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. 2014 Dec 3;9(3):740–748. doi: 10.1016/j.molonc.2014.11.009

Quantifying initial cellular events of mouse radiation lymphomagenesis and its tumor prevention in vivo by positron emission tomography and magnetic resonance imaging

Sumitaka Hasegawa 1,✉, Yukie Morokoshi 1, Atsushi B Tsuji 1, Toshiaki Kokubo 2, Ichio Aoki 1, Takako Furukawa 1, Ming-Rong Zhang 1, Tsuneo Saga 1
PMCID: PMC5528708  PMID: 25510653

Abstract

Radiation‐induced thymic lymphoma (RITL) in mice is induced by fractionated whole‐body X‐irradiation (FX) and has served as a useful model for studying radiation carcinogenesis. In this model, the initial postirradiation cellular events in the thymus and bone marrow (BM) are critically important for tumorigenesis, and BM transplantation (BMT) prevents RITL. However, direct assessment of these events is so far restricted by the lack of noninvasive monitoring techniques. Here, we have developed positron emission tomography (PET) and magnetic resonance imaging (MRI) methods to quantify the events critical for RITL development and the effects of BMT in living animals. Apparent diffusion coefficients (ADCs) were calculated from diffusion‐weighted MRI to evaluate the changes in the BM of mice receiving FX. ADC values dramatically changed in the irradiated BM, corresponding to pathological findings of the irradiated BM, returning to normal levels following BMT sooner than with spontaneous recovery. PET with 4ʹ‐[methyl‐11C]thiothymidine, a novel tracer for cell proliferation, revealed that the irradiated thymus showed significantly higher tracer uptake than the unirradiated thymus 1 week after FX. Interestingly, its increased uptake was completely abolished by BMT, even with very few donor‐derived cells in the thymus. Thereafter, the thymus receiving BMT had significantly increased tracer uptake. These findings suggest that BMT first suppresses FX‐induced aberrant thymocyte proliferation and then accelerates thymic regeneration. This study demonstrates the feasibility of using PET and MRI for noninvasive monitoring of tumorigenic cellular processes in an animal model of radiation‐induced cancer.

Keywords: Radiation carcinogenesis, MRI, PET, Bone marrow, Thymus

Highlights

  • We quantify the in vivo process of mouse radiation‐induced lymphomagenesis by PET and MRI.

  • Diffusion‐weighted MRI reveals bone marrow changes after irradiation.

  • [11C]4DST‐PET reveals radiation‐induced thymic changes.

  • We directly assess the effects of bone marrow transplantation on tumor prevention for radiation lymphomagenesis.

1. Introduction

Radiation‐induced thymic lymphoma (RITL) in mice is one of the most intensively studied mouse models of radiation‐induced cancer and has unique tumor development features (Kominami and Niwa, 2006). In this model, fractionated whole‐body X‐irradiation (FX) induces thymic lymphoma in nearly 100% of C57BL/6 mice after a latency period of several hundred days from the first irradiation (Kaplan and Brown, 1952). FX causes cell death and tissue regeneration in the bone marrow (BM) and thymus in the initial stage of postirradiation, and eventually lymphoma arises in the regenerating thymus (Sado et al., 1991; Siegler et al., 1966). Postirradiation damage of the BM is assumed to be critical for the development of RITL because protection of the BM from irradiation prevents tumor development and BM transplantation (BMT) of unirradiated BM cells reduces the incidence of RITL (Humblet et al., 1989; Kaplan et al., 1953). Prevention of RITL by BMT critically depends on the timing of the BMT: BMT 1 day after FX rescues most of the affected mice from RITL, whereas BMT 30 days after FX has little or no effect on prevention (Sado et al., 1991). In addition, we and others recently reported that increased apoptosis in irradiated BM accelerated RITL development (Hasegawa et al., 2012; Labi et al., 2010; Michalak et al., 2010). In the thymus receiving FX, the cycles of cell attrition and its restoration are repeated (Kominami and Niwa, 2006). These findings strongly suggest that early cellular events of post‐FX in the thymus and BM are critical events, and probably the most important, for RITL. Assessment of the spatiotemporal in vivo dynamics of the events in living mice is therefore important for elucidating the pathogenesis of RITL, but these methods have yet to be developed.

