on WBC counts. BALB/c mice were injected i.p. with rhG-CSF alone (5 µg/d), Defibrotide alone (15 mg/d), or a combination of rhG-CSF (5 µg/d) with increasing doses of Defibrotide (1–15 mg/d). Mice were treated for 5 days and killed after 3 and 5 days of treatment, as well as 3 days after cessation of therapy. The following symbols were used to represent each group of mice: Defibrotide 15 mg ({diamondsuit}), rhG-CSF ({circ}), rhG-CSF+Defibrotide 1 mg ({blacktriangleup}), rhG-CSF+Defibrotide 10 mg ({square}), and rhG-CSF+Defibrotide 15 mg ({bullet}). Mock-injected mice received i.p. injections of buffered saline supplemented with 0.1% MSA (PBS/MSA; data not shown). The average WBC count in PBS/MSA control mice was 3.09 ± 0.3 x 106/ml blood. Data are expressed as mean; bars, ±SE. *, P <= 0.0001 and **, P <= 0.006 as compared with PBS/MSA-treated control mice.

 
Effects of Defibrotide and rhG-CSF on the Mobilization of Committed Progenitors.
Whereas Defibrotide alone did not affect CFC frequency, rhG-CSF treatment resulted in a significant increase of the mean (±SE) frequency of blood CFCs (per 105 MNCs), which resulted in an average 13-fold increase after 5 days of therapy as compared with control values (54 ± 5 versus 4 ± 1 CFC per 105 MNCs, P <= 0.0001; Fig. 2Citation ). As compared with mock-injected mice, addition of Defibrotide (1–15 mg/day) to rhG-CSF induced an average 27-, 32-, and 38-fold increase of circulating CFCs (P <= 0.0001), respectively. The simultaneous Defibrotide/rhG-CSF treatment increased the frequencies of mobilized CFCs up to 3-fold above the levels observed with rhG-CSF alone (P <= 0.0007; Fig. 2Citation ).



View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Frequency of total blood CFCs after 5 days of treatment with Defibrotide (Def) and/or rhG-CSF. BALB/c mice were injected i.p. with rhG-CSF alone (5 µg/d), Defibrotide alone (15 mg/d), or a combination of rhG-CSF (5 µg/d) with increasing doses of Defibrotide (1–15 mg/d). Total CFCs include CFU-GM, BFU-E, and multipotent CFC (CFU-Mix). Data are expressed as mean CFC per 105 MNCs from duplicate cultures on samples from each animal; bars, ± SE. The average CFC frequency in PBS/MSA control mice was 4 ± 1. *, P <= 0.0001 and **, P >= 0.05 as compared with PBS/MSA-treated control mice.

 
The kinetics of absolute numbers of circulating CFCs per ml of blood during treatment are shown in Fig. 3Citation . The absolute number of circulating CFCs in blood is a function of the frequency of CFC multiplied by the total number of MNCs per ml blood. After Ficoll separation, the mean recovery of blood MNCs in control mice and Defibrotide/rhG-CSF-treated mice was 42 ± 16% and 36 ± 11%, respectively. As compared with PBS/MSA-treated mice (data not shown), Defibrotide alone did not affect CFC mobilization, whereas a 5-day treatment with rhG-CSF alone induced a 27-fold increase of circulating CFCs (1,391 ± 157 versus 52 ± 9, P <= 0.0001). The combined 5-day treatment with Defibrotide and rhG-CSF induced a dose-dependent and statistically significant enhancement of circulating CFCs (P <= 0.0001; Fig. 3Citation ). As compared with mock-injected mice, addition of 1–15 mg/day Defibrotide to a 5-day rhG-CSF treatment was associated with a mean 52-, 60-, and 119-fold increase of circulating CFCs, respectively. Injection of the highest dose level of Defibrotide (15 mg/day) plus rhG-CSF increased the total number of CFCs by 4.5-fold above the level observed with rhG-CSF alone (6218 ± 681 versus 1391 ± 157, P <= 0.0001). The total number of circulating CFCs detected 3 days after cessation of mobilization therapy was significantly higher in the Defibrotide/rhG-CSF group as compared with the rhG-CSF group (819 ± 104 versus 237 ± 46, P <= 0.05).



