Molecular imaging with the PET tracer 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) allows assessment of the proliferative state of organs in vivo. Although used primarily in the oncology clinic, it can also shed light on the proliferation of other tissues, as demonstrated here for monitoring hematopoietic organs that recover after myelosuppressive chemotherapy. In the NMRI nude mouse model, we observed up to a 4.5-fold increase in [18F]FLT uptake in bone marrow and spleen on days 2, 3, and 5 after treatment with gemcitabine, a chemotherapeutic agent that is powerfully myelosuppressive in the model. Specifically, we observed (i) a reduced spleen weight; (ii) reduced bone marrow cell counts and proliferation (BrdUrd flow cytometry, spleen IHC; 6 hours/day 1); and (iii) reduced leukocytes in peripheral blood (day 5). In conclusion, our results show how [18F]FLT PET can provide a powerful tool to noninvasively visualize the proliferative status of hematopoietic organs after myelosuppressive therapy. Cancer Res; 76(24); 7089–95. ©2016 AACR.

Chemotherapy of malignant cancers is frequently accompanied by several side effects. One of the most common adverse effects is the impact of the chemotherapeutic agent on bone marrow cells, subsequently affecting the numbers of white blood cells (leukopenia or neutropenia; ref. 1), platelets (thrombocytopenia; ref. 2), and red blood cells (anemia; ref. 3). For instance, neutropenia may result in secondary infections, ultimately leading to the death of the patient. It is well recognized that the hematopoietic organs are able to regenerate after insult due to the presence of hematopoietic stem cells. Therefore, it is of importance, not to employ chemo- or radiotherapy in the phase of recovery of the hematopoietic organs, as this might result in the destruction of hematopoietic stem cells with permanent impact on the blood composition. A range of models exists that describes the relation of the application of a chemotherapeutic agent and the impact on bone marrow and other tissues (4). To know this relation is of crucial importance for dosing and timing of a chemotherapeutic drug. Unfortunately, changes in the blood cellular components, which are easily measurable in clinical routine, are not directly timely linked to changes in the proliferation of hematopoietic stem cells.

PET is an attractive tool to noninvasively and longitudinally visualize molecular changes within a living organism. It is widely used in the fields of oncology, cardiology, and neuroscience. Depending on the radiotracer used, it can monitor specific molecular events. 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a thymidine analogue that is transported into cells primarily via the human equilibrative nucleoside transporter 1 (hENT1; ref. 5). Within a cell, it is phosphorylated by thymidine kinase 1 (TK1), which results in trapping of the tracer. Hence, accumulation of [18F]FLT resembles the thymidine salvage pathway and therefore proliferation. An alternative thymidine-to-DNA pathway is the de novo synthesis pathway, with the key enzyme thymidylate synthase (TS). In various studies, [18F]FLT has been proven to be useful in monitoring response to anticancer treatments (6, 7). There are only limited reports on [18F]FLT PET imaging concentrating on other proliferative tissues than tumors like the hematopoietic compartments. One study showed that [18F]FLT PET can visualize bone marrow recovery after bone marrow transplantation in rats (8), and Ye and colleagues described that [18F]FLT PET is capable of imaging the proliferative state of cells in aortic plaques and hematopoietic organs in mice, rabbit, and men (9).

Here, we employed [18F]FLT PET to noninvasively and longitudinally visualize the recovery of hematopoietic organs after chemotherapeutic treatment with gemcitabine in a mouse model.

Animal model

Animal procedures were performed within the multi-centered QuIC-ConCePT study in accordance with the German Laws for Animal Protection and were approved by the animal care committee of the local government (North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection). During the experiments, general health and body weight of the mice were monitored. 8- to 12-week-old female NMRI nude mice (Janvier Labs) were used for the experiments. A total of 47 mice were employed in this study, which were initially used to monitor the response of subcutaneous lung cancer xenografts to chemotherapy (10). Mice were treated with intraperitoneal injections of 100 mg/kg gemcitabine in 100 μL 0.9% NaCl or 0.9% NaCl vehicle control on days 0 and 3. Analyses of day 3 samples were performed after a single injection on day 0. PET imaging was conducted according to the experimental schedule depicted in Supplementary Fig. S1. When performing bromodeoxyuridine (BrdUrd) flow cytometry or IHC, 50 mg/kg BrdUrd (5-bromo-2′-deoxyuridine, B5002, Sigma-Aldrich) was injected intraperitoneally 6 to 8 hours prior to cervical dislocation of the animal. Analysis of heparinized blood samples was performed with a Vet ABC Animal Blood Counter (scil).