Molecular imaging using positron emission tomography (PET) and magnetic resonance imaging (MRI) has become a powerful tool for studying biological processes (Massoud and Gambhir, 2003). These techniques have been applied to animal models of cancer (Hasegawa et al., 2011; Ishikawa et al., 2010; Nielsen et al., 2010; Saito et al., 2013). Diffusion‐weighted MRI (DWI) provides image contrast by measurement of water diffusion, and because the apparent diffusion coefficient (ADC) calculated from DWI has been shown to be a quantitative marker of cellularity, this imaging modality has been applied to the in vivo evaluation of cell death and regeneration (Chenevert et al., 2000; Chinnaiyan et al., 2000; Shang et al., 2011). Recently, PET with [18F]fluorothymidine (FLT), which is a thymidine analog used for evaluating cell proliferation, has been shown to be useful for assessing lymphocyte activation (Aarntzen et al., 2011). 4ʹ‐[methyl‐11C]thiothymidine ([11C]4DST, formally referred to as [methyl‐11C]S‐dThd), was developed as an alternative PET tracer for cell proliferation (Toyohara et al., 2008). [18F]FLT is hardly incorporated into DNA (McKinley et al., 2013), whereas [14C]4DST was rapidly incorporated into newly synthesized DNA in proliferative tissues such as the thymus and spleen (Toyohara et al., 2006), suggesting that 4DST may more precisely reflect thymidine metabolism than FLT. A comparative study revealed that [11C]4DST showed higher uptake than [18F]FLT in proliferating tumors, particularly in the spleen, a lymphoid tissue, in mice (Tsuji et al., 2009).

We therefore hypothesized that ADC mapping and [11C]4DST‐PET could be applied to quantify spatiotemporal changes in the initial postirradiation cellular events in the BM and thymus that are critical for the tumorigenesis of RITL.

2. Materials and methods

2.1. Mice

C57BL/6 mice of both sexes (C57BL/6NJcl or C57BL/6JJcl in some cases) and syngeneic EGFP transgenic (tg) mice were purchased from Clea Japan (Tokyo, Japan) and Japan SLC (Hamamatsu, Japan), respectively. EGFP tg mice ubiquitously express EGFP protein in the whole body, which is transcriptionally controlled by a CAG promoter composed of the CMV enhancer, a fragment of the chicken β‐actin promoter, and rabbit β‐globin exons (Ikawa et al., 1998). Mice were housed under a specific pathogen‐free condition with a 12‐h light/dark cycle at 22 ± 1 °C and a relative humidity of 45%–50%. All animal experiments were approved by the Institutional Animal Care and Use Committee of Experimental Animals of the National Institute of Radiological Sciences, Japan.

2.2. FX

Unanesthesized C57BL/6 mice were X‐irradiated with four 1.6‐Gy fractions (total dose: 6.4 Gy) at weekly intervals using a PANTAK HF‐320 X‐ray generator (Shimadzu Mectem, Otsu, Japan). The dose rate was 0.63–0.65 Gy/min.

2.3. BMT

Irradiated mice were injected intravenously with more than 2 × 107 BM cells isolated from the femur of 4–5‐week‐old C57BL/6 or EGFP tg mice. This is a sufficient number of transplanted BM cells to prevent RITL (Humblet et al., 1989). BMT immediately after FX (referred to as iBMT) was conducted within 6 h after the last FX. Delayed BMT (referred to as dBMT) was performed 4 weeks after the completion of FX.

2.4. Flow cytometry

Cells were prepared from the thymus and BM by mincing the tissues and isolating a single cell suspension. GFP expression was detected by a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

