Bone Marrow Cell Trafficking Analyzed by 89Zr-oxine Positron Emission Tomography in a Murine Transplantation Model

Abstract

Purpose: The success of hematopoietic stem cell transplantation (HSCT) depends on donor cell homing to the bone marrow. However, there is no reliable method of noninvasively monitoring the kinetics and distribution of transferred cells. Using zirconium-89 (⁸⁹Zr)-oxine cell labeling combined with PET imaging, we sought to visualize and quantify donor cell homing in a mouse bone marrow transplantation model.Experimental Design: The effect of ⁸⁹Zr-oxine labeling on bone marrow cell viability and differentiation was evaluated in vitro⁸⁹Zr-labeled bone marrow cells (2 × 10⁷ cells, 16.6 kBq/10⁶ cells) were transferred intravenously, and serial microPET images were obtained (n = 5). The effect of a CXCR4 inhibitor, plerixafor (5 mg/kg) and G-CSF (2.5 μg) on bone marrow homing and mobilization were examined (n = 4). Engraftment of the transferred ⁸⁹Zr-labeled cells was evaluated (n = 3).Results:⁸⁹Zr-oxine-labeled bone marrow cells showed delayed proliferation, but differentiated normally. Transferred bone marrow cells rapidly migrated to the bone marrow, spleen, and liver (n = 5). Approximately 36% of donor cells homed to the bone marrow within 4 hours, irrespective of prior bone marrow ablation. Inhibition of CXCR4 by plerixafor alone or with G-CSF significantly blocked the bone marrow homing (P < 0.0001, vs. nontreated, at 2 hours), confirming a crucial role of the CXCR4-CXCL12 system. Mobilization of approximately 0.64% of pretransplanted bone marrow cells induced a 3.8-fold increase of circulating bone marrow cells. ⁸⁹Zr-labeled donor cells engrafted as well as nonlabeled cells.Conclusions:⁸⁹Zr-oxine PET imaging reveals rapid bone marrow homing of transferred bone marrow cells without impairment of their stem cell functions, and thus, could provide useful information for optimizing HSCT. Clin Cancer Res; 23(11); 2759-68. ©2016 AACR.

Figure 1. ⁸⁹Zr-oxine labeling does not alter cellular phenotype and survival

A. ⁸⁹Zr-labeling (13.7–22.2 kBq/10⁶ cells) did not alter the expression of lineage marker in BM cells or Sca-1 and CD117 expression of lineage marker⁻ cells. Representative flow cytometry data of 3 independent experiments and the average of all data (white: control, black: ⁸⁹Zr-labeled) are shown. Approximately 4.2% of lineage negative cells were HSCs expressing both sca-1 and CD117 (n=6). B. BM cells not labeled (open circle) and labeled at 13.7 (filled circle) and 28.8 (cross) kBq/10⁶ cells were cultured without exogenous cytokines. Live cell number decreased in a similar manner in all the groups (n=3, 13.7 kBq/10⁶ cellls: p=0.0156 and 0.0016 at day 1 and 2, 28.8 kBq.10⁶ cells : p<0.0001, p=0.0186 and 0.0011 on day 2, 3 and 4, vs control). C. Cell associated ⁸⁹Zr activity, with decay correction, showed slight decrease at the early time points and then almost plateaued (n=3). No significant difference was observed between the two labeling doses. D. The ⁸⁹Zr-labeled cells (13.7 kBq/10⁶ cells, black) showed slightly higher fraction of apoptotic and/or necrotic cells (i.e. annexin V⁺ and/or PI⁺ cells) by flow cytometry analysis compared to non-labeled control (white) on day 2, but the difference was not significant (70–83% of total cells, n=3). E. Expression of Ki67 in live cells on day 2 was negligible (n=3).