PET imaging

Synthesis of [18F]FLT was performed as described previously (11). Emission scans were acquired with a quadHIDAC dedicated small-animal PET camera (Oxford Positron Systems; ref. 12) 70 to 90 minutes after injection of 10 MBq of the tracer. Mice were anesthetized with isoflurane inhalation (2% in oxygen), and temperature was maintained by using a heating pad. A multimodal animal bed was used that was transferred to a CT (Siemens Inveon) or a magnetic resonance (MR) tomograph (9.4 T, Biospec, Bruker, T2w imaging). This allowed coregistration with anatomic data with the help of fiducial markers.

After OPL-EM reconstruction (13), PET images were fused with either CT or MR images with the software Inveon Research Workspace 3.0 (Siemens Medical Solutions). Volumes of interest (VOI) were drawn on manubrium sterni, femur, spleen, muscle, and liver guided by CT/MR images. [18F]FLT accumulation was determined as %IDmax/mL. We also calculated %IDmean/mL, SUVmax, SUVmean, ratio to muscle [(%IDmax/mL)/(%IDmeanmuscle/mL)], and ratio to liver [(%IDmax/mL)/(%IDmeanliver/mL)] to confirm that the mode of analysis does not alter the results. The respective data are depicted in Supplementary Table S1.

IHC

Spleen and femur were excised and fixed in 4% paraformaldehyde. The bones underwent a decalcification procedure (incubation in 20% EDTA for 4 weeks at 37°C) prior to embedding in paraffin. Five-micron sections were cut from paraffin-embedded tissue and stained overnight at 4°C for either BrdUrd (AbD Serotec, OBT0030G, 1:100) or Ki67 (Abcam, ab16667, 1:100). Antibody binding was visualized with a respective secondary antibody labeled with 3,3′-diaminobenzidine (DAB). Images were acquired with a Nikon Eclipse 90i microscope and the NIS-Elements software package (Nikon). ImageJ (NIH, Bethesda, MD) was used for quantification of the specifically stained area after color deconvolution. The average of the DAB-positive area of 5 to 8 images at 20× resolution per section was determined.

Bone marrow cell isolation and BrdUrd flow cytometry

Bone marrow cells were isolated by flushing the cells of the femur in 5 mL PBS/2% BSA. The total cell number was determined by counting the cells in a Z2 coulter particle count and size analyzer (Beckman Coulter). Thereafter, cells were pelleted by centrifugation, and erythrocytes were lysed by incubation for 5 minutes with AKC lysis buffer (0.15% NH4Cl, 0.1 mol/L EDTA, 1 mmol/L KHCO3, pH 7.4). After addition of 5-mL washing buffer (PBS/2% BSA), cells were centrifuged again. The cell pellet was then fixed in 70% cold ethanol in PBS and stored at −20°C. After washing, DNA was denatured by incubation with 400 μL 2 mol/L HCl for 20 minutes. The cells were washed again and incubation for 2 minutes with 600 μL 0.1 mol/L sodium tetraborate (pH 8.5) ensured a neutralization of the pH. After an additional washing step, the cell pellet was incubated with 10 μL of anti-BrdUrd antibody (BD Pharmingen, #556028) for 1 hour. Propidium iodide (250 μL, 50 μg/mL) was added 1 hour before measuring the cells in a flow cytometer (FACSCalibur, Becton Dickinson). All steps were performed at room temperature and were followed by centrifugation (400 × g, 5 minutes).

