Docetaxel-induced mobilization of hematopoietic stem cells in a murine model

Docetaxel-induced mobilization of hematopoietic stem cells in a murine model

Experimental Hematology 28 (2000) 451–459 Docetaxel-induced mobilization of hematopoietic stem cells in a murine model: Kinetics, dose titration, and...

2MB Sizes 0 Downloads 1 Views

Experimental Hematology 28 (2000) 451–459

Docetaxel-induced mobilization of hematopoietic stem cells in a murine model: Kinetics, dose titration, and toxicity John O. Ojeifoa, Aiguo G. Wua, Yihong Miaoa, Herbert B. Herscowtizb, and Kenneth R. Meehana a Division of Hematology and Oncology, Bone Marrow Transplantation Program, and Department of Microbiology and Immunology, Georgetown University Medical Center, and Vincent T. Lombardi Cancer Center, Washington, DC, USA


(Received 1 July 1999; revised 15 December 1999; accepted 27 December 1999)

Objective. Docetaxel (DXT) is an anticancer agent that has demonstrated therapeutic efficacy against solid tumors, particularly breast cancer. Based on the use of hematopoietic stem cell (HSC) transplantation to restore hematopoietic reconstitution after myeloablative therapy, this study was performed to determine if DXT could mobilize HSCs in vivo. Materials and Methods. C57Bl/6 mice were injected intraperitoneally with varying doses of DXT (equivalent to human doses of 40 to 120 mg/m2). Spleens were harvested on days 2, 4, 6, 8, 10, and 12 after DXT administration for recovery of mononuclear cells (MNCs). The number of HSCs present within the MNCs was determined by clonogenic assay for colony-forming units in culture (CFU-C) and by FACS analysis for CD34⫹ cells. Peripheral blood samples were obtained at the time of spleen harvest to determine the hematologic profile. Liver and renal function tests were performed to monitor toxicity. Results. DXT mobilized HSCs in a dose- and time-dependent manner. When measured by the CFU-C assay, maximal mobilization of HSC (⬎10-fold increase over control; p ⬍ 0.01) was observed at a dose of 30 mg/kg (equivalent to human dose of 75 mg/m2) on day 7. The number of mobilized HSCs peaked on days 6 to 8 at all doses of DXT tested. There was no evidence of weight loss, liver, or renal toxicity at any of the DXT doses tested. Conclusion. These results indicate that DXT efficiently mobilizes HSCs in a murine model and provide the rationale for similar studies in a clinical trial. © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Hematopoietic stem cells—Priming chemotherapy—Docetaxel—Animal model— Mobilization

Introduction Hematopoietic stem cells (HSCs) have the capacity to selfrenew as well as the ability to generate all cell types of myeloid, lymphoid, and erythroid lineages [1–5]. These properties are exploited in experimental stem cell mobilization models to generate cytotoxic effector cells for immunotherapeutic purposes [6] and clinically to reconstitute hematopoiesis after high-dose chemotherapy [4,5,7–9]. Cyclophosphamide, an antineoplastic agent commonly used to mobilize HSCs in cancer patients, inhibits cytotoxic T-cell function [6,10], an effect that may exacerbate the tumor- induced immunosuppression that preexists in these patients [11–13]. In addition, HSC populations mobilized by

Offprint requests to: Kenneth R. Meehan, M.D., Division of Hematology and Oncology, Bone Marrow Transplantation Program, Georgetown University Medical Center, 3800 Reservoir Road, NW, Washington, DC 20007 USA; E-mail: [email protected]

myeloid growth factors, such as granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, alone or in combination with chemotherapy, are not significantly enriched in quality and quantity of immunocompetent cells, which could allow for eradication of tumor cells within the graft or minimal residual disease within the treated host [6]. Consequently, tumor recurrence remains a major problem after HSC transplantation. Identification of HSCmobilizing agents or regimens that can generate large number of stem cells with effective antitumor activity is desirable. Docetaxel (DXT) is a potent antineoplastic agent belonging to the taxoid family. It has demonstrated therapeutic efficacy against anthracycline- and paclitaxel-resistant breast, ovarian, and other solid tumors [14–19]. Although DXT has been extensively investigated for its therapeutic effects, little is known about its effects on HSC mobilization. The goal of the present study was to determine whether DXT could mobilize HSCs in vivo as well as to define the optimal priming dose, kinetics, and toxicity profile in a murine model system.

0301-472X/00 $–see front matter. Copyright © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(00)0 0 1 3 0 - 2