2.5. DWI and ADC measurement

DWI was obtained on a 7.0‐T MRI scanner (Magnet: Kobelco and JASTEC, Kobe, Japan; Console: Bruker Biospin, Ettlingen, Germany) with a volume coil for transmission (Bruker) and a 2‐channel phased‐array coil for reception (Rapid Biomedical, Rimpar, Germany). The images were acquired using a sagittal single‐slice SE echo‐planar imaging sequence (TR = 3800ms, TE = 23ms, slice thickness = 0.8 mm, FOV = 19.2 × 19.2 mm2, matrix = 128 × 128, number of segments = 4, fat suppression = on, NA = 1) with motion‐probing gradients (b = 1.62, 670 s/mm2, δ = 5ms, Δ = 10ms). ADC maps (i.e., mean diffusibility) were calculated from the DWI images acquired at two b‐values (b = 1.62, 670 s/mm2) and in three diffusion directions. We chose these two b‐values for the following two reasons: When we tested the signal‐to‐noise ratio (SNR) preliminarily, SNR of b = 1000 s/mm2 was not enough at our MRI system. We therefore used b = 670 s/mm2 as a maximum strength for getting appropriate SNR. In addition, these values are initial settings of ParaVision system (Bruker Biospin) we used for image reconstruction and analysis. Regions of interest (ROIs) were placed on the BM regions and the surface of the region was eliminated to minimize partial‐volume effects between the bone and BM. ParaVision and MRVision (MRVision Co., Winchester, MA) were used for image reconstruction and analysis.

2.6. PET

[11C]4DST was synthesized as described previously (Toyohara et al., 2006). Mice were injected with 11–12 MBq of [11C]4DST into the tail vein. Fifty minutes after injection, PET data acquisition was conducted for 10 min using a small‐animal PET system (Inveon; Siemens Medical Solutions, Malvern, PA) under 1.5%–2% isoflurane anesthesia during the entire scanning period. Images were reconstructed using a 3D maximum a posteriori algorithm without attenuation correction. Image analysis was performed with ASIPro software (Siemens Medical Solutions). For radioactivity quantification in the thymus, an ROI encompassing the whole tissue area on each of the coronal slices was automatically created with a fixed size (10 pixels) and placed over the thymus area in four or more coronal images. All ROIs were linked to form a 3D volume of interest (VOI) using the 3D VOI dimensionality tool to calculate the mean standardized uptake value (SUVmean).

2.7. Immunohistochemistry and histopathological analysis

Tissues were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 4 μm thickness. BM tissues were decalcified in a solution of 10% EDTA‐3Na for 2 weeks before paraffin embedding. Sections were stained with hematoxylin and eosin. Incorporation of BrdU in the thymus was evaluated with the BrdU Labeling and Detection Kit II (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's instructions. BrdU‐positive nuclei were automatically counted using a NanoZoomer slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and Tissue Studio software (Definiens, München, Germany).

2.8. Experimental design

Our experimental schedule is shown in Figure 1. Mice were X‐irradiated from 4 to 7 weeks of age as described above. iBMT and dBMT (see “BMT” in the Materials and Methods section) was conducted as indicated in Figure 1. DWI and PET images were acquired as shown in Figure 1 and Supplementary Table 1. For DWI experiments, the images were acquired 3 days after each irradiation during FX and thereafter once a week at 6–7‐day intervals. For PET experiments, the images were acquired once a week after FX at 5–7‐day intervals. Time points of BrdU measurement, histological analyses of the BM, and flow cytometrics are also shown in Figure 1.

Figure 1.

Figure 1

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Experimental design. DWI, PET, BrdU measurement, BM histology, and flow cytometry are shown under the mouse age timeline (weeks). Irradiations and BMT are indicated on the mouse age timeline (yellow arrows). Unirradiated (black arrows), FX‐only (blue arrows), FX‐iBMT (red arrows), FX‐dBMT (purple arrows). Unfilled arrows indicate the times of BrdU measurement, BM histology, and flow cytometry. The numbers in parentheses indicate the number of mice measured in each group.

2.9. Statistical analysis

All statistical analysis was performed using JMP version 9 software (SAS Institute Japan, Tokyo). ADC values were compared using an unpaired t‐test in the case of two groups and one‐way analysis of variance (ANOVA) with Tukey‐Kramer multiple comparison in the case of three groups. The values of the SUVmean and the numbers of BrdU‐incorporated cells were compared using one‐way ANOVA with Tukey‐Kramer multiple comparison. A P value of less than 0.05 was considered statistically significant.