Statistical analysis

Box plots were used to visualize the median and the 25th and 75th percentiles. Whiskers represent minimum and maximum values. Mean and SD were calculated as well and can be found in the Supplementary Table S2, including numbers of samples analyzed. P < 0.05 calculated by Mann–Whitney rank sum test (Sigma Plot 13.0) was considered statistically significant.

When imaging tumor-bearing nude mice after gemcitabine therapy by [18F]FLT PET, we noticed an atypical pattern of [18F]FLT distribution within the body with clear accumulation in bone marrow and spleen (Fig. 1). In untreated or vehicle-treated mice, these organs could not be discriminated from background, whereas 2 to 3 days after a single dose of gemcitabine, or day 5 of gemcitabine therapy (i.e., 2 days after two doses of gemcitabine on days 0 and 3), these organs clearly showed an enhanced uptake of [18F]FLT (e.g., [18F]FLT uptake in femur in %IDmax/mL: baseline: 3.52 ± 0.59, day 2: 6.25 ± 2.35†††,***, day 3: 15.46 ± 6.58†††,*, day 5: 9.99 ± 1.12††; ††, P < 0.05; †††, P < 0.01 relative to baseline; *, P < 0.05; ***, P < 0.001 relative to NaCl; see Supplementary Table S2 for all numbers).

Figure 1.

PET imaging of gemcitabine-treated mice revealed increased [18F]FLT uptake in hematopoietic organs. Maximum intensity projections of emission scans acquired 70 to 90 minutes after tracer injection showed that the tracer accumulated in bone marrow and spleen starting from about day 2 after drug administration. Quantification verified that this increased uptake pattern is reproducible. The black dots adjacent to the animals represent the coregistration landmarks. Subcutaneous tumors were also visible on the PET images. bl, baseline; d, day; white, NaCl control; gray, gemcitabine; *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to NaCl; †, P < 0.05; ††, P < 0.01; †††, P < 0.001 relative to baseline.

Figure 1.

PET imaging of gemcitabine-treated mice revealed increased [18F]FLT uptake in hematopoietic organs. Maximum intensity projections of emission scans acquired 70 to 90 minutes after tracer injection showed that the tracer accumulated in bone marrow and spleen starting from about day 2 after drug administration. Quantification verified that this increased uptake pattern is reproducible. The black dots adjacent to the animals represent the coregistration landmarks. Subcutaneous tumors were also visible on the PET images. bl, baseline; d, day; white, NaCl control; gray, gemcitabine; *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to NaCl; †, P < 0.05; ††, P < 0.01; †††, P < 0.001 relative to baseline.

Close modal

The bone marrow compartment and the spleen represent organs that are important for hematopoiesis, that is, the production of new blood cellular components. Many chemotherapeutic agents, including gemcitabine, affect hematopoietic compartments, as the cells of these tissues are highly proliferating. After a while, the hematopoietic organs recover, giving rise to new blood cells. We hypothesized that increased [18F]FLT uptake represents recovery of hematopoietic organs after initial myelosuppression.

To analyze whether gemcitabine exerts a myelosuppressive effect in our mouse model, we first performed blood count analysis (Fig. 2). We noticed a decrease of leukocytes (103 cells/mL NaCl vs. day 5 gemcitabine: white blood cells: 6.5 ± 3.7 vs. 2.8 ± 1.3*, monocytes: 0.53 ± 0.43 vs. 0.05 ± 0.05**, granulocytes: 3.82 ± 2.84 vs. 0.95 ± 0.26**; *, P < 0.05; **, P < 0.01 relative to NaCl), indicating that gemcitabine indeed affects the cells responsible for the formation of peripheral blood cellular components. Other blood cells were not affected at the time points investigated (see Supplementary Table S3).

Figure 2.

Hemogram analysis showed onset of low blood count after gemcitabine therapy. Heparinized blood was measured in a blood counter. d, day. *, P < 0.05; **, P < 0.01 relative to NaCl control.

Figure 2.

Hemogram analysis showed onset of low blood count after gemcitabine therapy. Heparinized blood was measured in a blood counter. d, day. *, P < 0.05; **, P < 0.01 relative to NaCl control.