J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459

Materials and methods Animal and treatment groups Seven to nine-week-old (body weight 18 to 21 g) female C57BL/6 mice (H-2b) (Taconic, Germantown, MD) were housed in groups of five per cage in laminar flow cabinets and fed standard peletted food and water ad libitum. Five study groups were used: 1) DXT (Rhone- Poulenc-Rorer, Collegeville, PA)-treated, 2) recombinant human granulocyte colony-stimulating factor (rhG-CSF) (Amgen, Thousand Oaks, CA.)-treated, 3) DXT ⫹ rhG-CSF-treated, 4) mice treated with intraperitoneal (IP) injection of docetaxel diluent, and 5) untreated mice. Because similar results were obtained in mice in groups 4 and 5, their values were pooled and the average used as the control. Animal protocols used were approved by the Georgetown University Animal Care and Use Committee (GUACUC). Ethical guidelines for conducting research on laboratory animals were followed throughout these studies. Mobilization regimen On day 0, DXT was administered by IP injection in doses ranging from 16 to 48 mg/kg (equivalent to human dose levels of 40 to 120 mg/m2). DXT, supplied as a concentrated solution in polysorbate, was mixed in the supplied diluent (13% ethanol in water) to produce a premix solution of 10 mg/mL. This solution was diluted in 5% aqueous dextrose solution to give a working concentration of 0.8 mg/mL. Forty-eight hours after DXT administration, rhG-CSF was administered and continued throughout the study period. The rhG- CSF was diluted in 5% aqueous dextrose solution to produce a working concentration of 25 ␮g/mL, which then was administered at a concentration of 125 ␮g/kg twice a day by subcutaneous injection. Because more HSCs are mobilized to the spleen than to peripheral blood [20], splenic mononuclear cells (MNCs) were used as the source of HSCs for these studies. Immediately afer blood collection, the mice were killed and their spleens were removed and processed for recovery of MNCs using standard method [21]. Briefly, a nick was made at one end of the spleen with a pair of sterile scissors. The plunger of a 3-mL syringe was applied to the organ at the opposite end of the cut, and cells were gently squeezed out. Thereafter, a 27-gauge needle tip was used to tease the connective tissue into tiny pieces to release the cells still lodged within the tissue. Extraction of total cells from each spleen and single cell suspension were further ensured by repeated gentle aspirations and flushing through a 19-gauge needle. Cells recovered from each spleen were subjected to density centrifugation on Ficoll-Hypaque (density 1.086 g/mL, Fico/Lite LM; Atlanta Biologicals, Norcross, GA). Light-density MNCs were washed twice in culture medium (CM), which consisted of RPMI 1640 (Biofliuds, Rockville, MD) supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 5 ⫻ 10⫺5M 2-mercaptoethanol, 100 units/mL penicillin, and 100 ␮g/mL streptomycin (Biofluids, Rockville, MD). Cells were counted and tested for viability by trypan blue exclusion. Quantification of HSCs The number of HSCs mobilized following DXT treatment was assessed by a clonogenic (colony-forming unit in culture [CFU-C]) assay. Briefly, fresh light-density splenic MNCs obtained from

each of the groups were added at a concentration of 1 ⫻ 105 cells/ mL to semisolid methyl cellulose medium (HCC 3434; Stem Cell Technology, Vancouver, Canada). One-milliliter cultures were established in duplicate 35-mm culture dishes (Costar, Cambridge, MA) and incubated at 37⬚C in a humidified atmosphere of 5% CO2. After 7 days, the number of colonies with more than 40 cells (CFU-C), including CFU-granulocyte-macrophage (CFU-GM), CFU-erythroid (CFU-E), and CFU-mixed (CFU-GEMM), was enumerated using a a dissecting microscope. The different colonies were identified and counted based on their morphologic appearances. Colonies with irregular shape consisting of small, refractile, light-brown, or pinkish- brown cells were considered CFU-E. Colorless colonies with dark, dense core or consisting of granular or foamy cells were counted as CFU-GM. Colonies consisting of a dark central core and “hazy” appearance were enumerated as CFU-GEMM [22]. The total number of CFU-C mobilized to the spleen was calculated using the number of MNCs harvested from each spleen. Flow cytometric analysis of splenic MNCs Fluorescence-activated cell sorting (FACS) was used to determine the number of cells expressing CD34 in the splenic MNC population harvested from the DXT-treated and control mice using the method described by Verma et al. [6]. Essentially, light-density MNCs were stained with FITC-conjugated antibodies to mouse stem cell (CD34⫹) marker (PharMingen, San Diego, CA), washed, and analyzed by one-color flow cytometry on a FACS Star Plus (Beckon-Dickinson, Mountain View, CA). The percentage of CD34-expressing cells was calculated using Reproman software (TrueFacts Software Inc., Seattle, WA). Evaluation of neuromotor function, hematologic profile, and liver and renal function tests Because bone marrow, liver, and kidney functions may be altered by the administration of DXT, peripheral blood leukocyte (PBL) count, total bilirubin, alkaline phosphatase, serum transaminases (aspartate aminotransferase [AST], serum glutamic oxaloacetic transaminase [SGOT], alanine aminotransferase [ALT], serum glutamic-pyruvic transaminase [SGPT]), blood urea nitrogen, and creatinine were evaluated. After DXT administration, the mice were closely monitored three times daily for evidence of gastrointestinal toxicity, such as loss of appetite and dehydration, and stool examination for diarrhea, blood, or mucous. The mice also were examined for neuromotor abnormalities, including limb paralysis, hunching, or lethargy. Additionally, each mouse was weighed at the beginning and end of each experimental period. Each dose of DXT was evaluated using five mice, and each test was conducted three separate times. On days 2, 4, 6, 8, and 10 after DXT administration or at the end of each experiment, peripheral blood samples were collected by terminal cardiac puncture under metophane anesthesia to determine hematologic profile, liver, and kidney function by standard methods (Antec Diagnostics, Inc. Rockville, MD). Statistical analysis Comparison between the different groups was made using the Student’s t-test.