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Kinetics of mobilization of total CFCs per ml of blood in mice under treatment with Defibrotide and/or rhG-CSF. BALB/c mice were injected i.p. with rhG-CSF alone (5 µg/d), Defibrotide alone (15 mg/d), or a combination of rhG-CSF (5 µg/d) with increasing doses of Defibrotide (1–15 mg/d). Total CFCs include CFU-GM, BFU-E, and CFU-Mix. Mice were treated for 5 days and killed after 3 and 5 days of treatment, as well as 3 days after cessation of therapy. The following symbols were used to represent each group of mice: Defibrotide 1 mg ({diamondsuit}), rhG-CSF ({circ}), rhG-CSF+Defibrotide 1 mg ({blacktriangleup}), rhG-CSF+Defibrotide 10 mg ({square}), and rhG-CSF+Defibrotide 15 mg ({bullet}). Mock-injected mice received i.p. injections of PBS/MSA (data not shown). The average CFC count in mock-injected mice was 52 ± 9 per ml blood. Data are expressed as mean derived from duplicate cultures on samples from each animal at each time point; bars, ±SE. *, P <= 0.0001 as compared with PBS/MSA-treated control mice.

 
In separate experiments, a dose-response for rhG-CSF was performed (Table 1)Citation . As compared with mock-injected mice, treatment with rhG-CSF at 5 µg/day and 10 µg/day increased circulating CFCs by 32- and 92-fold, respectively. Addition of Defibrotide (15 mg/day) increased the total number of CFCs mobilized with 5 or 10 µg/day of rhG-CSF by 4.5- (5779 ± 440 versus 1299 ± 198, P <= 0.001) and 2.6-fold (9496 ± 1201 versus 3,686 ± 482, P <= 0.001), respectively.


View this table:
[in this window]
[in a new window]

  		Table 1 WBC and CFC counts in mice treated with Defibrotide and/or increasing doses of rhG-CSF

 
Effects of Defibrotide and rhG-CSF on the Mobilization of Primitive Progenitor Cells and Radioprotective Cells.
The frequency of circulating LTC-IC was assessed by limiting dilution assay. In two separate experiments, administration of Defibrotide and rhG-CSF resulted in a significant increase (P <= 0.001) of the mean frequency of circulating LTC-IC, as compared with rhG-CSF alone (Table 2)Citation . We next studied whether the combined Defibrotide/rhG-CSF treatment could preclude the mobilization of radioprotective cells. To test this hypothesis, recipient mice were lethally irradiated (1050 cGy) and rescued with blood MNCs obtained from donor mice that had been treated for 5 days with PBS/MSA, rhG-CSF (5 µg/day), or rhG-CSF (5 µg/day) plus Defibrotide (15 mg/day; Fig. 4Citation ). There was no survival in the irradiation control group and 15% survival in the group injected with PBS/MSA MNCs (5 x 105 cells). Injection of rhG-CSF-mobilized MNCs at 5 x 105 or 2 x 105 cells/mouse rescued 87% and 43% of recipient mice, respectively. Defibrotide/rhG-CSF-mobilized MNCs rescued 90% and 71% of mice injected with 5 x 105 and 2 x 105 cells, respectively. There was significantly greater survival in mice reconstituted with 5 x 105 or 2 x 105 MNCs from rhG-CSF- or Defibrotide/rhG-CSF-treated mice, when compared with the irradiation control group (P <= 0.0001, 0.0128, 0.0001, and 0.0002, respectively) or when compared with the PBS/MSA MNC group (P <= 0.0001, 0.05, 0.0001, and 0.0006, respectively). The survival of mice reconstituted with 2 x 105 MNCs from rhG-CSF- or Defibrotide/rhG-CSF-treated mice was not statistically different (P <= 0.07), although a trend in favor of a better survival in the Defibrotide/rhG-CSF group was evident.