Close modal

Spleen weights as well as bone marrow cell counts were reduced 6 hours or 1 day after gemcitabine (spleen-to-body weight: NaCl: 4.4 ± 1.3, 6-hour gemcitabine: 3.1 ± 0.6*, day 1 gemcitabine: 2.8 ± 0.7**; 106 bone marrow cells: NaCl: 20.2 ± 4.9, 6-hour gemcitabine: 14.9 ± 1.4*, day 1 gemcitabine: 7.8 ± 3.2***; *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to NaCl; Fig. 3), underlining that gemcitabine negatively effects these hematopoietic compartments. On days 2, 3, and 5 of gemcitabine therapy, spleen weight was undistinguishable from NaCl-treated controls, indicating recovery of this organ. The number of bone marrow cells was again decreased on day 5.

Figure 3.

Numbers of spleen and bone marrow cells were reduced early after gemcitabine therapy. Excised spleens were weighted, and bone marrow (bm) cells flushed from the femur were counted as indicators for myelosuppression. d, day. *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to NaCl control.

Figure 3.

Numbers of spleen and bone marrow cells were reduced early after gemcitabine therapy. Excised spleens were weighted, and bone marrow (bm) cells flushed from the femur were counted as indicators for myelosuppression. d, day. *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to NaCl control.

Close modal

Also, IHC of spleen tissue confirmed that gemcitabine exerted an effect on the cells, giving rise to blood cellular components: The proliferation was significantly impaired 6 hours and 1 day after drug administration (%Ki67-positive area: NaCl: 41.5 ± 9.8, 6-hour gemcitabine: 5.6 ± 3.0*, day 1 gemcitabine: 7.9 ± 4.4*; BrdUrd-positive area: NaCl: 36.6 ± 11.7, 6-hour gemcitabine: 18.8 ± 9.8*, day 1 gemcitabine: 5.5 ± 2.5**; *, P < 0.05; **, P < 0.01 relative to NaCl), and the proliferation receded to baseline levels on days 2, 3, and 5 (Fig. 4). Proliferation of the bone marrow cells of the femur after gemcitabine treatment was affected in a similar manner (see Supplementary Fig. S2).

Figure 4.

IHC of spleen tissue revealed that proliferation was severely impaired after gemcitabine application. The DAB-positive area was quantified to assess the proliferative status of the tissue. d, day. *, P < 0.05; **, P < 0.01 relative to NaCl control. Scale bar, 100 μm.

Figure 4.

IHC of spleen tissue revealed that proliferation was severely impaired after gemcitabine application. The DAB-positive area was quantified to assess the proliferative status of the tissue. d, day. *, P < 0.05; **, P < 0.01 relative to NaCl control. Scale bar, 100 μm.

Close modal

To more closely evaluate the proliferative status of the hematopoietic tissue upon gemcitabine therapy, we performed BrdUrd flow cytometry of the bone marrow cells to assess the number of cells in the S-phase and performed propidium iodide staining to determine the cellular DNA content (Fig. 5). Analogue to the IHC of BrdUrd, a significant drop in BrdUrd staining could be noted after 6 hours (%BrdUrd-positive cells: NaCl: 32.8 ± 4.7, 6-hour gemcitabine: 6.9 ± 4.2, P < 0.01). Furthermore, propidium iodide incorporation revealed that the DNA of bone marrow cells 6 hours after gemcitabine treatment appeared to be more fragmented, indicating apoptosis (%apoptotic cells: NaCl: 0.4 ± 0.4, 6-hour gemcitabine: 1.6 ± 0.8, P < 0.01). On days 2, 3, and 5 of gemcitabine therapy, BrdUrd incorporation exceeded that of vehicle-treated bone marrow cells up to 65%, implying an increase of proliferation.

Figure 5.

Flow cytometry confirmed decreased proliferation early after gemcitabine application, which was accompanied by increased cell death after 6 hours. BrdUrd-positive nuclei represent cells within the S-phase. Propidium iodide incorporation provides information on DNA content. Cells in the G2–M phase possess duplicated DNA. Six hours after gemcitabine, an increased population of cells with fragmented DNA could be noted representing cells in sub-G1, that is, cells undergoing cell death. d, day. **, P < 0.01 relative to NaCl control.