J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459


Results Splenic MNC and PBL recovery after docetaxel administration Figures 1 and 2 show the numbers of splenic MNCs and PBLs recovered from control and DXT-treated mice on various days after the administration of 30 mg/kg of DXT, equivalent to a human dose of 75 mg/m2. A decrease in the number of splenic MNCs was most prominent on day 2 (56% reduction from baseline; [p ⬍ 0.01]), but returned to normal by day 4 (Fig. 1). The number of PBLs also decreased after DXT administration, reaching a nadir on day 2 (46% of control), with normalization by day 4 (Fig. 2). These findings of a maximum decrease in the number of both splenic MNCs and PBLs on day 2 after DXT treatment was observed at all doses tested (data not shown). A peak increase in the mean number of splenic MNCs and PBLs occurred on day 8 and 4, respectively (Figs. 1 and 2). Because chemotherapeutic agents frequently are combined with myeloid growth factors to mobilize peripheral blood stem cells, we also evaluated the effects of docetaxel plus granulocyte colony-stimulating factor administration on the number of splenic MNCs and PBL in mice. Administration of rhG-CSF (125 ␮g/kg) to DXT-treated mice reversed the decrease in the number of splenic MNCs and PBLs observed on day 2 and produced a significant increase in the number of splenic MNCs and PBLs on days 4 through 10 (Table 1). Mice treated with rhG-CSF alone, at a concentration of 125 ␮g/kg twice daily, had a 14- and 11-fold higher number of splenic MNCs and PBLs, respectively, compared to control mice on day 10 (Table 1). Docetaxel dose and splenic MNC recovery To determine the effect of various doses of DXT on the number of splenic MNCs recovered, mice were treated with different doses of DXT ranging from 16 to 48 mg/kg (equivalent to human dose levels of 40 to 120 mg/m2) and spleens were harvested from the animals on the eighth day after treatment, the time of maximal splenic hypercellularity. Figure 3 demonstrates that DXT mobilization of MNCs was dose dependent, with a maximal number mobilized at 30 mg/kg (equivalent to human dose level of 75 mg/m2 ). Kinetics of docetaxel mobilization of HSCs To determine the kinetics of DXT mobilization of HSCs, the number of progenitors in MNC populations harvested from control and DXT-treated mice was assessed by a clonogenic assay for CFU-C and by FACS analysis for cells expressing CD34. DXT was administered at 30 mg/kg, and the HSC content of the splenic MNC population was evaluated on days 3, 7, and 10 after treatment. Figure 4A shows the total number of CFU-C per spleen in each of the four groups of animals in the study. A large increase in both

Figure 1. Splenic mononuclear cell (MNC) recovery following docetaxel (DXT) administration. Mice were treated with 30 mg/kg of DXT (equivalent to a human dose of 75 mg/m2) on day 0. Results are expressed as number of light-density MNCs per spleen at times indicated after DXT treatment. Values are given as mean ⫾ SD of three independent experiments, with a minimum of five mice at each time point.

CFU-C per 105 plated MNCs and the total splenic CFU-C (⬎8-fold increase over control) was observed on days 6 through 8, with a peak on day 7, when a 10-fold increase was observed. Thereafter, the magnitude of the DXT-induced increase in the total number of CFU-C per spleen slowly declined to fivefold greater than baseline on day 10. The number of CFU-C per 105 plated MNCs and the total splenic CFU-C per spleen obtained on day 3 after DXT administration was not significantly different from that obtained in control mice. Treatment of DXT-primed mice with rhG-CSF, at a concentration of 125 ␮g/kg twice daily, resulted in a 45-fold increase in the total splenic CFU-C on days 7 and 10 com-

Figure 2. Peripheral blood leukocyte (PBL) count following docetaxel treatment. Mice were treated with 30 mg/kg of DXT (equivalent to a human dose of 75 mg/m2) on day 0. Results are expressed as number of PBLs (⫻ 103/mm3) at times indicated after DXT treatment. Values are given as mean ⫾ SD of three independent experiments, with a minimum of five mice at each time point.


J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459

Table 1. Splenic mononuclear cell and peripheral blood leukocyte levels in mice treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone or with docetaxel plus rhG-CSF


Duration (days)

MNCs (mean ⫾ SD) (⫻106)

PBL (103/mm3)

2–10 2 2 4 4 6 6 8 8 10 10

149.2 ⫾ 18.73 283.1 ⫾ 6.61 223.5 ⫾ 8.4 487.5 ⫾ 5.2 481.3 ⫾ 11.2 536.8 ⫾ 12.8 1372.3 ⫾ 10.2 581 ⫾ 3.2 1460.2 ⫾ 9.1 2041.3 ⫾ 15.4 1788 ⫾ 12.3

6.3 ⫾ 1.5 12.6 ⫾ 2.1 4.2 ⫾ 1.1 24.6 ⫾ 3.2 18.9 ⫾ 2.2 26.56 ⫾ 1.4 49.8 ⫾ 5.6 38.43 ⫾ 3.4 51.66 ⫾ 2.0 70.2 ⫾ 8.8 13.84 ⫾ 1.04

Mice were treated with subcutaneous (SC) injections of rhG-CSF alone, at a concentration of 125 ␮g/kg twice daily for 3, 7, or 10 days, or a combination of the SC injections of rhG-CSF for the same duration and a single intraperitoneal dose of 30 mg/kg of docetaxel (DXT; equivalent to a human dose of 75 mg/m2) on day 0, or the vehicle of the agents. Spleens and blood were harvested and processed for mononuclear cells (MNCs) and peripheral blood leukocytes (PBLs), respectively, as described in the Materials and methods. Results are given as number of light-density MNCs per spleen or as number of PBLs (⫻ 103/mm3) at times indicated. Values are given as mean ⫾ SD of three independent experiments, with a minimum of five mice at each time point.

pared to control values (Fig. 4A). Results of FACS analysis of the splenic MNCs for the percentage of cells expressing CD34 paralleled the results observed in the clonogenic assay (Fig. 4B). However, the number of CD34-expressing cells did not correlate with total splenic CFU-Cs.