3. Results

3.1. Quantitative evaluation of FX‐induced BM changes by ADC

To quantitatively evaluate FX‐induced BM changes, we longitudinally acquired ADC maps of the tibia BM (Figure 2A). In the unirradiated mice used as an age‐matched control, BM ADCs barely changed from 4 to 15 weeks of age and were at approximately 0.5 × 10−3 mm2/s (Figure 2B). In contrast, mice treated with FX (FX‐only) showed a markedly different pattern (Figure 2B): the BM ADCs in FX‐only mice increased during the course of FX compared with those in unirradiated mice. The values continuously decreased after the second irradiation until 3 weeks after FX. The BM ADCs in FX‐only mice again increased and then returned to levels comparable with those of unirradiated mice 5–8 weeks after FX. Statistical analyses revealed that BM ADCs were significantly higher in FX‐only mice than unirradiated mice during FX and conversely were lower 2–4 weeks after FX. There were no significant differences in BM ADCs between the two groups 5–8 weeks after FX.

Figure 2.

Figure 2

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ADC measurements and flow cytometry in the BM. (A) ADC maps of the BM. A diagram of tibia BM is shown. Black dotted lines in the ADC maps indicate the outer boundary of tibia bone. Arrowheads indicate the BM. The color scale indicates the ADC, shown at the right side of the images. (B) Changes in the BM ADC values of FX‐only (blue, round), FX‐iBMT (red, triangle), FX‐dBMT (purple, diamond), and unirradiated (black, square) mice. The experimental schedule of FX (blue arrows), iBMT (red arrow), and dBMT (purple arrow) is indicated in the graph. Means ± standard deviation (SD) are given (N = 3–4). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with unirradiated mice. (C) Flow cytometric analyses of BM cells after iBMT and dBMT. Upper: Histogram of representative BM cells sampled from individual mice after iBMT (unirradiated 11‐week‐old C57BL/6 mice, white; iBMT 1 week, yellow; iBMT 2 weeks, red; iBMT 4 weeks, green). The ratios of donor‐derived EGFP‐positive cells in each sample are shown in the histogram. Lower: The ratios of donor‐derived EGFP‐positive cells after BMT, including the data plotted in the upper histogram (N = 4). Sampling points are shown as weeks after BMT.

3.2. Rapid recovery of the BM ADC by iBMT but not dBMT

As described above, iBMT efficiently prevents RITL development, whereas dBMT has little effect on prevention (Sado et al., 1991). We next investigated the effects of iBMT and dBMT on the BM ADC. In the mice treated with iBMT following FX (FX‐iBMT), BM ADCs were rapidly restored to normal levels 3 and 4 weeks after iBMT (Figure 2B). In contrast, BM ADCs in the mice treated with dBMT (FX‐dBMT) showed no apparent differences from those of FX‐only mice (Figure 2B).

3.3. Engraftment of donor cells in the BM after BMT

We conducted BMT by injection of BM cells derived from EGFP tg mice and determined the percentages of EGFP‐positive cells in the BM by flow cytometry (Figure 2C). One week after iBMT, 40%–60% of the BM cells were EGFP positive. Over 80% of the BM cells were EGFP positive 2 and 4 weeks after iBMT. The rates of EGFP‐positive cells varied widely in the BM 4 weeks after dBMT, ranging from 0.1% to 72.4% (Figure 2C).

3.4. Pathological analyses of the BM

We conducted pathological analyses of the BM in FX‐only, FX‐BMT, and unirradiated mice (Figure 3). No apparent histological abnormalities were found in unirradiated BM at 4–7 weeks of age (Figure 3H–K). In contrast, the BM exhibited various pathological changes during and after FX in FX‐only mice (Figure 3A–G). Sinusoids were slightly dilated and congestion was observed 3 days after the first irradiation (Figure 3A). Reduced BM cellularity with loss of hematopoietic cells was observed 3 days after the first irradiation and the cellularity was more progressively reduced 3 days after the second irradiation (Figure 3A and B). Three days after the third irradiation, severe hemorrhage was observed and very few hematopoietic cells existed in the BM (Figure 3C). Fatty change occurred 3 days after the last irradiation in the BM (Figure 3D). Three weeks after FX, the BM was completely converted to fatty marrow (Figure 3E). Regenerative responses were observed as the hematopoietic cell population increased 4 weeks after FX (Figure 3F), and the amount of fatty marrow was considerably decreased 8 weeks after FX (Figure 3G). The BM had already been regenerated 4 weeks after iBMT (Figure 3L), which was distinctly different from the BM in age‐matched FX‐only mice (Figure 3F). BM treated with dBMT showed no apparent differences from the BM of age‐matched FX‐only mice (Figure 3G and M).