Figure 2. ⁸⁹Zr-oxine-labeled BM cells retain differentiation function

A. BM cells were labeled with ⁸⁹Zr at 10.9 (8.14–13.7, filled circle) and 28.5 (28.1–28.8, cross) kBq/10⁶ cells and cultured with SCF, FLT3-L and TPO (100 ng/ml each, n=6). The cells labeled at the lower dose slightly proliferated after day 4, while the cells labeled at the higher dose decreased in number (10.9 kBq/10⁶ cells vs control (open circle): P = 0.0305, 0.0015 and 0.0001, 28.1 kBq/10⁶ cells vs control: P = 0.0068, 0.0002, and 0.00001, at 2, 4, and 7 days, respectively). B. Decay corrected specific activity of the ⁸⁹Zr-oxine labeled BM cells decreased in the cells labeled with the lower dose, reflecting the cellular proliferation, but remained constant in cells labeled with the higher dose (n=6) (P = 0.00197, 0.00035 at day 4 and 7, respectively). C. Higher fractions of annexin V⁺ and/or PI⁺ cells were detected by flow cytometry in the ⁸⁹Zr-labeled cells (13.7 kBq/10⁶ cells, black, n=3) on day 4 and 7 than the non-labeled cells (white). D. The labeled cells proliferated as indicated by Ki67 staining (n=3). E. BM cells were labeled with ⁸⁹Zr at 13.7 (filled circle) or 28.8 (cross) kBq/10⁶ cells and cultured with 20 ng/ml GM-CSF (n=6). Compared to the non-labeled control (open circle), the labeled cells showed delayed increase in cell number (13.7 kBq/10⁶ cellls: p<0.0001 on day 6 and 8, p=0.0168 on day 12, 28.8 kBq.10⁶ cells: p=0.0121 and 0.0001 on day 3 and 12, p<0.0001 on day 6, 8 and 10, vs control). F. Decay corrected cell associated ⁸⁹Zr activity gradually decreased in cells labeled at the lower dose, but was plateaued with the higher dose until around day 8 (p=0.0177, 0.0008 and 0.0006 on day 8, 10, 12, 13.7 vs 28.8 kBq/10⁶ cells). G. Higher fraction of annexin V⁺ and/or PI⁺ cells were detected in the ⁸⁹Zr-labeled cells (13.7 kBq/10⁶ cells, black) on day 6 and in the non-labeled cells (white) on day 8 (p=0.0176 on day 6 and 8). H. Proliferation of the cells indicated by the expression of Ki67 was delayed in the labeled cells reaching the peak on day 8, whereas the peak in non-labled cells was observed on day 6 (p=0.0041 and p<0.0001 on day 8 and 10). I. ⁸⁹Zr-oxine-labeled BM cells (13.7–22.2 kBq/10⁶ cells, black) differentiated to DCs (CD11c⁺) comparable to the non-labeled cells (white) after a 10 day-culture in GM-CSF (20 ng/ml). Around 40% of CD11c+ cells expressed a maturation marker CD86 (n=6). J. When cultured with IL-15 (25 nM), ⁸⁹Zr-labeled BM cells (black) differentiated to NK1.1⁺ NK/NK-T cells in 10 days similar to the non-labeled cells (white) (n=6). K. Colony forming cell assay of non-labeled (white) and labeled (14.8 kBq/10⁶ cells, black) BM cells showed no significant difference in the number of various hematopoietic cell colonies on day10 (n=3, BFU-E: burst forming unit-erythroid, CFU-G: colony forming unit-granulocyte, CFU-GM: colony forming unit-granulocyte macrophage, CFU-M: colony forming unit-macrophage, CFU-GEMM; Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte).