Figure 3. Effect of different doses of docetaxel (DXT) on splenic mononuclear cell (MNC) recovery. DXT was administered in doses that range from 16 to 48 mg/kg (equivalent to human dose levels of 40 to 120 mg/m2, respectively) by intraperitoneal injection on day 0. Light-density MNCs were harvested on day 8 after DXT administration. Results are expressed as number of light density MNCs per spleen. Values are given as mean ⫾ SD of a minimum of five mice.

Docetaxel concentration and clonogenic potential of mobilized splenic MNCs To determine the optimal dose of DXT for HSC mobilization, groups of mice were primed by IP injection of 16, 24, 30, 40, and 48 mg/kg, equivalent to human doses of 40, 60, 75, 100, and 120 mg/m2, respectively. In these studies, splenic MNCs were harvested on day 8 after DXT administration and assayed for CFU-C. As is evident from the results shown in Figure 5, a dose of 30 mg/kg of DXT mobilized the maximum number of CFU-C colonies per the spleen. The total number of CFU-C colonies observed per spleen in response to the administration of 16 mg/kg (equivalent to human dose level of 40 mg/m2) was not significantly greater than that of the control value. There was a substantial variation in the number of CFU-C colonies obtained per spleen in mice treated with DXT doses higher than 30 mg/kg. Growth pattern of docetaxel-mobilized HSCs Figure 6 shows photomicrographs of the colonies of splenic MNCs obtained from DXT- treated and control mice. Colonies of CFU-C from control mice were generally small in size and displayed clusters of round, adherent cells suggestive of limited growth potential [23–25]. Most of the CFU-C colonies of the splenic MNCs from DXT-treated mice were large in size, with a diameter from 0.3 to 0.6mm. These colonies exhibited uniformly dispersed nonadherent, blast-like cells, suggesting high proliferative, self-renewing, and multilineage-generating growth potential [20,23–25]. The CFU-C colonies of splenic MNCs from mice treated with DXT plus rhG-CSF showed a mixture of dispersed and clustered cells (Fig. 6C). Toxicity DXT toxicity was evaluated at doses ranging from 24 to 40 mg/kg (equivalent to human doses 60 to 100 mg/m2). These doses were chosen based on their ability to mobilize HSCs during the preliminary studies and because they parallel doses recommended for human chemotherapy. No death or significant weight loss occurred in DXT-treated mice. There were no hepatic, renal, or neuromotor abnormalities observed with any of the doses tested at the different time points. Table 1 shows the results of a representative experiment conducted on days 2, 4, 6, 8, and 10 after administration of 30 mg/kg of DXT. The number of PBL decreased on day 2 after DXT treatment but quickly returned to normal by day 4 at all doses tested (Fig. 2 and Table 2), suggesting that DXT induced transient bone marrow suppression. DXT administration also was associated with a rapid decrease in the percentage of lymphocytes in peripheral blood and a corresponding increase in the percentage in polymorphonuclear cells on days 2 and 4 (Table 2). Thereafter, the proportions of lymphocytes and polymorphonuclear cells gradually returned to baseline values by day 10.

J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459


Figure 4. Kinetics of hematopoietic stem cell (HSC) mobilization after treatment with docetaxel (DXT) or granulocyte colony-stimulating factor (G-CSF), alone or in combination. The time course of HSC mobilization after administration of 30 mg/kg (equivalent to human dose level of 75 mg/m2 ) of DXT or recombinant human G-CSF (rhG-CSF; 125 ␮g/kg body weight, twice daily), alone or in combination, was assessed by enumerating the number of colonyforming units in culture (CFU-C) (A) and by FACS analysis for CD34-expressing cells (B) in the splenic mononuclear cells harvested from mice at times indicated. Values are givne as total number (mean ⫾ SD; n ⫽ 5) of CFU-C colonies/spleen (A) or CD34-expressing cells/spleen (B) and are representative of three experiments.

Discussion Autologous bone marrow and peripheral blood stem cells have been used successfully to reconstitute hematopoiesis after myeloablative therapy [26–29]. Previous studies showed that HSC mobilization regimens that use chemotherapeutic agents alone or in combination with hematopoietic growth factors may be superior to those using hematopoietic growth factors alone because of the capacity of the former to induce rebound increases in peripheral blood stem cells [20,30–33].

Essential properties of an ideal HSC-mobilizing cytotoxic agent must include the ability to consistently mobilize a high yield and superior quality of HSCs, rapid onset of action, minimal toxicity, and absence of immunosuppressive properties. Studies of paclitaxel, the natural analog of DXT, indicate that it possesses many of these qualities and may be superior to cyclophosphamide when used alone or in combination with other agents to mobilize HSCs [10,25,34]. However, paclitaxel is innately toxic to lymphocytes [35]. The promising chemotherapeutic potency of DXT for treatment


J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459

Figure 5. Dose response of docetaxel (DXT) on hematopoietic stem cell (HSC) mobilization. The total number of HSCs mobilized after administration of varying doses of DXT alone was assessed by enumerating the number of colony-forming units in culture (CFU-C) in the splenic mononuclear cells harvested from mice on day 7 after treatment. Values are given as total number (mean ⫾ SD; n ⫽ 5) of CFU-C per spleen and are representative of three experiments.