View this table:
[in this window]
[in a new window]

  		Table 2 Frequency of LTC-IC mobilized by rhG-CSF or Defibrotide plus rhG-CSF

 


View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Survival of lethally irradiated BALB/c mice at 33 days after transplantation. Recipient mice were lethally irradiated (1050 cGy) and transplanted with blood MNCs from PBS/MSA-, rhG-CSF- or rhG-CSF/Defibrotide-treated mice. Donor mice were treated once daily for 5 days with PBS/MSA, rhG-CSF (5 µg/mouse), or rhG-CSF (5 µg/mouse) plus Defibrotide (15 mg/mouse). Control mice received irradiation only. Donor MNCs (5 x 105 or 2 x 105 cells/mouse) were injected i.v. 2 h after irradiation into recipient mice (10–25 mice/group). Survival data are expressed as absolute percentages of four experiments.

 
Defibrotide Injection Reduces Marrow Homing of PBPCs.
To test the hypothesis that the mobilizing activity of Defibrotide might be because of an effect on HSC/HPC trafficking, CFC homing was analyzed. Freshly prepared BM cells (1 x 107/mouse) were transplanted into lethally irradiated recipients that were treated with PBS/MSA, Defibrotide (15 mg/mouse 48, 24, and 2 h before reinfusion), or anti-VCAM-1 (300 µg/mouse 3 h before reinfusion). Anti-VCAM-1 treatment was chosen because VCAM-1 has been implicated in marrow homing of hematopoietic progenitors (12) . At 14 h after transplantation, single-cell suspensions were prepared from the blood, spleen, and marrow of recipient mice, and CFCs were cultured. As shown in Fig. 5Citation , homing of hematopoietic progenitors to the marrow was reduced almost 3-fold in Defibrotide-treated animals as compared with PBS/MSA-injected mice (P <= 0.001), and the numbers of CFCs were increased >2-fold in the spleen (P <= 0.02). As compared with mock-injected mice, Defibrotide-treated animals revealed a 3-fold increase of CFCs per ml peripheral blood (3 ± 1 versus 9 ± 0.5, P <= 0.001). CFC homing behavior in Defibrotide-treated animals was indeed similar to that observed in anti-VCAM-1-treated mice, supporting the hypothesis that Defibrotide-enhanced mobilization is mediated by disruption of the recirculation of mobilized progenitors back into BM, as a consequence of reduction of CFC adhesion to the vasculature. To bolster this interpretation, the kinetic decline in circulating progenitors was analyzed by treating mice with rhG-CSF plus Defibrotide for 5 days, then discontinuing rhG-CSF while maintaining the animals on Defibrotide for 3 additional days. The total number of circulating CFCs detected on day 8 was significantly higher in the setting of continued Defibrotide administration (1819 ± 218, P <= 0.001) as compared with a 5-day treatment with rhG-CSF alone (237 ± 46) or rhG-CSF plus Defibrotide (819 ± 104), demonstrating that the continued Defibrotide administration was associated with a marked delay in the kinetic decline of circulating progenitors.



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Homing of CFCs in Defibrotide-treated mice. Freshly prepared BM cells (1 x 107) were transplanted into lethally irradiated recipients that had been injected with either PBS/MSA, Defibrotide (15 mg/mouse 48, 24, and 2 h before transplant), anti-VCAM-1 (300 µg/mouse 3 h before transplant) or a combination of anti-VCAM-1 and Defibrotide. Fourteen h after transplantation, single-cell suspensions were prepared from the spleen and marrow of recipient mice, and the percentages of injected CFC homing per organ, i.e., BM or spleen, were evaluated. Data are expressed as mean percentages of CFC migrating to BM ({blacksquare}) or spleen ({square}) calculated according to the following formula: Homed CFCs = (number of CFCs per organ in transplanted mice - number of CFCs per organ of background controls)/input CFCs. *, P <= 0.001, **, P <= 0.03, #, P <= 0.02, and ##, P <= 0.04 as compared with PBS/MSA-treated control mice; bars, ±SE.

 
Because irradiation induces significant alterations of endothelial trafficking molecules (26) , homing experiments were also performed in nonirradiated animals. CFDASE-stained Sca-1+lin- cells were infused in nonirradiated recipients that had been treated with PBS/MSA or Defibrotide (15 mg/mouse 48, 24, and 2 h before reinfusion). Mice treated with Defibrotide and transplanted with rhG-CSF-mobilized Sca-1+lin- cells showed a 3-fold decrease of CFDASE+ cells migrating into BM and a 2-fold increase of CFDASE+ cells migrating into the spleen with a significant increase of CFDASE+ cells in the blood (164 ± 14 versus 111 ± 9, P <= 0.05; Table 3Citation ).


