361

ORIGINAL
Early Prediction of Radiotherapeutic Efficacy in a Mouse Modelof Non-Small Cell Lung Carcinoma Using 18F-FLT and 18F-FDG
PET / CT
Tamaki Otani1, Hitoshi Ikushima2, Yoshimi Bando3, Michiko Yamashita4, Kenmei Kuwahara5, Hideki Otsuka6,
Kazuya Kondo7, and Hirokazu Miyoshi1
1Advance Radiation Research, Education, and Management Center, Tokushima University, Tokushima, Japan, 2Department of Therapeutic
Radiology, Institute of Biomedical Science, Tokushima University, Tokushima, Japan, 3Department of Pathology and Laboratory Medicine,
Institute of Biomedical Science, Tokushima University, Tokushima, Japan, 4Department of Analytical Pathology, Institute of Biomedical Science, Tokushima University, Tokushima, Japan, 5Faculty of Health Science, Tokushima University Graduate School of Medicine, Tokushima
University, Tokushima, Japan, 6Department of Medical Imaging/Nuclear Medicine, Tokushima University Graduate School of Biomedical
Sciences, Tokushima University, Tokushima, Japan, 7Department of Oncological Medical Services, Institute of Biomedical Science, Tokushima
University, Tokushima, Japan

Abstract : This study investigated the usefulness of [18F]-3’-deoxy-3’-fluorothymidine (18F-FLT) and [18F]-fluoro-2deoxy-D-glucose (18F-FDG) positron emission tomography (PET) / computed tomography (CT) imaging for predicting the therapeutic efficacy of non-small cell lung cancer (NSCLC) irradiation at an early stage after radiation
treatment. Mice were xenografted with the human lung adenocarcinoma line A549 or large cell lung cancer line
FT821. Tumour uptake of 18F-FLT and 18F-FDG was imaged using PET / CT before and 1 week after irradiation. In
A549 tumours, 18F-FLT uptake was significantly decreased, and 18F-FDG uptake was unchanged post-irradiationcompared with pre-irradiation. In FT821 tumours, uptake of both 18F-FLT and 18F-FDG uptake was substantiallydecreased post-irradiation compared with pre-irradiation. In both xenografts, tumour volumes in the irradiatedgroups were significantly decreased compared with those in the control group. 18F-FLT is expected to contributeto individual NSCLC therapy because it accurately evaluates the decrease in tumour activity that cannot be captured by 18F-FDG. 18F-FDG may be useful for evaluating surviving cells without being affected by the inflammatory reaction at an extremely early stage, approximately 1 week after irradiation. Combined use of 18F-FLT and
18
F-FDG PET / CT imaging may increase the accurate prediction of radiotherapy efficacy, which may lead to improved patient outcomes and minimally invasive personalised therapy. J. Med. Invest. 70 : 361-368, August, 2023
Keywords : 18F-fluorodeoxyglucose, 18F-fluorothymidine, irradiation, inflammation, non-small cell lung cancer

INTRODUCTION
Several randomised controlled trials have investigated theusefulness of preoperative induction radiotherapy for non-small
cell lung cancer (NSCLC), but no significant improvement insurvival with neo-adjuvant chemoradiotherapy was confirmed(1, 2). Phase II trials of neo-adjuvant chemoradiation therapy for
non-small cell lung cancer have reported variable response rates
of 39% to 88% and complication rates of 0% to 67% (3-5). Thus, it
is conceivable that the efficacy and side effects of treatment differ
from case to case. In recent years, advances in testing technology have made it possible to provide individualised therapy that
selects the optimal treatment method according to the patient’s
constitution. If the effect of neo-adjuvant chemoradiation therapy can be determined accurately, individualised treatment will
be possible. This might include changing the treatment policy
to radical chemoradiotherapy according to the treatment effectof each case, leading to improved treatment outcomes and areduced burden on patients. Accurate diagnostic imaging by molecular imaging is essential for individualised cancer therapy. In
Received for publication October 26, 2022 ; accepted April 11, 2023.
Address correspondence and reprint requests to Tamaki Otani,
Advance Radiation Research, Education, and Management Center,
Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 7708503, Japan and Fax : +8188-633-9417. E-mail : otani.tamaki@
tokushima-u.ac.jp