of several cancers [36] and its low toxicity toward lymphocytes [37] make it an attractive agent for HSC mobilization. The results of this study demonstrate that DXT effectively mobilizes HSCs in a dose- and time-dependent manner in mice. A single dose of 30 mg/kg of DXT (equivalent to a human dose level of 75 mg/m2) stimulated 85 ⫾ 16 ⫻ 103 HSCs (assessed by CFU-C assay) in the spleen compared to 7.8 ⫾ 1 ⫻ 103 HSCs in untreated animals (p ⬍ 0.01). The mean 10-fold increase in the peak CFU-C level over baseline compares well with values frequently reported after administration of paclitaxel [25] and cyclophosphamide [6,20,30]. DXT administration also was associated with a rapid induction of HSCs, maintenance of a high level (⬎5-fold) of HSCs in the periphery over a period of 7 days, and a brief 2-day period of myelosuppression. Interestingly, the 4-day period between the time of DXT administration and the rebound increase of peripheral blood HSC level observed in this study is similar to the time period observed after paclitaxel treatment [25], but it is shorter than the 6 days often observed after cyclophosphamide administration [6,20,25,29,38]. The extended period of HSC and peripheral blood MNC induction by DXT provides an excellent opportunity for serial aphereses to obtain a high yield of these cells for autologous transplantation Murine and human studies have shown that bone marrow, unstimulated (normal) peripheral blood, and mobilized peripheral blood contain two types of stem cells [20,25, 30,39–41], namely, committed (late or mature) and primitive (early or pluripotent) stem cells. Committed stem cells exhibit limited growth capacity and can only maintain short-term hematopoietic reconstitution [20,24]. In contrast, primitive stem cells display a dispersed pattern of growth, extensive proliferative, self-renewing, and multilineage-

generating capacity, as well as the ability to sustain longterm hematopoietic reconstitution [20,24,30,38,40]. Neben et al. [20] reported that 98% of the cyclophosphamidemobilized HSCs were committed stem cells and had minimal self-renewing capacity. Although the state of maturation of the DXT-mobilized stem cells was not directly investigated in this study, the observation that most CFU-C colonies of DXT-mobilized HSCs were large in size and exhibited a dispersed growth pattern suggests that they possess a multilineage, high proliferative growth potential [23,24] and may be able to sustain long-term hematopoietic reconstitution. Toxicity of chemotherapeutic agents may limit their application for HSC mobilization. For example, cyclophosphamide is associated with cumulative toxicity, neutropenia, inhibition of T-cell function, pulmonary, and gonadal toxicity [30,34], and sometimes death [42]. Spindle poisons, such as etoposide or vinorelbin, may induce hematologic and oropharyngeal toxicities, requiring hospitalization [43,44]. Paclitaxel effectively mobilizes HSCs without the cumulative toxicity, thrombocytopenia, or damage to the stem cell pool or urinary bladder observed with cyclophosphamide [34,45], but its administration has been associated with severe degenerative changes in the brain, gonads, liver, kidneys, and heart, and death in mice and rabbits [46]. In contrast, studies of DXT toxicity by Tampellini et al. [44] show that it induces moderate reversible hematologic and gastrointestinal damage after repeated intravenous (IV) administration at 20 mg/kg/wk for 6 weeks or 30 mg/kg/wk for 5 weeks. In an earlier study, Bissery et al. [47] also reported that single or repeated IV administration of DXT at a concentration of 15 mg/m2 (equivalent to 6 mg/kg) produced hematologic, gastrointestinal, and neuromotor toxicities in mice and dogs. The results presented herein show that DXT, at a dose of 30 mg/kg (equivalent to human dose level of 75 mg/m2), which induced the maximal number of HSCs, had no significant toxic effects on tissues other than transient effects on the bone marrow. Gastrointestinal and neuromotor toxicities observed by Tampellini et al. [44] and Bissery [47] may be due to the IV route of administration or cumulative toxicity. Although DXT is used clinically at a standard dose of 100 mg/m2, a recent phase II study by Salminen et al. [48] demonstrated that advanced breast cancer patients with previous chemotherapy treated with 75 mg/m2 of DXT show better responses and less toxicity than patients treated with 100 mg/m2. In the present study, there was a substantial variation in the number of CFU-C colonies obtained per spleen in mice treated with a dose of 40 mg/kg (equivalent to100 mg/m2), resulting in an overall decrease in the total number of HSCs mobilized to the spleen compared to the number mobilized by a dose of 30 mg/kg (equivalent to 75 mg/m2). This suggests that a dose of 30 mg/kg (equivalent to 75 mg/m2 ) of DXT could be safely used in the induction phase of the treatment of advanced breast cancer with high-dose chemotherapy and HSC transplantation.

J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459


Figure 6. Growth patterns of splenic mononuclear cells (MNCs) from control, docetaxel (DXT) alone, or docetaxel plus granulocyte colony-stimulating factor (G-CSF)-treated mice. Splenic MNCs 1 ⫻ 105 from each experimental group were cultured as described in the Materials and methods. (A) Colony-forming units in culture (CFU-C) colonies from control mice. (B) CFU-C colonies from DXT-treated mice. (C) CFU-C colonies from DXT plus G-CSF-treated mice (original magnification ⫻ 60.5).