Figure 3.

Figure 3

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Pathological changes in the BM. Histopathological changes in the BM in FX‐only (A–G), unirradiated (Un) (H–K), FX‐iBMT (L), and FX‐dBMT (M) mice. Sampling time points after each X‐irradiation (X‐irra) and weeks after FX or BMT are shown below the images. Mouse age is also shown below the images of unirradiated mice. Two‐way arrows indicate that the linked upper and lower images are those from age‐matched mice. Scale bar in (A) = 50 μm.

3.5. [11C]4DST‐PET of thymic changes

We investigated thymic cell proliferation by [11C]4DST‐PET in FX‐only, FX‐BMT, and unirradiated mice (Figure 4A–E and Supplementary Figure 1). The SUVmean, a quantitative value for tracer uptake, significantly increased in FX‐only mice compared with that of age‐matched unirradiated and FX‐iBMT mice 1 week after FX (P < 0.01 and P < 0.001, respectively; Figure 4A). Two weeks after iBMT, tracer uptake was significantly higher in FX‐iBMT mice than in unirradiated and FX‐only mice (P < 0.01 and P < 0.001, respectively; Figure 4B). FX‐iBMT mice still had a higher tracer uptake compared with unirradiated and FX‐only mice 3 weeks (P < 0.05 and P < 0.01, respectively; Figure 4C) and 4 weeks after iBMT (P < 0.01; Figure 4D). Figure 4E shows a time course plot of the SUVmean. There were no significant differences in the SUVmean among FX‐only, FX‐dBMT, and unirradiated mice (Figure 4F).

Figure 4.

Figure 4

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[ 11 C]4DST‐PET and flow cytometry in the thymus. (A–D) PET images and SUVmean in the thymus in unirradiated, FX‐only, and FX‐iBMT mice. Representative coronal slices (upper) and plots (lower). (A) One week after FX (FX‐only, N = 10) or iBMT (FX‐iBMT, N = 5) and in age‐matched unirradiated mice (N = 10). (B) Two weeks after FX (FX‐only, N = 12) or iBMT (FX‐iBMT, N = 6) and in age‐matched unirradiated mice (N = 11). (C) Three weeks after FX (FX‐only, N = 4) or iBMT (FX‐iBMT, N = 4) and in age‐matched unirradiated mice (N = 4). (D) Four weeks after FX (FX‐only, N = 10) or iBMT (FX‐iBMT, N = 6) and in age‐matched unirradiated mice (N = 10). Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns; not significant. A color scale indicates the SUV, shown on the right side of the images. (E) Changes in the SUVmean within the period from (A) to (D). Mean ± SD. (F) Changes in the SUVmean after dBMT. Mean ± SD. N = 3–4. (G) Flow cytometric analyses of thymocytes after iBMT and dBMT. Left: Representative histogram of thymocytes sampled from individual mice after iBMT (unirradiated 11‐week‐old C57BL/6 mice, white; iBMT 1 week, yellow; iBMT 2 weeks, red; iBMT 4 weeks, green). The ratios of donor‐derived EGFP‐positive cells at each sampling time are shown in the histogram. Right: The ratios of donor‐derived EGFP‐positive cells after BMT, including the data plotted in the left histogram (N = 4).

3.6. Distribution of donor‐derived thymocytes after BMT

We performed flow cytometry to determine the ratio of donor cells in the thymus after BMT of EGFP‐positive BM cells (Figure 4G). Very few thymocytes were EGFP positive 1 week after iBMT (0.5%–1.3%). Two weeks after iBMT, approximately 60% of thymocytes were positive for EGFP in two of the four mice, whereas nearly 90% were positive in the other two mice. Over 90% of thymocytes were donor‐derived GFP‐positive cells in all mice 4 weeks after iBMT. There were few EGFP‐positive thymocytes 4 weeks after dBMT.

