Insights into the biology of mobilized hematopoietic stem/progenitor cells through innovative treatment schedules of the CXCR4 antagonist AMD3100

Abstract

Objective: The CXCR4 antagonist AMD3100 mobilizes hematopoietic stem/progenitor cells (HSPC) in several species. Few data are available on the biology of HSPC mobilized with AMD3100 as single agent. To further study the kinetics and properties of AMD3100-mobilized HSPC, and to explore the size of mobilizable pools of HSPC targeted by AMD3100, we studied the effect of a continuous infusion scheme with saturating doses of AMD3100 [AMDi].

Materials and methods: Using established procedures, we evaluated mice mobilized with AMD3100, or those transplanted with AMD3100-mobilized HSPC.

Results: Relative to single-bolus AMD3100 [AMDb], the number of circulating CFU-C or CRU was dramatically higher after [AMDi]. During [AMDi], circulating CFU-C accumulated slowly, but after its discontinuation, CFU-C disappeared rapidly. Compared to bone marrow (BM)-c-kit(+) cells, AMD3100-mobilized (AMDb or AMDi) c-kit(+) cells showed reduced expression of several cytoadhesion molecules, similar to granulocyte colony-stimulating factor-mobilized c-kit(+) cells. In contrast to the latter, expression of CXCR4 and CD26 were not reduced on AMD3100-mobilized c-kit(+) cells. BM homing of [AMDi]-mobilized CFU-C was >50% increased over normal BM-CFU-C. Hematopoietic recovery after transplantation of [AMDi]-mobilized peripheral blood was comparable to that of continuous infusion granulocyte colony-stimulating factor-mobilized peripheral blood. AMD3100-mobilized HSPC were predominantly in G(0), and partial bromodeoxyuridine-labeling experiments documented underrepresentation of labeled cells (<5%) among [AMDb]-mobilized c-kit(+) cells, suggesting that cycling cells in BM, or those that recently completed cell cycle, are not targeted for mobilization by AMD3100.

Conclusions: Our data demonstrate that [AMDi] is an efficacious mobilization scheme fully supporting transplantation demands and expands previous knowledge about properties and size of AMD3100-sensitive BM-HSPC pools.

Fig. 1. Mobilization of progenitor cells and stem cells by AMD3100

A: Mobilization by AMD3100 bolus injection: Mobilization kinetics (left): Mice received i.p. bolus injections of AMD3100 (100 μg) at 0 hrs. CFU-C were enumerated by colony assay at the indicated times. The number of circulating CFU-C peaked after 1 hour and returned to normal within 4 hours. Asterisks indicate statistically significant differences compared to baseline. CFU-C after 1 hour were statistically significantly higher than at all other time points. Dose-response curve (right): Mice received i.p. injections of the indicated dose of AMD3100. CFU-C were enumerated by colony assay one hour after injection. Mobilization peaked after a dose of 100 μg, equivalent to ca. 4 mg/kg. Asterisks indicate statistically significant differences compared to baseline. B: Response to repeated AMD3100 stimulation: Mice received 100 μg boli of AMD3100 ([AMD b]) every 12 hours for 6 successive days (left) or at 0 and 4 hours (nadir according to Fig 1A, right), as indicated by arrows. At the time points indicated by grey bars, circulating CFU-C were enumerated (mean+SEM). Mobilization of the same magnitude was observed at all time points, without accumulation of circulating CFU-C. (All times significantly greater than baseline, as indicated by asterisks) C: Continuous infusion of AMD3100 [AMD i] dramatically improves mobilization efficiency; kinetics of mobilization with [AMD i]: During continuous infusion of AMD3100, CFU-C accumulated relatively slowly, achieving only half-maximal numbers after 24 hours, while clearance from blood after discontinuation of infusion was very rapid. This observation illustrates the previously demonstrated short biologic half-life of AMD3100 in vivo. Asterisks indicate CFU-C numbers in excess of baseline. D: Efficacious mobilization by continuous infusion of AMD3100: CFU-C contents in BM and MPB: Mobilization with [AMD i] was equivalent to approximately 3-4% of the total CFU-C content of a mouse and was not accompanied by a significant reduction of CFU-C content in femurs. G-CSF infusion primed BM [G-CSF i] contained reduced numbers of CFU-C, fewer than can be explained by mobilization alone. The highly efficacious mobilization with G-CSF infusion + AMD3100 bolus [G-CSF i + AMD b] resulted in mobilization of more than two femur equivalents and was accompanied by an additional reduction in CFU-C contents in BM relative to G-CSF infusion alone. E: Quantities administered during AMD3100 infusion are saturating: Mice received [AMD b], [AMD i], [AMD i] followed by [AMD b] on day 5, or [G-CSF i]. Where applicable, circulating CFU-C were enumerated 1 hour after [AMD b]. After [AMD i], CFU-C/mL were 10-fold increased over [AMD b] (p<0.001), and 30- to 50-fold increased over baseline, to numbers which compare favorably to those achieved with G-CSF. Administration of [AMD b] after [AMD i] did not result in additional mobilization compared to [AMD i], suggesting that satiating doses of AMD3100 were administered. Asterisks indicate statistically significantly increased CFU-C of [AMD i] relative to [AMD b] and [G-CSF] relative to [AMD i]. F: Mobilization of CRU: CRU frequency in [AMD b] or [AMD i] mobilized blood was compared by limiting dilution transplantation assays. Groups of 8-10 lethally irradiated mice (CD45.2) received transplants of 250,000 normal BM cells (CD45.2) plus the indicated volume of mobilized blood (CD45.1). Engraftment levels of CD45.1 cells were quantified 16 weeks after transplantation; mice with ≥1% CD45.1 cells were considered positive for mobilized blood engraftment. Dose dependent engraftment derived from mobilized blood was observed, (AMD b = black diamonds, AMD i = grey squares) (left). Mean CRU frequency was 77/mL for [AMD b] blood, 273/mL for [AMD i] blood (right) (mean+S.D.). This difference was statistically significant.