this study, we demonstrated the usefulness of positron emission
tomography / computed tomography (PET / CT) imaging to assess
the effects of radiotherapy.
[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) PET / CT is an excellent method for imaging tumours and the most commonly usedimaging modality for accurately delineating tumour lesions (6,7). However, when used to determine the effects of radiotherapy,
infiltration of inflammatory cells around the tumour lesions,such as macrophages, may cause false positive results (8, 9).
Inflammatory lesions present frequently at approximately 1month after radiotherapy, and it is difficult to determine the efficacy of radiotherapy using 18F-FDG PET / CT at an earlier stage.
[18F]-3’-deoxy-3’-fluorothymidine (18F-FLT) can be used to evaluate cell proliferation through the enzymatic activity of thymidine
kinase 1. 18F-FLT is a tumour proliferation imaging tracer thatis not affected by inflammation. Therefore, it is expected that 18FFLT will be highly accurate for the evaluation of radiotherapy
efficacy, even immediately after treatment (10, 11).
Owing to the inflammatory responses detected by 18F-FDG,
many reports have evaluated the effects of radiotherapy approximately 4 months after treatment. However, few studies havedetermined or predicted the therapeutic effects within 1-month
post-treatment. An early and precise diagnosis of lung cancer
response is essential because surgery or re-irradiation with curative intent may be feasible. In this study, we investigated theusefulness of 18F-FLT and 18F-FDG PET / CT for determining thetherapeutic efficacy of radiotherapy in NSCLC. We used mice

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T. Otani, et al. Predicting Therapeutic Effects using PET / CT

subcutaneously transplanted with human NSCLC cells to evaluate the effects of radiotherapy in the very early stage (1 week)
after irradiation.

equilibrated with 5% CO2 and 95% air. Subcutaneous implantation of A549 or FT821 cells (2.0 × 106 / mouse) was performed in
the lateral chest of the mice.

Irradiation

MATERIALS AND METHODS
Animals

Male nude mice (BA LB / cAJcl-nu / nu ; CLEA Japan
Inc. ; Tokyo, Japan) 6–8 weeks of age were used in this study
and maintained in the Laboratory for Animal Experiments at
our institution with a standardised 12-h light / dark cycle andaccess to food and water ad libitum. The protocols for all animal
experiments were approved by and performed in accordancewith the guidelines of the Institutional Animal Care and Use
Committee of the University of Tokushima. The humanitarian
endpoint for subcutaneously transplanted mice was euthanasia
when the tumour diameter reached 20 mm.

Cell culture and transplantation
We used two types of NSCLC cell lines : A549 (human adenocarcinoma lung cancer cells) and FT821 (human large celllung cancer cells). We established the FT821 cell line using a
primary culture from surgically resected tissue provided by
Tokushima University (Tokushima, Japan) (12, 13). A549 cells
were purchased from the Health Science Research Resources
Bank (Osaka, Japan). The cell lines were cultured in RPMI
1640 (Sigma Chemical Co. ; St. Louis, MO, USA) supplemented
with 10% heat-inactivated foetal bovine serum (BioWhittaker ; Walkersville, MD, USA) at 37°C in a humidified incubator

Local external beam irradiation was applied using an X-ray
irradiation unit (MBR-1520R-3 ; Hitachi ; Tokyo, Japan). A549
and FT821 were transplanted into mice, and the mice weredivided into X-ray irradiation (20 Gy / 2 fractions / 2 days) andnon-irradiation groups. Tumour irradiation was performed at 20
and 50 days after transplantation with A549 and FT821 cells,
respectively. Areas other than the tumour were shielded with alead barrier to prevent nonspecific radiation exposure.
18