View this table:
[in this window]
[in a new window]

  		Table 3 Quantification of CFDASE stained Sca-1+lin- cells detected within the bone marrow, spleen, and blood

 
Homing of Defibrotide/rhG-CSF-mobilized PBPCs.
Because in vivo injection of Defibrotide reduces the lodgment of transplanted cells in the BM, experiments were performed to ascertain whether Defibrotide/rhG-CSF-mobilized PBPCs might have alterations in their capacity of homing into BM. In a first set of experiments, mice were transplanted after lethal irradiation (1050 cGy) with syngeneic blood MNCs (5 x 106) mobilized with either rhG-CSF or Defibrotide/rhG-CSF. As shown in Fig. 6Citation , the percentages of rhG-CSF- or Defibrotide/rhG-CSF-mobilized PBPCs migrating into BM (24 ± 4 versus 23 ± 4, P >= 0.05) or spleen (4 ± 1 versus 5 ± 1, P >= 0.05) were not statistically different. Experiments performed in nonirradiated mice demonstrated that homing properties of Defibrotide/rhG-CSF-mobilized Sca-1+lin- cells were identical to those of Sca-1+lin- cells mobilized with rhG-CSF alone (Table 3)Citation .



View larger version (8K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Homing of rhG-CSF- or rhG-CSF/Defibrotide-mobilized PBPCs. Blood MNCs (5 x 106) mobilized with either rhG-CSF alone or rhG-CSF plus Defibrotide were transplanted into lethally irradiated recipients. Fourteen h after transplantation, single-cell suspensions were prepared from the spleen and marrow of recipient mice, and the percentages of injected CFC that homed per organ, i.e., BM ({blacksquare}) or spleen ({square}), were evaluated. Data are expressed as mean percentages of CFC migrating to marrow or spleen calculated according to the formula reported in Fig. 5Citation . *, P >= 0.05, as compared with mice injected with rhG-CSF-mobilized cells; bars, ±SE.

 
Effect of Defibrotide on in Vitro Progenitor Cell Proliferation.
To analyze whether Defibrotide treatment per se exerted any influence on the proliferation of hematopoietic progenitors, BM nucleated cells (n = 3) were plated (104/dish) in methylcellulose cultures without or with rm stem cell factor, rmIL-3, rhIL-6, and rh erythropoietin in the continuous presence of Defibrotide or control vehicle. No colony growth was detected by plating marrow cells in cytokine-free cultures, which were supplemented with Defibrotide (10–1000 µg/ml). Total CFC growth recorded in cultures supplemented with cytokines (24 ± 5, mean ± SD) was unaffected by adding Defibrotide at 10 (23 ± 5, P >= 0.05), 100 (26 ± 4, P >= 0.05), or 1000 (21 ± 4, P >= 0.05) µg/ml, demonstrating that Defibrotide has no direct proliferative effect on hematopoietic progenitors and no modulating activity on cytokine production by accessory cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data demonstrate that Defibrotide significantly enhances the frequency and absolute number of a broad spectrum of hematopoietic progenitors, including committed progenitors and primitive LTC-IC, which are mobilized into the circulation by rhG-CSF. As compared with rhG-CSF alone, the combined Defibrotide/rhG-CSF administration increases the LTC-IC frequency by 2-fold and the absolute numbers of committed CFCs by 4-fold. When added in vitro in semi-solid culture systems, Defibrotide per se has no proliferative effect on PBPCs. Moreover, in vivo administration of Defibrotide alone in mice is devoid of any mobilizing activity, suggesting that Defibrotide injection per se has no capacity to trigger cytokine production.

In mice, on day 3 of treatment, the combined Defibrotide/rhG-CSF injection results in WBC and PBPC counts equal those observed after 5 days of rhG-CSF injection, suggesting that Defibrotide addition may decrease the frequency of administration and total amount of rhG-CSF required for harvesting sufficient blood stem cells for transplantation. Alternatively, the combined Defibrotide/rhG-CSF mobilization regimen may significantly increase the total dose of PBPCs collected during a standard mobilization procedure in healthy donors or cancer patients. Increasing stem cell mobilization and collection may be crucial for a variety of clinical applications ranging from graft engineering procedures to gene therapy programs (27) . Additionally, a number of stem cell-based cell-replacement therapies requiring large amounts of progenitor cells may be envisioned on the basis of our study (28) .