Myelosuppression often is a side effect of a variety of antineoplastic agents. This chemotherapy-mediated effect on bone marrow generally is reflected by a significant decrease in PBL count, splenic weight, and cellularity [44]. In the

present study, DXT-induced leukopenia, low splenic weight, and low cellularity lasted for only 2 to 3 days. This observation is in agreement with the findings of Tampellini et al. [44] and suggests that the duration of DXT’s bone


J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459

Table 2. Effects of docetaxel treatment on peripheral blood leukocyte count and liver and renal functions in mice

Days after treatment

Total bilirubin (mg/dL)

Alkaline phosphatase (U/L)

Serum transaminases AST (U/L)

Reference values 0 (control) 2 4 6 8 10

0.1–2.1 0.4 ⫾ 0.2 0.2 ⫾ 0.1 0.3 ⫾ 0.1 0.3 ⫾ 0.1 0.2 ⫾ 0 0.2 ⫾ 0.1

12–192 71.2 ⫾ 3 25 ⫾ 3.1 86 ⫾ 4.9 87 ⫾ 5 61 ⫾ 9 68 ⫾ 3

54–234 176 ⫾ 8 205 ⫾ 9 129 ⫾ 3 151 ⫾ 7 124 ⫾ 10 166 ⫾ 6


Blood urea nitrogen (mg/dL)

Creatinine (mg/dL)

PBL (103/mm3)

PMNs (%)

Lymphocytes (%)

15–93 64 ⫾ 3 78 ⫾ 5 43 ⫾ 2.6 28 ⫾ 6 42 ⫾ 3 50 ⫾ 2

18–60 26 ⫾ 4 21 ⫾ 4.5 30 ⫾ 0.9 31 ⫾ 1.8 34 ⫾ 3 33 ⫾ 4

0.6–1.5 0.5 ⫾ 0.1 0.6 ⫾ 0 0.62 ⫾ 0.2 0.76 ⫾ 0.1 0.84 ⫾ 0.17 0.8 ⫾ 0.1

5–10 6.3 ⫾ 1.5 3.34 ⫾ 2.34 8.83 ⫾ 1.54 7.63 ⫾ 1.27 6.56 ⫾ 1.55 6.34 ⫾ 1.3

10.2–14.1 13.2 ⫾ 3 29.4 ⫾ 4 36.2 ⫾ 3 26 ⫾ 4 23 ⫾ 3 17 ⫾ 4.8

78.5–88.4 83 ⫾ 4 67 ⫾ 4 60 ⫾ 3 71 ⫾ 7 74 ⫾ 3 79 ⫾ 9

Peripheral blood samples were collected from animals by terminal cardiac puncture on days 2, 4, 6, 8, and 10 after administration of 30 mg/kg of docetaxel (equivalent to a human dose of 75 mg/m2). The various measurements were performed by Antec Diagnostics Inc. (Rockville, MD) using standard methods. Values are given as mean ⫾ SD of mean, n ⫽ 5. ALT ⫽ alanine aminotransferase (serum glutamic-pyruvic transaminase [SGPT]); AST ⫽ aspartate aminotransferase (serum glutamic oxaloacetic transaminase [SGOT]); PBL ⫽ peripheral blood leukocyte; PMNs ⫽ polymorphonuclear cells.

marrow suppression may be relatively short, regardless of the route of administration. The ability of DXT to rapidly and efficiently mobilize HSCs in healthy mice and its low toxicity profile compared to other chemotherapeutic agents currently used to mobilize HSCs suggest that there is a potential for use of DXT in HSC mobilization and a need for similar evaluation in a clinical trial. Studies are in progress in our laboratory to determine the functional capacity and immunocompetence of lymphocytes derived from DXTmobilized HSCs.




Acknowledgments This work was supported by a grant from Rhone-Poulenc Rorer, Collegeville, PA. We are grateful to Karen Creswell for flow cytometric analysis, Ann Murray and Tina Wilson for their veterinary technical assistance provided through the Lombardi Cancer Center animal core grant P30 CA-51008, and Carmela Matias for her excellent secretarial assistance. We also are grateful to Dr. Solveig G. Ericson, MD, Ph.D for reviewing this manuscript.

References 1. Keller G (1992) Hematopoietic stem cells. Curr Opin Immunol 4:133 2. Ogawa M (1993) Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844 3. Spangrude GJ, Smith L, Uchida N, Ikuta K, Heimfeld S, Friedman J, Weissman IL (1991) Mouse hematopoietic stem cells. Blood 78:1395 4. Sheridan WP, Begley GG, Turner CA, Szer T, To LB, Maber D, McGrath KM, Mortstyn G, Fox RM (1992) Effect of peripheral-blood progenitor cells mobilized by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy. Lancet 339:640 5. Negram RS, Kusnierz-Glaz CR, Still B, Schriber TR, Chao NF, Long GD, Hovie C, Hu WW, Horning SF, Brown RW, Blume KG, Strober S (1995) Transplantation of enriched and purged peripheral blood progenitor cells from a single apheresis product in patients with nonhodgkin’s lymphoma. Blood 85:3334 6. Verma UN, Yankelevich B, Hodgson J, Mazumder A (1997) Effect of the in vitro priming for peripheral blood stem cells (PBSC) mobiliza-



12. 13.