F-FLT and 18F-FDG PET / CT measurements

18
F-FLT and 18F-FDG PET / CT measurements were takenfrom mice in the control group prior to radiation treatment of
the irradiation group, and from the irradiation group 1 week
after radiation treatment (Fig. 1). Fourteen mice transplantedwith A549 (control group : 7, irradiation group : 7) and 10 micetransplanted with FT821 (control group : 5, irradiation group : 5)
were used for 18F-FLT and 18F-FDG PET / CT measurements. Allscans were performed with a Siemens Inveon small-animal PET
scanner (Siemens Healthcare ; Knoxville, TN, USA). Mice monitored by 18F-FDG PET / CT were fasted for 18–24 h with accessto water only. Mice used for 18F-FLT PET / CT were not fasted.
Body weights were measured, and mice were anaesthetised by
1.5%–2.0% isoflurane inhalation and injected via a tail-vein
catheter with 10 MBq / 0.1–0.2 mL 18F-FLT or 18F-FDG. The entire mouse body was scanned by CT and PET (field of view : 99.0

Fig 1. Schematic presentation of the study time course.
Non-small cell lung cancer cell lines A549 and FT821 were transplanted into mice, and the mice were divided into irradiationand control (non-irradiation) groups. [18F]-3’-deoxy-3’-fluorothymidine (18F-FLT) and [18F]-fluoro-2-deoxy-D-glucose (18F-FDG)
positron emission tomography / computed tomography (PET / CT) measurements were performed on mice in the control group prior
to radiation treatment of the irradiation group. 18F-FLT and 18F-FDG PET / CT measurements were performed on mice in theirradiation group 1 week after radiation treatment. Irradiation was performed at 18 and 45 days after transplantation of A549
and FT821 tumours, respectively. The same mice could not be evaluated using PET / CT pre- and post-irradiation because theirremoval from the PET / CT facility was prohibited to prevent radioactive contamination. Therefore, non-irradiation in the control
group was defined as pre-irradiation (0 week), and 1 week after irradiation in the irradiated group was defined as post-irradiation
for comparisons between the groups. At 3 weeks after irradiation, 18F-FDG PET / CT measurements were performed on the control
and irradiation groups. There was no data for 18F-FDG PET / CT imaging of A549 tumours in the control group at 3 weeks because
mice in the control group reached a humane endpoint regarding tumour size within 3 weeks and were sacrificed.

The Journal of Medical Investigation Vol. 70 August 2023
× 99.0 × 126.2 mm). PET data were acquired in list mode for 20
min following a delay of 40 min to allow for 18F-FLT and 18F-FDG
uptake. PET images were reconstructed using three-dimensional ordered-subject expectation maximisation followed by maximum a posteriori reconstruction The image matrix was 128 ×
128 pixels, and the slice thickness of the PET images was 0.796
mm. Only the 18F-FDG PET / CT measurement was performed
again 3 weeks after irradiation.

363

Statistical analysis
The quantitative values in the control-irradiation groups werecompared using the Mann-Whitney U-test (SPSS software,version 20 ; IBM Corp. ; Armonk, NY, USA). Comparisons ofquantitative values between 1- and 3-week irradiation groups
were performed using the Wilcoxon signed rank test. A p-valueof < 0.05 was considered statistically significant.

PET image analysis
The PET / CT images were analysed using PMOD software(version 4.201 ; PMOD Technologies LLC. ; Zürich, Switzerland). The window levels of all PET images were displayed at
0–3 based on the standardised uptake value (SUV). For all
PET / CT datasets, the volume of interest was defined manuallyaround the area of 18F-FLT or 18F-FDG uptake by the tumour.
Measurements included the maximum SUV (SUVmax), the averaged SUV (SUVmean), and the metabolic tumour volume (MTV).
The total lesion glycolysis (TLG) was the product of MTV and
SUVmean (total lesion proliferation [TLP] was used for 18F-FLT
imaging). The MTV and TLG (TLP), which reflect changes
in tumour size, were recently used as indices of whole tumour
uptake and might be useful for evaluate the therapeutic effect ofradiotherapy more accurately. The clinical usefulness of these indicators for prognosis and treatment response was demonstrated
in many cancers, including lung, head-and-neck, and gynaecological cancer (14, 15).