Defibrotide has been reported to decrease the expression or the functional activity of adhesion molecules on endothelial cells, thus reducing CFC adhesion to endothelial layers (18 , 19) . In addition, Defibrotide exerts an antifibrin activity by increasing fibrinolysis, thereby participating in a general antiadhesive mechanism (29) . In vivo homing experiments performed in irradiated and nonirradiated mice show that Defibrotide administration reduces BM homing of transplanted CFCs or CFDASE-stained Sca-1+lin- cells while inducing their accumulation in the blood. Analysis of the kinetic decline in circulating progenitors after stopping rhG-CSF but maintaining Defibrotide injection shows a marked delay in the decline of progenitors, which are returning to BM. Taken together, these data strongly suggest that Defibrotide-enhanced mobilization is mediated by disruption of the recirculation of mobilized progenitors back into BM and not by an additive effect on the physiology of rhG-CSF administration.

A variety of polyanions, such as dextran sulfate (30) , polymethacrylic acid (31) , and fucoidan (32) have been shown to exhibit mobilizing effects through mechanisms such as increased release of matrix metalloproteinases, cytokines, and chemokines, which do not necessarily imply a direct interference with PBPC adhesion to the vasculature (33) . Whether or not these mechanisms might be involved in Defibrotide-induced PBPC mobilization remains to be investigated.

In contrast to function-blocking antibodies to VLA-4 or VCAM-1, which can induce peripheralization of PBPCs when used alone (12 , 34) , Defibrotide has no mobilizing activity when used as a single agent, thus suggesting that it has no specific activity in favoring the deadhesion of PBPCs from BM microenvironment. This finding may be explained by the fact that monoclonal antibodies, such as anti-VLA-4 or anti-VCAM-1, are capable of competing with their natural receptors and actively displace PBPCs from their homing sites, whereas Defibrotide may only be able to decrease PBPC adhesion by "masking" unoccupied homing sites and rendering them no longer available for CFC adhesion. Whether a single adhesion molecule or several classes of molecules are targeted by Defibrotide will require additional analysis.

The use of molecules interfering with PBPC adhesion to improve stem cell mobilization will only be useful if antiadhesion molecules also mobilize radioprotective cells, and if they do not interfere with homing and engraftment of transplanted progenitors. Indeed, Defibrotide/rhG-CSF-mobilized PBPCs are capable of radioprotective activity, as shown by experiments in which lethally irradiated recipient mice were rescued by reinfusion with Defibrotide/rhG-CSF-mobilized cells. In addition, Defibrotide/rhG-CSF-mobilized PBPCs efficiently home to the BM niches, as shown by the percentages of Defibrotide/rhG-CSF- or rhG-CSF-mobilized CFCs that home to the marrow of lethally irradiated mice.

In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. In vivo homing experiments allow us to hypothesize that Defibrotide-enhanced mobilization occurs through the modulation of the expression pattern of adhesion receptors involved in stem cell trafficking. Additional studies are required to elucidate the cellular and molecular mechanism(s) involved in Defibrotide-induced mobilization. Evaluation of the in vivo mobilizing effects of Defibrotide plus rhG-CSF is currently ongoing in nonhuman primates to evaluate potential species-related differences in potency before the start of human clinical trials. Because patients have been treated with Defibrotide at doses lower than that used in the present study, clinical trials aimed at exploring the capacity of Defibrotide to enhance rhG-CSF-induced mobilization should initially evaluate the safety and tolerability of increasing doses of Defibrotide.