3.7. BrdU incorporation in the thymus after FX and BMT

We conducted a BrdU assay in the thymus for comparison with PET results (Figure 5). BrdU labels cells in the S phase of the cell cycle. The numbers of BrdU–positive cells were approximately 1.2‐fold higher in FX‐only mice than in unirradiated and FX‐iBMT mice 1 week after FX, although there were no significant differences among the three groups. At 2 weeks after iBMT, the numbers of BrdU‐incorporated cells were 2.1‐ and 1.7‐fold higher in FX‐iBMT mice than in unirradiated and FX‐only mice, respectively, with a statistically significant increase.

Figure 5.

Figure 5

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Cell proliferation in the thymus assessed by BrdU staining. Left: BrdU staining. Brown spots indicate cell nuclei incorporating BrdU. Scale bar = 50 μm. Right: The number of BrdU‐incorporated cells per 1 mm2. The numbers of samples are indicated in the figure. Mean ± SD. **P < 0.01, ***P < 0.001. ns, not significant.

4. Discussion

Here, we describe novel PET and MRI combination approaches to the quantification of spatiotemporal changes in in vivo cellular events critical for RITL development. These quantitative, noninvasive methods allowed us to analyze the in vivo postirradiation processes of cell damage and tissue regeneration, which are critical events for RITL development, in the thymus and BM.

As described above, the initial postirradiation events that occur in the thymus and BM are critically important for the development of RITL. Given that there is a critical period for RITL prevention by BMT (Sado et al., 1991), it is clear that the cellular events that occur in the BM and thymus within several weeks of FX are decisively important for RITL development. Therefore, our work focused on early events in the BM and thymus within 8 weeks of FX.

In ADC mapping of the BM, we found dramatic changes in the ADC values in FX‐only mice. These changes correlated well with the histopathological changes in cell density. ADC values significantly increased in FX‐only BM compared with unirradiated BM during FX. Because the BM is the organ that is most sensitive to radiation, exposure to a sublethal dose of X‐ray or photon irradiation induces BM failure that is accompanied by cell damage or apoptosis of hematopoietic cells in the rodent BM (Fliedner et al., 1961; McCulloch and Till, 1962). In accordance with this notion, our histopathological analyses revealed that reduced cellularity and loss of hematopoietic cells were evident in the FX‐only BM during FX. A reduction in cell numbers in tissue and expansion of extracellular space lead to faster water diffusion and increased ADC values in tissue (Chenevert et al., 2000; O'Flynn and DeSouza, 2011). In fact, accumulating evidence suggests a positive correlation between ADC values and cell death (Chenevert et al., 2000; Mardor et al., 2003; Moffat et al., 2005; Theilmann et al., 2004). Therefore, we speculate that the increased BM ADCs observed during FX reflect reduced cellularity in the BM.

In addition, significant sinusoidal vasodilation was also histologically apparent in the FX‐only BM. Altered capillary perfusion in the voxel affects the signal in the DWI (Lemke et al., 2010). Thus, besides reduced cellularity, increased perfusion may also have contributed to the increased ADC values in FX‐only BM. Thereafter, the ADC values dramatically decreased until 3 weeks after FX. Severe fatty changes were observed 3 weeks after FX in the BM by histopathological analyses. Given that yellow marrow (fatty marrow) results in lower ADC values than red marrow (Ward et al., 2000), the decrease in the ADC may be accounted for by restricted water diffusion by lipid molecules in the fatty altered BM. BM ADC values were restored to the levels of unirradiated BM 5–8 weeks after FX as cellularity was recovered and BM regeneration proceeded. Importantly, BM ADCs rapidly returned to normal levels following iBMT, but not dBMT, sooner than for spontaneous recovery. These findings strongly suggest that BM is rapidly regenerated and fully restored by iBMT, which may be a cellular basis of RITL prevention by BMT.