Measurement of tumour volumes
The major and minor axes of the tumours were measured for
3 weeks after irradiation using a calliper. Tumour volume
wasMajor axis × (Minor axis)2
determined by the formula : Tvol = ——————————————————————————.
2

Histological examination

Cell division, vascularisation, and radiation-induced inflammation were examined histologically by measuring levels of thecell proliferation marker Ki67 and number of viable neutrophils.
Mice were sacrificed, and tumour specimens were removed at
the same time point as PET / CT measurements in the control
and irradiation groups 1 week after irradiation and resecteden bloc. Each tumour was fixed in 10% phosphate-buffered formalin for 24–48 h, embedded in paraffin blocks, and processed
for histological analysis. Sections were stained with haematoxylin and eosin or immunohistochemically using standard
protocols. Briefly, tissue sections were heated in a pressure
cooker in Tris-EDTA buffer for antigen retrieval. The sections
were blocked using goat serum in phosphate-buffered saline
followed by incubation with anti-Ki67 antibody (rabbit, clone
SP6 ; Nichirei Bioscience Inc. ; Tokyo, Japan). Then, the sections
were incubated with a biotinylated secondary antibody (Nichirei
Bioscience, cat. #424031) and streptavidin-horseradish peroxidase followed by colorimetric detection using 3,3’-diaminobenzidine. Histology slides were observed under light microscopy at
×400 (high-power-field with a 0.2376 mm2 field of view), and theKi67 labelling index was quantified using the hot spot method(16). Viable neutrophils were counted within eight high-power
fields at the surface and central area of each section. It was notpossible to perform a pathological examination of mice that were
subjected to PET / CT because their removal from the PET / CT
facility was prohibited to prevent radiation exposure and / orradioactive contamination. Therefore, mice were prepared forpathological analysis separately from the mice used for PET. Fifteen (control group : 7, irradiation group : 8) and 18 (control : 10,
irradiation : 8) mice xenografted with A549 and FT821 cells,
respectively, were used for pathological analysis.

RESULTS

Prediction of the radiotherapeutic effect using 18F-FLT and 18F-FDG
PET  /  CT
Figure 2 shows representative 18F-FLT and 18F-FDG PET
images from control (pre-irradiation) and irradiation (post-irradiation) groups. A marked decrease in A549 tumour uptake wasobserved visually with 18F-FLT imaging after irradiation compared with that in the control group. Figure 3 shows the quantitative indexes obtained from 18F-FLT and 18F-FDG PET / CTscans of mice in the control (pre) and irradiation (post) groups.
A significant decrease in the SUVmax was observed in the A549
tumours imaged with 18F-FLT after irradiation compared withthose in the control (pre) group, but there was no change in
FT821 tumours. In A549 tumours, the MTV and TLP weredecreased in the irradiation (post) group only with 18F-FLT imaging compared with those in the control (pre) group. In FT821
tumours, the MTV and TLG (TLP) were decreased in the irradiation (post) group compared with those in the control (pre) group
after 18F-FLT and 18F-FDG imaging.

Evaluation of the therapeutic effect using tumour volume
measurements
The A549 tumour volumes in the irradiated group weresignificantly decreased compared with those in the control
group ; however, the volumes appeared to increase gradually(Fig. 4a). The FT821 tumour volumes in the irradiated group ofmice were significantly decreased compared with those in thecontrol group, and there was no tendency towards an increaseover time (Fig. 4b). For the A549 tumours, mice in the control
group reached a humane endpoint in tumour size within 3 weeks
and were sacrificed (Fig. 4c) survival curves). No mice died in the
FT821 group.