   FOOTNOTES
 
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’Università e della Ricerca Scientifica e Tecnologica" (MURST, Rome, Italy) and "Associazione Italiana per la Ricerca sul Cancro" (AIRC, Milan, Italy). Back

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{at}unimi.it Back

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. Back

Received 12/17/01. Accepted 8/26/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Demirer T., Bensinger W. I., Buckner C. D. Peripheral blood stem cell mobilization for high-dose chemotherapy. J. Hematother., 8: 103-113, 1999.[Medline]
  2. Demirer T., Bensinger W. I. Optimization of peripheral blood stem cell collection. Curr. Opin. Hematol., 2: 219-226, 1995.[Medline]
  3. Engelhardt M., Bertz H., Afting M., Waller C. F., Finke J. High-versus standard-dose filgrastim (rhG-CSF) for mobilization of peripheral-blood progenitor cells from allogeneic donors and CD34+ immunoselection. J. Clin. Oncol., 17: 2160-2172, 1999.[Abstract/Free Full Text]
  4. Shpall E. J., Wheeler C. A., Turner S. A., Yanovich S., Brown R. A., Pecora A. L., Shea T. C., Mangan K. F., Williams S. F., LeMaistre C. F., Long G. D., Jones R., Davis M. W., Murphy-Filkins R., Parker W. R., Glaspy J. A. A randomized phase 3 study of peripheral blood progenitor cell mobilization with stem cell factor and filgrastim in high-risk breast cancer patients. Blood, 93: 2491-2501, 1999.[Abstract/Free Full Text]
  5. Craddock C. F., Nakamoto B., Andrews R. G., Priestley G. V., Papayannopoulou T. Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice. Blood, 90: 4779-4788, 1997.[Abstract/Free Full Text]
  6. Whetton A. D., Graham G. J. Homing and mobilization in the stem cell niche. Trends Cell. Biol., 9: 233-238, 1999.[Medline]
  7. Verfaillie C. M. Adhesion receptors as regulators of the hematopoietic process. Blood, 92: 2609-2612, 1998.[Free Full Text]
  8. Levesque J. P., Simmons P. J. Cytoskeleton and integrin-mediated adhesion signaling in human CD34+ hemopoietic progenitor cells. Exp. Hematol., 27: 579-586, 1999.[Medline]
  9. Kansas G. S. Selectins and their ligands: current concepts and controversies. Blood, 88: 3259-3287, 1996.[Free Full Text]
  10. Frenette P. S., Subbarao S., Mazo I. B., von Andrian U. H., Wagner D. D. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow. Proc. Natl. Acad. Sci. USA, 95: 14423-14428, 1998.[Abstract/Free Full Text]
  11. Frenette P. S., Mayadas T. N., Rayburn H., Hynes R. O., Wagner D. D. Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins. Cell, 84: 563-574, 1996.[Medline]
  12. Papayannopoulou T., Craddock C., Nakamoto B., Priestley G. V., Wolf N. S. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc. Natl. Acad. Sci. USA, 92: 9647-9651, 1995.[Abstract/Free Full Text]
  13. Levesque J. P., Takamatsu Y., Nilsson S. K., Haylock D. N., Simmons P. J. Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood, 98: 1289-1297, 2001.[Abstract/Free Full Text]
  14. Palmer K. J., Goa K. L. Defibrotide. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in vascular disorders. Drugs, 45: 259-294, 1993.[Medline]
  15. Pescador R., Porta R., Ferro L. An integrated view of the activities of defibrotide. Semin. Thromb. Hemost., 22(Suppl. 1): 71-75, 1996.
  16. Bianchi G., Barone D., Lanzarotti E., Tettamanti R., Porta R., Moltrasio D., Cedro A., Salvetti L., Mantovani M., Prino G. Defibrotide, a single-stranded polydeoxyribonucleotide acting as an adenosine receptor agonist. Eur. J. Pharmacol., 238: 327-334, 1993.[Medline]
  17. Evangelista V., Piccardoni P., de Gaetano G., Cerletti C. Defibrotide inhibits platelet activation by cathepsin G released from stimulated polymorphonuclear leukocytes. Thromb. Haemostasis, 67: 660-664, 1992.[Medline]
  18. Scalia R., Kochilas L., Campbell B., Lefer A. M. Effects of defibrotide on leukocyte-endothelial cell interaction in the rat mesenteric vascular bed: role of P-selectin. Methods Find. Exp. Clin. Pharmacol., 18: 669-676, 1996.[Medline]