15. 16. 17.

tion on in vitro generation of cytotoxic effectors by IL-2 activation of PBSC in a murine model. Bone Marrow Transplant 19:265 Engelhardt M, Winkler J, Waller C, Lange W, Mertelsmann R, Henschler R (1997) Blood progenitor cell (BPC) mobilization studied in multiple myeloma, solid tumor, and non-hodgkin’s lymphoma patients after combination chemotherapy and G-CSF. Bone Marrow Transplant 19:529 Demire T, Rowley S, Bucker CD, Appelbaum FR, Lilleby K, Starb R, Bensinger WI (1995) Peripheral-blood stem cell collections after paclitaxel, cyclophosphamide, and recombinant human granulocyte colonystimulating factor in patients with breast cancer. J Clin Oncol 13:1714 Fennelly D, Schneider J, Spriggs D, Bengala C, Hakes T, Reich L, Barakat J, Curtin J, Moore MAS, Hoskins W, Norton L, Crown J (1995) Dose escalation of paclitaxel with high dose cyclophosphamide, with analysis of progenitor cell mobilization and hematologic support of advanced ovarian cancer patients receiving rapidly sequenced high-dose carboplatin/cyclophosphamide courses. J Clin Oncol 13:1160 Rajagopal C, Areman E, Verma UN, Meehan K, Cahill R, Frankel SR, Kotula P, Hancock S, Sacher R, Mazunder A (1995) Paclitaxel as a single agent is superior to cyclophosphamide for mobilizing CD34⫹ cells for stem cell transplantation. Blood 86:405a (abstr) Kaklamanis L, Leek R, Koukourakis M, Gatter KC, Harris AL (1995) Loss of transporter in antigen processing I transport protein and major histocompatibility complex class I molecules in metastatic versus primary breast cancer. Cancer Res 55:5191 Ogmundsdottir HM, Petursdottir I, Gudmundsdottir I (1995) Interactions between the immune system and breast cancer. Acta Oncol 34:647 Mizoguchi H, O’Shea JJ, Longo DL, Loeffler CM, McVicar DW, Ochoa AC (1992) Alteration in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795 Marie-Christine B, Guenard D, Guerittte-Voegelein F, Lavelle F (1991) Experimental antitumor activity of Taxotere (RP 56976, NSC 628503), a Taxol analogue. Cancer Res 51:4845 Israel R, Horwitz SB (1991) Studies with RP 56976 (Taxotere): a semisynthetic analogue of Taxol. J Natl Cancer Inst 83:288 Von Hoff DD (1997) The Taxoids: same roots, different drugs. Semin Oncol 24:S13-3 Valero V, Holmes FA, Walters RS, Theriault RL, Esparza L, Fraschini G, Fonseca GA, Bellet RE, Buzdar AU, Hartobagyi GN (1995) Phase II trial of docetaxel: a new, highly effective antineoplastic agent in the management of patients with anthracycline-resistant metastatic breast cancer. J Clin Oncol 13:2886

J.O. Ojeifo et al./Experimental Hematology 28 (2000) 451–459 18. Ravdin PM, Burris HA III, Cook G, Eisenberg P, Kane M, Bierman WA, Mortimer J, Genevois E, Bellet RE (1995) Phase II trial of docetaxel in advanced anthracycline- resistant or anthracenedione-resistant breast cancer. J Clin Oncol 13:2879 19. Chan S, Fredrichs K, Noel D, Pinter T, Van Belle S, Voroblof D, Duarte F, Yelle L, Germa JR, Bodrogi I, Murray E, Walpole E, et al, on hehalf of the international 303 study group (1997) A phase III study of docetaxel versus doxorubicin in patients with metastatic breast cancer who have failed an alkylating-containing regimen. Breast Cancer Res Treat 46:23 (abstr) 20. Neben S, Marcus K, Mauch P (1993) Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor. Blood 81:1960 21. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Stober W (eds.) (1993) Isolation and fractionation of mononuclear cell populations. In: Current protocols in immunology, vol. 1, suppl. 8. New York: John Wiley & Sons, Inc., p. 3.1.3 22. Stem Cell Technologies (1999) Colony assay of murine cells using methylcellulose- based medium. In: Instructional booklet manual version 2.0.0. Vancouver, Canada: Stem Cell Technologies, p. 6 23. Bradley TR, Hodgson GS (1979) Detection of primitive macrophage progenitor cells in mouse bone marrow. Blood 54:1446 24. Huang S, Testappen LW (1994) Lymphoid and myeloid differentiation of a single human CD34⫹, HLA-DR⫹, CD38⫺ hematopoietic stem cell. Blood 83:1515 25. Verma UN, Van den Blink B, Pilla R, Chawla J, Mazumder A, Herscowitz HB, Meehan KR (1999) Paclitaxel versus cyclophosphamide in peripheral blood stem cell mobilization: comparative studies in a murine model. Exp Hematol 27:553 26. Rajagopal C, Verma UN, Meehan K, Areman E, Bagg A, Cahill R, Mazumder A (1995) Il-2-activated peripheral blood stem cell (PBSC) transplantation in breast cancer patients: patients with metastatic disease generate less potent cytotoxic effectors. Proc ASCO 14:104 (abstr) 27. Areman EM, Mazumder A, Kotula PL, Verma UN, Rajagopal C, Hancock C, Guevarra C, Djahanmir M, Sacher RA, Meehan KR (1996) Hematopoietic potential of IL-2-cultured peripheral blood stem cells from breast cancer patients. Bone Marrow Transplant 18:521 28. Antman KH, Rawlings PA, Vaughan PW, Pelz CJ, Fay JW, Fields KK, Freytes CO, Gale RP, Hillner BE, Holland HK, Kenedy MJ, Klein JP, et al (1997) High-dose chemotherapy with autologous hematopoietic stemcell support for breast cancer in North America. J Clin Oncol 15:1870 29. Chabannon C, Cornetta K, Lotz J-P, Rosenfeld C, Shiomchik M, Yanovitch S, Marolleau J-P, Sledge G, Novakovitch G, Srour EF, Burtness B, Camerio J, et al (1998) High-dose chemotherapy followed by infusion of selected CD34⫹ peripheral blood cells in patients with poor-prognosis breast cancer: a randomized multicenter study. Br J Cancer 78:913 30. To LB, Shepherd KM, Haylock DN, Dyson PG, Charles P, Thorp DL, Dale BM, Dart GW, Roberts MM, Sage RE, Juttner CA (1990) Single high doses of cyclophosphamide enable the collection of high numbers of hematopoietic stem cells from the peripheral blood. Exp Hematol 18:442 31. Webb IJ, Eickhoff CE, Elias AD, Ayash LJ, Wheeler CA, Schwartz GN, Demetri GD, Anderson KC (1996) Kinetics of peripheral blood mononuclear cell mobilization with chemotherapy and/or granulocytecolony-stimulating factor: implications for timing and yield of hematopoietic progenitor cell collection. Transfusion 36:160 32. Cremer FW, Kiel K, Wallmeier M, Haas R, Goldschmidt H, Moos M