Evaluation of the therapeutic effect using 18F-FDG PET  /  CT
Table 1 shows the quantitative indexes obtained from 18F-FDG
PET / CT at 3 weeks after irradiation. No changes in the SUVs
were observed in A549 tumours at the 1- and 3-week time points
after irradiation ; however, the MTV and TLG were increased
at 3 weeks compared with 1 week after irradiation. In FT821
tumours, a significant increase in the SUV, MTV, and TLG
was observed in the control group at 3 weeks after irradiationcompared with pre-irradiation, but no significant changes wereobserved within the irradiation group between 1 and 3 weeks
after irradiation. In FT821 tumours, comparisons of values
between the control and irradiation groups at the 3-week time
point showed that the irradiation group had significantly lower
SUV, MTV, and TLG.

Evaluation of the therapeutic effect using histopathological analysis
Figure 5 shows the haematoxylin-eosin staining of tumour
tissues and Table 2 shows the percentage of Ki67+ cells andnumbers of viable neutrophils 1-week post-irradiation. In the
A549 and FT821 xenografts, necrotic lesions and degenerative
tumour tissue were more common in the irradiated group thanin the control group. Ki67 staining was significantly decreased

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T. Otani, et al. Predicting Therapeutic Effects using PET / CT

Fig 2. 18F-FLT and 18F-FDG PET images of mice.
Representative 18F-FLT and 18F-FDG PET images of mice in the control (pre-irradiation) and irradiation (postirradiation) groups. The yellow arrows show 18F-FLT and 18F-FDG uptake by the xenografted tumours. All
images were displayed with a window level of 0 to 3 based on standardised uptake volume.

Fig 3. Quantitative indexes measured using PET / CT.
The maximum standardised uptake value (SUVmax), the metabolic tumour volume (MTV), and the total lesion glycolysis(TLG) of the control (pre) and irradiation (post) groups were measured using 18F-FLT and 18F-FDG PET / CT. (a) A549 and (b)FT821 tumours. The horizontal bar in the boxplot indicates the median. Comparisons of the quantitative indexes between
control (pre) and irradiation (post) groups were performed using the Mann-Whitney U-test. *: p < 0.05, **: p < 0.01.

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The Journal of Medical Investigation Vol. 70 August 2023

Fig 4. Change in tumour volumes.

Tumour volumes of xenografted tissue in control and irradiation groups. (a) A549 and (b) FT821 tumours.
(c) In A549, mice in the control group reached a humane endpoint in tumour size within 3 weeks and weresacrificed, and the results are shown as survival curves. Comparisons of tumour volumes between control andirradiation groups were performed using the Mann-Whitney U-test. *: p < 0.05, **: p < 0.01.

Table 1. Quantitative indexes obtained from 18F-FDG PET / CT before and at 1 and 3 weeks after irradiation
A549
Control

SUVmax

FT821

Post-irradiation

Control

Post-irradiation

0 week

1 week3 weeks

0 week3 weeks

1 week3 weeks

1.37 ± 0.28

1.27 ± 0.17

1.27 ± 0.13

0.96 ± 0.06

1.62 ± 0.23

0.84 ± 0.09

0.80 ± 0.11

Comparisonsof quantitativevalues between
control andirradiatedFT821 tumoursat 3 weeks
p < 0.01

SUVmean

0.84 ± 0.10

0.79 ± 0.14

0.82 ± 0.08

0.73 ± 0.03

0.97 ± 0.09

0.63 ± 0.07

0.66 ± 0.06

p < 0.01

MTV (mm3)734.7 ± 214.8

805.3 ± 166.9

1051.8 ± 433.5*

68.4 ± 42.6

393.5 ± 116.5

27.2 ± 6.3

16.9 ± 9.1

p < 0.01

TLG (mm3)

611.4 ± 167.1

628.0 ± 119.6

870.4 ± 424.6*

50.7 ± 33.0

384.8 ± 126.8

17.1 ± 4.5

11.5 ± 7.1

p < 0.01*Comparisons of quantitative values between 1- and 3-week post-irradiation groups were performed using the Wilcoxon signed rank test

(p < 0.05).
18
F-FDG, [ F]-fluoro-2-deoxy-D-glucose ; PET / CT, positron emission tomography / computed tomography ; MTV, metabolic tumour volume ; SUV, standardised uptake value ; TLG, total lesion glycolysis.
18

T. Otani, et al. Predicting Therapeutic Effects using PET / CT366

Fig 5. Histological sections of tumour tissue.