  19. Pellegatta F., Lu Y., Radaelli A., Zocchi M. R., Ferrero E., Chierchia S., Gaja G., Ferrero M. E. Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion. Br. J. Pharmacol., 118: 471-476, 1996.[Medline]
  20. UK Coordinating Committee on Cancer Res UKCCCR guidelines for the welfare of animals in experimental neoplasia. Br. J. Cancer, 58: 109-113, 1998.
  21. Humphries R. K., Eaves A. C., Eaves C. J. Characterization of a primitive erythropoietic progenitor found in mouse marrow before and after several weeks in culture. Blood, 53: 746-763, 1979.[Free Full Text]
  22. Lemieux M. E., Rebel V. I., Lansdorp P. M., Eaves C. J. Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow "switch" cultures. Blood, 86: 1339-1347, 1995.[Abstract/Free Full Text]
  23. Moore K. A., Ema H., Lemischka I. R. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood, 89: 4337-4347, 1997.[Abstract/Free Full Text]
  24. Pruijt J. F., van Kooyk Y., Figdor C. G., Lindley I. J., Willemze R., Fibbe W. E. Anti-LFA-1 blocking antibodies prevent mobilization of hematopoietic progenitor cells induced by interleukin-8. Blood, 91: 4099-4105, 1998.[Abstract/Free Full Text]
  25. Chervenick P. A., Boggs D. R., Marsh J. C., Cartwright G. E., Wintrobe M. M. Quantitative studies of blood and bone marrow neutrophils in normal mice. Am. J. Physiol., 215: 353-360, 1968.[Free Full Text]
  26. Mazo I. B., Quackenbush E. J., Lowe J. B., von Andrian U. H. Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow. Blood, 99: 4182-4191, 2002.[Abstract/Free Full Text]
  27. Somia N., Verma I. M. Gene therapy: trials and tribulations. Nat. Rev. Genet., 1: 91-99, 2000.[Medline]
  28. Orlic D., Kajstura J., Chimenti S., Jakoniuk I., Anderson S. M., Li B., Pickel J., McKay R., Nadal-Ginard B., Bodine D. M., Leri A., Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature (Lond.), 410: 701-705, 2001.[Medline]
  29. Falanga A., Rickles F. R. Pathophysiology of the thrombophilic state in the cancer patient. Semin. Thromb. Hemost., 25: 173-182, 1999.[Medline]
  30. Nothdurft W., Steinbach K. H., Ross W. M., Fliedner T. M. Quantitative aspects of granulocytic progenitor cell (CFUc) mobilization from extravascular sites in dogs using dextran sulphate (DS). Cell Tissue Kinet., 15: 331-340, 1982.[Medline]
  31. van der Ham A. C., Benner R., Vos O. Mobilization of B and T lymphocytes and haemopoietic stem cells by polymethacrylic acid and dextran sulphate. Cell Tissue Kinet., 10: 387-397, 1977.[Medline]
  32. Frenette P. S., Weiss L. Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms. Blood, 96: 2460-2468, 2000.[Abstract/Free Full Text]
  33. Sweeney E. A., Priestley G. V., Nakamoto B., Collins R. G., Beaudet A. L., Papayannopoulou T. Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence. Proc. Natl. Acad. Sci. USA, 97: 6544-6549, 2000.[Abstract/Free Full Text]
  34. Papayannopoulou T., Nakamoto B. Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin. Proc. Natl. Acad. Sci. USA, 90: 9374-9378, 1993.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
C. S. Mitsiades, C. Rouleau, C. Echart, K. Menon, B. Teicher, M. Distaso, A. Palumbo, M. Boccadoro, K. C. Anderson, M. Iacobelli, et al.
Preclinical Studies in Support of Defibrotide for the Treatment of Multiple Myeloma and Other Neoplasias
Clin. Cancer Res., February 15, 2009; 15(4): 1210 - 1221.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Carlo-Stella, M. Di Nicola, R. Milani, A. Guidetti, M. Magni, M. Milanesi, P. Longoni, P. Matteucci, F. Formelli, F. Ravagnani, et al.
Use of recombinant human growth hormone (rhGH) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) for the mobilization and collection of CD34+ cells in poor mobilizers
Blood, May 1, 2004; 103(9): 3287 - 3295.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carlo-Stella, C.
Right arrow Articles by Gianni, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carlo-Stella, C.
Right arrow Articles by Gianni, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online