34. 35.






41. 42.





47. 48.


(1998) Leukapheresis products in multiple myeloma: lower tumor load after mobilization with cyclophosphamide plus granulocyte colony stimulating factor (G-CSf) compared with G-CSF alone. Exp Hematol 26:969 Talmadge JE, Reed EC, Kessinger A, Kuszynski CA, Perry GA, Gordy CL, Mills KC, Thomas ML, Pirruccello SJ, Letheby BA, Arneson MA, Jackson JD (1996) Immunologic attributes of cytokine mobilized peripheral blood stem cells and recovery following transplantation. Bone Marrow Transplant 17:101 Shea TC (1997) Mobilization of peripheral blood progenitor cells with paclitaxel-based chemotherapy. Semin Oncol 24(suppl. 2):S2-105 Chuang LT, Lotova E, Cook KR, Munkarah A, Morris M, Wharton JT (1994) Alteration of lymphocyte microtubule assembly, cytotoxicity, and activation by the anticancer drug Taxol. Cancer Res 54:1286 Braakhuis BJM, Hill BT, Dietel M, Kelland LR, Aapro MS, Zoli W, Lelieveld P (1994) In vitro antiproliferative activity of docetaxel (Taxotere), paclitaxel (Taxol), and cisplatin against human tumor and normal bone marrow cells. Anticancer Res 14:205 Munkarah A, Chuang L, Lotzova E, Cook K, Morris M, Wharton JT (1994) Comparative studies of Taxol and Taxotere on tumor growth and lymphocyte functions. Gynecol Oncol 55:211 Chaddock CF, Apperley JF, Wright EG, Healy LE, Bennett CA, Evans M, Grimsley PG, Gordon MY (1992) Circulating stem cells in mice treated with cyclophosphamide. Blood 80:264 Ogawa M, Shih JP, Katayama N (1994) Enrichment for primitive hematopoietic progenitors of marrow cells from 5-fluorouracil-treated mice and normal mice. Blood Cells 20:7 Moore T, Huang S, Terstappen LWMM, Bennett M, Kumar V (1994) Expression of CD43 on murine and human pluripotent hematopoietic stem cells. J Immunol 153:4978 Huang S, Testappen LWMM (1994) Analysis of bone marrow stem cells. Blood Cells 20:45 Rowlings PA, Bayly JL, Rawling CM, Juttner CA, To LB (1992) A comparison of peripheral blood stem cell mobilization after chemotherapy with cyclophosphamide as a single agent in doses of 4 g/m2 or 7 g/m2 in patients with advanced cancer. Aust N Z J Med 22:660 Kanfer EJ, McGuigan D, Samson D, Abboudi Z, Abrahamson G, Apperle JF, Chilcott S, Craddock C, Davis J, MacDonald C, Olavarria E, Philpott N, et al (1998) High-dose etoposide with granulocyte colonystimulating factor for mobilization of peripheral blood progenitor cells: efficacy and toxicity at three dose levels. Br J Cancer 78:928 Tampellini M, Filipski E, Liu XH, Lemaigre G, Li XM, Vrignaud P, Francois E, Bissery MC, Levi F (1998) Docetaxel chronopharmacology in mice. Cancer Res 58:3896 Fennelly DW, Aghajanian C, Shapiro F, O’Flaherty C, O’Connor K, Curtin JP, Crown JP, Hoskins WJ, Spriggs DR (1997) Dose escalation of paclitaxel with high-dose caboplatin using peripheral blood progenitor cell support in patients with advanced ovarian cancer. Semin Oncol 24(suppl. 2):S2-26 Gokhale PC, Newsome JT, Dritschilo A, Kasid U, Rahman A (1999) Toxicity, histopathologic and pharmacokinetic evaluation of liposomeencapsulated paclitaxel and Taxol in mice and rabbits. Proc AACR 40:417 (abstr) Bissery MC (1995) Preclinical pharmacology of docetaxel. Eur J Cancer 31A(suppl. 4):S1 Salminen E, Bergman M, Huhtala S, Ekholm E (1999) Docetaxel: standard recommended dose of 100 mg/m2 is effective but not feasible for some metastatic breast cancer patients heavily pretreated with chemotherapy—a phase II single-center study. J Clin Oncol 17:1127