Representative haematoxylin and eosin-stained histological sections from A549 (a–c) and FT821 (d–f)
tumours. Tumour tissues from the control (a, d) and irradiation (b, e) groups are shown at ×100 magnification.
The square indicates the area with viable neutrophils that were confirmed at the margin of the necrotic
lesions. This area is shown at ×400 magnification (c, f), and the arrows indicate the viable neutrophils.

Table 2. Histopathological results from A549 and FT821 tumour xenografts at 1 week after irradiation
Control
A549
FT821

Irradiation

p value*

Ki67 (%)

60.5 ± 16.4

31.6 ± 5.2

0.05

Viable Neutrophil Number

17.0 ± 4.9

20.8 ± 9.9

0.69

Ki67 (%)

48.9 ± 11.5

23.3 ± 6.5

0.02

Viable Neutrophil Number

20.5 ± 17.2

43.8 ± 34.9

0.12*Comparisons of quantitative values between control and irradiation groups were performed using the Mann-Whitney U-test.

in the irradiated group compared with that in the control groupfor the A549 and FT82 xenografts. The number of viable neutrophils was used to assess the inflammatory response caused byirradiation. Viable neutrophils were confirmed at the margin of
the necrotic lesions 1 week after irradiation (Fig. 5b and d) ; however, there were no significant changes in viable neutrophil numbers between the control and irradiation groups.

DISCUSSION
As a result of the multifaceted evaluation of the therapeutic
effects of irradiation using tumour volume measurements, 18FFDG PET / CT, and pathological analysis, multiple anticancer
effects were observed, which included a decrease in tumour
volume, 18F-FDG uptake, and Ki67 index, and an increase in radiation-induced necrotic tumour regions. From these results, weconcluded that a therapeutic effect of irradiation was obtained for
the A549 and FT821 xenografts. However, in the A549 tumours,
the tumour volume appeared to increase over time ; therefore,
the tumour suppressive effect of irradiation may have been temporary. In view of these therapeutic effects, 18F-FLT and 18F-FDG
PET / CT were analysed for their ability to predict radiotherapy
efficacy.

When the therapeutic effects of radiotherapy using PET were
investigated, the results differed depending on the quantitativeindexes SUV, MTV, and TLG. In FT821 tumours, the MTV and
TLG were more consistently altered than the SUV. Therefore,
the MTV and TLG, which reflect changes in tumour size, canbe used to evaluate the therapeutic effect of radiotherapy moreaccurately than the SUV. The usefulness of MTV and TLG wasshown in previous studies (15, 16). The tumour volumes of theFT821 xenografts were small ; therefore, the SUVmax may nothave been measured accurately because of the influence of thepartial volume effect (17).
Considering the SUVmax as well as the MTV and TLG (TLP),
18
F-FLT PET / CT showed a significant decrease in A549 andFT821 tumour uptake. Staining of Ki67, a histological index oftumour activity, was significantly decreased in the irradiatedgroup compared with that in the control group for the A549
and FT821 xenografts. Previous studies reported that 18F-FLT
uptake correlated with Ki67 levels (18, 21), and changes in
18
F-FLT uptake reflected tumour activity, which is useful for
judging therapeutic effects. The usefulness of 18F-FLT PET / CTfor predicting therapeutic effects has been proposed in manystudies, and the uptake of 18F-FLT may be better than 18F-FDG
for predicting the radiotherapeutic response of tumours (18-24).
FT821 tumours, in which the 18F-FLT and 18F-FDG uptake