Fig. 2. Effect of AMD3100 on HSPC in vitro migration and adhesion; effect of AMD3100 infusion on myelosuppression by 5-Fluorouracil

A: c-kit+ BM cells were isolated and incubated in vitro with AMD3100 (100 μg/mL) or medium, followed by transwell migration towards medium (grey bars) or SDF-1 (100 ng/mL, black bars) for three hours. As previously shown, AMD3100 incubation almost completely blocked migration towards SDF-1 (asterisk indicate statistically significant differences compared to not AMD3100 treated control cells). B: [AMD i] mobilized peripheral blood cells (grey) or steady-state BM cells (black) were allowed to migrate towards SDF-1 (100 ng/mL) for four hours, followed by incubation of migrated cells in colony assays. The fraction of migrated CFU-C was similar for [AMD i] MPB and ssBM. C: c-kit+ cells were treated in vitro with AMD3100 (100 μg/mL) for 20 minutes, followed by incubation on RetroNectin for 2 hours. Adhering cells were quantified by Crystal Violet staining. There was no difference in adhesion to RetroNectin between AMD3100 treated and untreated c-kit+ cells. D-F: No evidence for hematopoietic suppression by AMD3100-infusion: Mice received single injections of 250 mg/kg of 5-fluorouracil, followed by continuous infusion of [AMD i] or [PBS/BSA] for a total of 8 days, starting 24 hours after 5-FU. Hematopoietic regeneration was monitored. The recovery of hematocrit (Hct, D), white cell count (WBC, E) or platelet count (Plt, F) was no different in control and AMD3100 treated animals.

Fig. 3. BM homing and engraftment of AMD3100-mobilized HSPC in lethally irradiated recipients

A:Recipients received lethal irradiation conditioning, followed by transplantation of [AMD i] or [G-CSF i] mobilized peripheral blood cells ([AMD i MPB] or [G-CSF i MPB]), of BM cells from these mobilized animals ([AMD i BM] or [G-CSF i BM]), or of BM cells from untreated controls ([ssBM]). 20 hours after transplantation, BM homing was quantified relative to the number of injected CFU-C. Homing of [AMD i MPB] or [G-CSF i MPB] was significantly superior to that of [ssBM], while homing of [AMD i BM] or [G-CSF i BM] was significantly lower than that of [ssBM]. B, C, D: Engraftment kinetics of mobilized HSPC: Lethally irradiated hosts received transplants of 100 μl AMD3100 infusion-mobilized [AMD i] or G-CSF-mobilized [G-CSF] peripheral blood. CBC were followed as indicated. All recipients survived with this cell dose. Engraftment kinetics of erythrocytes (B) and leukocytes (C) were the same for both groups, while normalization of platelet counts was delayed in [AMD i MPB] recipients (asterisks) (D).

Fig. 4. CFU-C mobilization by AMD3100 in different mouse models

A: CFU-C mobilization by AMD3100 during hematopoietic stress: 8 days after transplantation of 5×10E6 normal BM c-kit+ cells, mice received injections of PBS/BSA or [AMD b]. [AMD b] treated mice responded with significant CFU-C mobilization of at least similar magnitude as during steady-state. This difference from baseline was statistically significant. B: CFU-C mobilization by AMD3100 in G-CSFR-/- mice: Untreated G-CSFR-/-mice (steady-state, left), or G-CSFR-/- mice treated 5 days prior with Cyclophosphamide (right), responded to [AMD b] with significant CFU-C mobilization from very low baselines, as indicated by asterisks. C: CFU-C mobilization by AMD3100 in mice treated with a Gi protein inhibitor: As described, Pertussis Toxin (PTX) mobilizes CFU-C. [AMD b] did not mobilize additional CFU-C, indicating that PTX and AMD3100 mobilize by the same molecular mechanism. D: CFU-C mobilization by AMD3100 in splenectomized mice: Splenectomized mice showed the expected elevation in baseline CFU-C. Mobilization by [AMD b, left] or [AMD i, right] was at least as efficient as in not splenectomized animals, indicating that AMD3100 mobilized cells do not originate in the spleen.

Fig. 5. Cell cycle status of HSPC mobilized by AMD3100

A-F: AMD3100 mobilized HSPC are quiescent: Five days after Cyclophosphamide-treatment, G-CSFR-/- mice received [AMD b] followed by exsanguination 1 hour later (A), or untreated wild-type mice were exsanguinated after a 5-day course of [AMD i] (B). BM cells from similarly treated (PBSBSA injection or sham-pumps, respectively) donors were used for comparison in each case. c-kit+ cells were isolated by flow sorting, and cell cycle status was analyzed by Acridine Orange staining. MPB c-kit+ cells were overwhelmingly in G0, in contrast to BM c-kit+ cells. Asterisks indicate statistical significance of the difference relative to BM. C,D show a representative DNA/RNA dot plot, E,F a representative DNA histogram from one of the samples represented in D, with mobilized blood c-kit+ cells on the left (C,E), steady-state BM c-kit+ cells on the right (D,F). G: Mice pulsed with BrdU 2, 23 or 47 hours before mobilization, or not pulsed with BrdU, received [AMD b] followed by exsanguination one hour later. While 3, 24 or 48 hours after BrdU injection 20->40% of BM c-kit+ cells were labeled with BrdU, i.e. had entered S-phase, the frequency of BrdU labeled cells among mobilized peripheral blood c-kit+ cells was very low. Asterisks indicate statistically significant differences to BM labeling.