Figure 3. Serial microPET/CT imaging reveals rapid trafficking of ⁸⁹Zr-oxine-labeled BM cells through the lungs to the BM, spleen and liver

A. ⁸⁹Zr-oxine-labeled BM cells (2×10⁷ cells at 16.6 kBq/10⁶ cells) were transferred via the tail vein to non-BM ablated (A) and BM ablated (9.5 Gy whole-body irradiation) (B) mice (n=5). PET images indicate that immediately after the injection (0 h), cells started to migrate to the lungs, liver, spleen and the BM (%ID/mL: percent of injected dose per mL of tissue). C. Quantitative analysis of the microPET/CT images revealed similar migration kinetics to the BM, lungs, liver and spleen between non-irradiated and irradiated recipient mice (n=3, blue squire: lungs, green triangle: liver, purple triangle: spleen, red circle: bone marrow). The example of quantitated area/regions of interest set on the images are indicated in the upper left (bone marrow) and right (lungs, liver, and spleen).

Figure 4. ⁸⁹Zr-oxine microPET/CT imaging demonstrates CXCR4 dependent BM homing of transferred BM cells

Groups of mice received ⁸⁹Zr-oxine-labeled BM cells (2×10⁷ cells at 16.6 kBq/10⁶ cells)); A. Control mice (n=4), B. mice receiving plerixafor (5 mg/kg) i.v. 15 min before the BM cell transfer, and C. mice receiving plerixafor (5 mg/kg) and G-CSF (2.5ug) i.v. 15 min before the BM transfer. All recipient mice received a lethal whole-body irradiation at 9.5 Gy 24h prior to cell transfer. PET images indicates that in comparison to the control, plerixafor alone or plerixafor/G-CSF inhibited the initial donor cell migration to the BM. Addition of G-CSF to plerixafor sustained the suppression of BM homing (%ID/mL: percent of injected dose per mL of tissue, Representative images of 4 experiments). D. Quantification of spinal ⁸⁹Zr activity demonstrated inhibition of BM homing with plerixafor at the 0–2 h time points and prolonged inhibition with plerixafor/G-CSF. An example of regions of interest set on the images are shown at the right. Asterisks indicate statistical significance within each time point. *:P < 0.05, **:P < 0.01, ***:P < 0.001, ****:P< 0.0001. White bar: control, patterned bar: plerixafor, and black bar: plerixafor/G-CSF.

Figure 5. ⁸⁹Zr-labeled GFP⁺ pre-transplanted BM cells are mobilized into the blood by plerixafor/G-CSF treatment

Two groups of mice were pre-transplanted with ⁸⁹Zr-labeled GFP⁺ BM cells (2×10⁷ cells at 37–111 kBq/10⁶ cells). One group of mice received i.v. injections of plerixafor/G-CSF at 3 h and 1 d and blood was collected 2h after the second mobilization treatment from both experimental and control groups. A. ⁸⁹Zr activity associated with cells in the blood of experimental group (black) increased by 3.8-fold compared to control (white) (n=4; P = 0.0009). B. Flow cytometry analysis confirmed that BM cell mobilization increased GFP⁺ cell fraction in the circulation (n=4, white: control, black: mobilized).

Figure 6. Donor BM cells labeled with ⁸⁹Zr-oxine engraft and differentiate into mature immune cells in the recipient mice

Flow cytometry analysis of BM cells and splenocytes was performed 10 weeks after the BM transfer (n=3). A. CD45.2⁺ donor BM cells, with or without ⁸⁹Zr-labeling, reconstituted the BM of the hosts (CD45.1⁺) that received BM ablation prior to the transplantation, but not that of the non-BM ablated hosts. B. ⁸⁹Zr-labeling did not impair the engraftment of CD45.2⁺lineage marker⁻sca-1⁺CD117⁻ hematopoietic stem/progenitor cells in the BM ablated hosts. C. Similarly, CD45.2⁺ donor derived cells reconstituted the spleen only in the BM ablated hosts. D. Analysis of splenocytes revealed that donor BM cells had differentiated into DCs (CD11c⁺), B cells (B220⁺), NK cells (CD3⁻NK1.1⁺), T cells (CD3⁺NK1.1⁻) in BM ablated recipient mice. (white: non-labeled BM transferred to BM ablated host, black: ⁸⁹Zr-labeled BM transferred to BM ablated hosts),