The Journal of Medical Investigation Vol. 70 August 2023
were decreased 1 week after treatment, underwent a significant
antitumor effect, which was present even 3 weeks after treatment. However, in A549 tumours, 18F-FLT PET / CT showed asignificant decrease in tumour uptake, and the 18F-FDG uptake
did not change. In A549 tumours, the tumour volume appeared
to increase over time ; therefore, the tumour suppressive effectof irradiation may have been temporary. Our data suggested
that cells with a more aggressive phenotype may have led toregrowth after irradiation, which affected 18F-FDG uptake 1
week after irradiation. When using 18F-FDG in radiotherapy,
the effect of radiation-induced inflammation should be considered because this inflammation causes an increase in glucose
uptake. The inflammatory reaction is substantial approximately
1 month after radiation treatment, and the earliest evaluationof therapeutic effectiveness is recommended at 6–8 weeks after
irradiation (25). Therefore, there have been few studies of the effect of inflammation on 18F-FDG uptake at an early stage, within
1 month after irradiation, regardless of whether it is normal
or tumour tissue. In this study, pathological analyses revealed
neutrophil infiltration at the margins of the necrotic tumour
lesions, and the possibility that 18F-FDG uptake was causedby acute radiation-induced inflammation 1 week after irradiation could not be disregarded. However, similar pathologicalfindings were obtained in FT821 tumours, which demonstrated
radiation-mediated antitumour effects without an increase in
18
F-FDG accumulation. Therefore, the effect of radiation-induced
acute inflammation on 18F-FDG accumulation was considered tobe small.
18

F-FLT is expected to contribute to individual therapy because this method accurately evaluates the decrease in tumour
activity that cannot be captured by 18F-FDG. However, 18F-FLT
cannot assess the presence or absence of surviving tumour cells
and is merely an indicator of proliferative activity. Everitt et al.
suggested that 18F-FLT appears to be a more sensitive tracer of
early treatment responses than 18F-FDG, although it is currently
unclear whether these changes predict eventual clinical outcomes (19), and our study supports this proposal. Furthermore,
18
F-FDG may be useful for evaluating surviving cells without
being affected by the inflammatory reaction at an extremely
early stage, approximately 1 week after irradiation. The combined use of 18F-FLT and 18F-FDG makes it possible to accurately
predict the effects of radiotherapy, which may lead to minimallyinvasive personalised therapy.
Our evaluation of 18F-FDG accumulation during the radiation-associated inflammatory response had several limitations.
Because of the laws pertaining to the use of radioisotopes andthe regulations of the facility, PET evaluations before and after
irradiation on the same individual were not possible. To matchthe experimental conditions, data were all obtained from model
mice using cell lines that were cultured and transplanted withthe same timing. However, evaluation in the same individual
will eliminate unnecessary bias and provide more reliable
results. The relationship between 18F-FDG accumulation andradioactive inflammation is an important factor and the degreeof radioactive inflammation was evaluated by changes in neutrophil counts by haematoxylin and eosin staining in this study.
Furthermore, the evaluation period was only 1 week after irradiation. Assessing data over a longer duration using immunohistochemical staining might be useful for evaluating inflammation
more objectively with more reproducible results.

367

CONCLUSION
To predict the therapeutic effect of irradiation using PET imaging, tumour cell proliferative activity and cell death need to beclearly separated. We demonstrated some of the limitations andfurther possibilities of using 18F-FLT and / or 18F-FDG PET / CTto evaluate radiotherapeutic efficacy in mouse models of NSCLC.
These findings may have implications for clinical tumour responses after radiotherapy in humans.

CONFLICTS OF INTERESTS AND SOURCE OF
FUNDING
This work was supported in part by the Japan Society for the
Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI), grant No. 20K07698. The authors declarethat they have no conflicts of interest.

ACKNOWLEDGEMENTS
We thank Dr Haruhiko Fujino (Tokushima University,
Tokushima, Japan) who established the FT821 cell line usinga primary culture from surgically resected tissue. We thankSusan Zunino, PhD, and J. Ludovic Croxford, PhD, from Edanz(https://jp.edanz.com/ac) for editing a draft of this manuscript.

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