Figure 5.

Flow cytometry confirmed decreased proliferation early after gemcitabine application, which was accompanied by increased cell death after 6 hours. BrdUrd-positive nuclei represent cells within the S-phase. Propidium iodide incorporation provides information on DNA content. Cells in the G2–M phase possess duplicated DNA. Six hours after gemcitabine, an increased population of cells with fragmented DNA could be noted representing cells in sub-G1, that is, cells undergoing cell death. d, day. **, P < 0.01 relative to NaCl control.

Close modal

Conversely, proliferation of bone marrow cells as measured by flow cytometry on day 1 of therapy was comparable with that of vehicle-treated mice. This was in contradiction to the results obtained by the other experimental approaches employed, which demonstrated that also on day 1, the myelosuppressive effect of gemcitabine is very prominent. This discrepancy can probably be explained by the fact that flow cytometry does not account for the absolute number of cells, but for the number of positive cells relative to the number of cells present. As can be seen on the immunohistochemical sections (Supplementary Fig. S2), the absolute cell number is severely impaired, and the few remaining cells indeed are BrdUrd positive. Low bone marrow cell numbers could also be directly noted by counting of bone marrow cells (Fig. 3).

In summary, with a panel of different ex vivo analyses, we showed that bone marrow and spleen tissue were impaired early after gemcitabine administration, which was followed by a later recovery of these compartments.

Here, we demonstrate that gemcitabine exerts a myelosuppressive effect in a mouse model of cancer and that [18F]FLT PET is able to noninvasively image the subsequent recovery of the hematopoietic organs in vivo.

Our [18F]FLT PET imaging results showed substantial proliferation of the hematopoietic compartment if there was enough time for the tissue to recover after gemcitabine therapy (i.e., at least 2 days, Fig. 1). In a pilot study, we also observed the accumulation of [18F]FLT in bone marrow and spleen in mice 3 days after two doses of paclitaxel (see Supplementary Fig. S3). The latter finding implies that the preclinical observations made here for gemcitabine can also be transferred to other therapeutic approaches.

Our observations were corroborated by extensive ex vivo analyses of the proliferative status of the hematopoietic organs. The mass and proliferation of hematopoietic compartments was severely impaired early after application of a single dose of gemcitabine (after 6 hours and 1 day), and apoptosis was increased. Hence, we directly showed that gemcitabine exerts a myelosuppressive effect. After 5 days, we were able to show reduced leukocyte cell counts (Fig. 2). This indicator of myelosuppression, which is also frequently assessed in the clinic, is not directly temporally linked to degradation of the bone marrow compartment. Lymphocytes are extremely sensitive and die in interphase. Hence, in a mouse model of radiotherapy, their number was reduced within the first week of therapy. In that experimental approach, thrombocytopenia could be noted after 2 to 3 weeks and anemia after 2 to 3 months (14). Our results of reduced numbers of leukocytes are well in line with these data, whereas reduction of platelets or red blood cells can probably only be measured at later time points.

In our study, the hematopoietic organs recovered from day 2 onwards, as indicated by increased proliferation, spleen weight, and bone marrow cell number, respectively. However, it is interesting to note that the proliferation on days 2, 3, and 5 as determined by ex vivo measurements was comparable with the proliferation state at baseline. On the other hand, [18F]FLT PET imaging implied that the proliferation should be remarkably increased up to 4.5-fold in comparison with baseline. These results underline that [18F]FLT should not be taken as a proliferation tracer per se. It is well recognized that its uptake can be influenced by a variety of factors, like presence of competing thymidine (15, 16) or the balance of thymidine salvage and de novo pathway (17). It will be interesting to investigate whether the latter differs between hematopoietic organs in an equilibrium state and in a recovery state. Analysis of the expression of TK1, TS, and hENT1 at baseline and on day 3 after gemcitabine did not point to an involvement of these proteins in the observed changes in [18F]FLT uptake (Supplementary Fig. S4). However, it should be considered that expression levels are not necessarily directly related to enzyme or transporter activity.