We applied [11C]4DST‐PET for the quantification of thymic changes after FX and BMT. We found that the thymus receiving FX showed a transient but significant increase in tracer uptake 1 week after FX. These findings are consistent with previous reports showing a growth expansion of residual T cell subsets in the cortex of the murine thymus within 1 week of irradiation (Mizutani et al., 2002; Nishimura et al., 2004). Therefore, our data supports the notion that FX induces compensatory proliferation of radioresistant T cell subsets in the regenerating thymus. Interestingly, our PET data showed that such cell proliferation was suppressed by iBMT. The mechanism underlying the suppression is currently unknown. However, we propose two possible mechanisms. One possibility is that transplanted donor cells localized in the BM suppress thymic cell proliferation via systemically secreted regulatory factors such as cytokines. The other possibility is that, given that very few but surely existing donor‐derived cells migrated into the thymus, such migrating donor‐derived cells could inhibit thymocyte proliferation through direct cell‐to‐cell interaction or regulatory paracrine actions in the affected thymus. Two to four weeks after iBMT, thymic tracer uptake in the FX‐iBMT thymus was significantly higher than that of the unirradiated and FX‐only thymus. The increased tracer uptake is probably due to cell proliferation in the regenerating thymus, where over 80% of the thymocytes were derived from donor cells, as revealed by flow cytometric analyses.

BrdU incorporation assays were conducted for comparison with PET results. At 2 weeks after FX or iBMT, the BrdU assay results strongly supported the [11C]4DST‐PET results. At 1 week after FX or iBMT, the BrdU data agreed well with the PET results. However, unlike in the PET results, there were no significant differences in BrdU measurements among the three groups. The reason for this statistical discrepancy is currently unknown. There may be different effects of BrdU and thymidine incorporation into DNA on cell proliferation (Duque and Rakic, 2011). In addition, Zhang et al. reported that [18F]FLT uptake was not always consistent with BrdU incorporation (Zhang et al., 2012).

Flow cytometric analyses revealed that iBMT, but not dBMT, rapidly induces hematologic reconstitution. Over 80% of cells were replaced by donor cells in the thymus and BM within 4 weeks of iBMT. The quantitative ADC values and SUVmean in our studies suggest that donor‐derived cells will be grafted and differentiate and proliferate in the regenerating BM and thymus 2–4 weeks after iBMT. In the case of dBMT, donor cells do not appear to proliferate in the BM because of spontaneous regeneration by host‐derived BM cells.

There are several limitations in this study. First, no intra‐individual comparison was performed before and after BMT in ADC and PET studies. Second, there were no ADC and PET measurements before FX in FX‐only mice. Further research will use more carefully‐designed cohorts of mouse and time points optimized. Third, there was a lack of correlation between BrdU incorporation and [11C]4DST uptake. Thus, further studies are needed to ascertain the correlation between [11C]4DST uptake and BrdU incorporation.

In conclusion, the PET and MRI approaches described here proved useful in the direct and noninvasive assessment of the in vivo spatiotemporal dynamics of cellular changes critical for RITL development. The proposed method is feasible for determining the relationship between thymic or BM responses to FX and RITL onset in individual mice and will contribute to a better understanding of RITL.

Supporting information

The following are the supplementary data related to this article:

Supplementary data

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Supplementary data

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Acknowledgments

We thank Dr. Hiroaki Kanda (The Cancer Institute of the Japanese Foundation for Cancer Research) for histopathological analyses; Drs. Tatsuhiko Imaoka and Yoshiya Shimada for the quantification of BrdU‐positive cells; Nobuhiro Nitta, Yoshikazu Ozawa, Sayaka Shibata, and Aiko Sekita for assistance with the MRI studies; Aya Sugyo and Hidekatsu Wakizaka for assistance with the PET studies; Hisashi Suzuki for the production of [11C]4DST; Takeshi Maeda and the FACS support team of National Institute of Radiological Sciences for assistance with the flow cytometric analyses; and Drs. Takayuki Obata and Maki Okada for many helpful discussions. This work was performed with the aid of a research fund from the National Institute of Radiological Sciences, Japan.

Supplementary data 1.

1.1.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2014.11.009

Hasegawa Sumitaka, Morokoshi Yukie, Tsuji Atsushi B., Kokubo Toshiaki, Aoki Ichio, Furukawa Takako, Zhang Ming-Rong, Saga Tsuneo, (2015), Quantifying initial cellular events of mouse radiation lymphomagenesis and its tumor prevention in vivo by positron emission tomography and magnetic resonance imaging, Molecular Oncology, 9, doi: 10.1016/j.molonc.2014.11.009.

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