Up to now, there are only limited possibilities to evaluate the proliferative state of hematopoietic organs. For instance, myelosuppressive effects of therapeutic approaches can be assessed by colony-forming cell assays, employing hematopoietic stem cells isolated from bone marrow, cord blood, or peripheral blood (18, 19). Even though good correlations to clinically observed myelosuppression can be observed with regards to drug dose, no clear timely relations can be concluded from these in vitro studies. Also, measurement of peripheral blood components does not provide temporal information about the status of hematopoietic compartments. Another approach to assess myelosuppression is histologic examination of bone marrow, requiring the collection of biopsies (20). Hence, there is need for a reliable, noninvasive method to assess the proliferation of hematopoietic compartments, especially when employing novel therapy approaches. Therefore, we propose that [18F]FLT PET should be considered and further explored in the clinical situation for that purpose.

There are a few clinical studies implying that [18F]FLT accumulation indeed reflects proliferation of hematopoietic organs. Reduced [18F]FLT uptake in the spinal bone marrow has been observed within the radiation field in head and neck cancer patients 5 days after 10 Gy radiotherapy (21) and in laryngeal carcinoma patients 1 month after 68 Gy radiotherapy (22). Moreover, [18F]FLT reduction was shown to be related to radiation dose in head and neck cancer patients undergoing one week of chemoradiation (23). These reductions in tracer uptake presumably reflect reduced proliferation and can assist in further therapy planning. Furthermore, after platinum-based chemotherapy, a substantial decrease in [18F]FLT uptake could be noted in week 2, which was followed by recovery in week 4, reflecting the absence of chemotherapy between these time points. Uptake in spleen after 4 weeks even exceeded uptake prior to therapy, implying substantial proliferation (24). Also, our data suggest that there needs to be a lag phase without therapy for the tissue to regenerate and accumulate high amounts of [18F]FLT. This time frame might differ substantially between the different therapy approaches. Most likely, the highest proliferation can be seen at the end of a treatment cycle. To further elucidate this issue, a retrospective systematic analysis of [18F]FLT PET scans acquired for the assessment of tumor treatment response with respect to alterations in the hematopoietic compartments could be performed. Ideally, these imaging findings should be related to other established laboratory parameters, like peripheral blood counts. Better knowledge of the status of hematopoietic organs can help in improved understanding of the side effects of the applied treatment and thus facilitate future treatment planning in taking hematologic toxicity into account. Doing so with established therapies will help to determine the value of [18F]FLT PET for monitoring the proliferative state of hematopoietic organs. This will enable [18F]FLT PET to be used for the visualization of myelosuppressive side effects of novel therapy approaches in a prospective manner.

Of note, even though there are clinical studies implying that [18F]FLT PET is capable of visualizing the proliferation of hematopoietic compartments, these studies did not validate their observations by ex vivo tissue analyses. On contrary, our study indicates that changes in [18F]FLT PET reflect changes in hematologic tissue biology. Therefore, our study provides the necessary link to employ [18F]FLT PET for noninvasive assessment of hematopoietic proliferation and recovery after therapy.

No potential conflicts of interest were disclosed.

Conception and design: S. Schelhaas, C. Müller-Tidow, A.H. Jacobs

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Schelhaas, A. Held, N. Bäumer

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Schelhaas, A. Held, N. Bäumer, T. Viel, S. Hermann, C. Müller-Tidow

Writing, review, and/or revision of the manuscript: S. Schelhaas, S. Hermann, C. Müller-Tidow, A.H. Jacobs

Study supervision: S. Schelhaas, A.H. Jacobs

We acknowledge Christine Bätza, Stefanie Bouma, Irmgard Hoppe, Nina Kreienkamp, Sarah Köster, Christa Möllmann, and Dirk Reinhardt for excellent technical assistance. We also acknowledge the Interdisciplinary Centre for Clinical Research (IZKF, core unit PIX), Münster, Germany for conducting the MR measurements, namely Prof. Dr. Cornelius Faber and Dr. Lydia Wachsmuth, and the PET measurements, namely Roman Priebe.

The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was also supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC1003 – CiM), University of Münster.

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