Quantitative evaluation of oxygen metabolism in the intratumoral hypoxia: 18F-fluoromisonidazole and 15O-labelled gases inhalation PET
Watabe et al. EJNMMI Research
Quantitative evaluation of oxygen metabolism in the intratumoral hypoxia: 18 15 F-fluoromisonidazole and O-labelled gases inhalation PET
Tadashi Watabe 0 1
Yasukazu Kanai 0 2
Hayato Ikeda 1
Genki Horitsugi 1
Keiko Matsunaga 0 2
Hiroki Kato 0 1
Kayako Isohashi 0 1
Kohji Abe 0 2 4
Eku Shimosegawa 0 1 2
Jun Hatazawa 0 1 3
0 Medical Imaging Center for Translational Research, Osaka University Graduate School of Medicine , Suita , Japan
1 Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine , Suita , Japan
2 Department of Molecular Imaging in Medicine, Osaka University Graduate School of Medicine , Suita , Japan
3 Research Laboratory for Development, Shionogi & Co., Ltd.; Immunology Frontier Research Center, Osaka University , Suita , Japan
4 Department of Drug Metabolism & Pharmacokinetics, Osaka University Graduate School of Medicine , Suita , Japan
Background: Intratumoral hypoxia is one of the resistant factors in radiotherapy and chemotherapy for cancer. Although it is detected by 18F-fluoromisonidazole (FMISO) PET, the relationship between intratumoral hypoxia and oxygen metabolism has not been studied. The purpose of this study was to evaluate the intratumoral perfusion and oxygen metabolism in hypoxic regions using the rat xenograft model. Ten male Fischer rats with C6 glioma (body weight = 220 ± 15 g) were investigated with 18F-FMISO PET and steady-state inhalation method of 15O-labelled gases PET. The tumoral blood flow (TBF), tumoral metabolic rate of oxygen (TMRO2), oxygen extraction fraction (OEF), and tumoral blood volume (TBV) were measured under artificial ventilation with 15O-CO2, 15O-O2, and 15O-CO gases. Multiple volumes of interest (1-mm diameter sphere) were placed on the co-registered 18F-FMISO (3 h post injection) and functional 15O-labelled gases PET images. The TBF, TMRO2, OEF, and TBV values were compared among the three groups classified by the 18F-FMISO uptake as follows: group Low (L), less than 1.0; group Medium (M), between 1.0 and 2.0; and group High (H), more than 2.0 in the 18F-FMISO standardized uptake value (SUV). Results: There were moderate negative correlations between 18F-FMISO SUV and TBF (r = −0.56 and p < 0.01), and weak negative correlations between 18F-FMISO SUV and TMRO2 (r = −0.38 and p < 0.01) and 18F-FMISO SUV and TBV (r = −0.38 and p < 0.01). Quantitative values were as follows: TBF, (L) 55 ± 30, (M) 32 ± 17, and (H) 30 ± 15 mL/100 mL/min; OEF, (L) 33 ± 14, (M) 36 ± 17, and (H) 41 ± 16%; TMRO2, (L) 2.8 ± 1.3, (M) 1.9 ± 1.0, and (H) 2.1 ± 1. 1 mL/100 mL/min; and TBV, (L) 5.7 ± 2.1, (M) 4.3 ± 1.9, and (H) 3.9 ± 1.2 mL/100 mL, respectively. Intratumoral hypoxic regions (M and H) showed significantly lower TBF, TMRO2, and TBV values than non-hypoxic regions (L). OEF showed significant increase in the severe hypoxic region compared to non-hypoxic and mild hypoxic regions. Conclusions: This study demonstrated that intratumoral hypoxic regions showed decreased blood flow with increased oxygen extraction, suggesting the need for a treatment strategy to normalize the blood flow for oxygen-avid active tumor cells in hypoxic regions.
PET; Fluoromisonidazole; Hypoxia; Blood flow; Oxygen consumption
It is well known that the hypoxic regions in tumors can be
resistant to radiotherapy as well as chemotherapy . PET
imaging of a hypoxic tracer, such as
18F-fluoromisonidazole (FMISO), can be used to detect hypoxia in patients
with head and neck cancer and predict an adverse
prognosis against hypoxia-targeting agents . 18F-FMISO is
a 2-nitroimidazole, which accumulates in the tissue
environment at low oxygen concentrations by binding
to macromolecules after reductive activation . The
rate of its accumulation increases as partial pressure
of oxygen (pO2) falls below 10 mmHg . Clinical
hypoxia thresholds based on pO2 measurements (2.5
and 5.0 mmHg) corresponded to the 18F-FMISO
standardized uptake value (SUV) (2.0 ± 0.6 and 1.4 ± 0.5,
respectively) . However, 18F-FMISO PET cannot
reflect regional oxygen metabolism, and in vivo
quantitative imaging of oxygen consumption in the hypoxic
tumor has not been investigated.
Tumor hypoxia is divided into two types, acute
hypoxia and chronic hypoxia. Acute hypoxia is the result of
pronounced heterogeneity of the tumor blood flow
distribution, whereas chronic hypoxia is observed at the
limits of oxygen diffusion (100–200 μm) away from
blood vessels . It has been suggested that intratumoral
oxygen metabolism is heterogeneous depending on the
tumor microenvironment and quantitative measurement
of oxygen consumption is essential to elucidate the
mechanism of tumor hypoxia as it is the shortage of
oxygen supply by blood flow against oxygen demand. In
the hypoxic regions, two conditions were assumed, one
is the shortage of blood supply with reduced blood flow
and maintained oxygen consumption; the other is
increased oxygen demand with a maintained blood flow
and increased oxygen consumption. In this study, we
aimed to evaluate the relationship between intratumoral
oxygen demand and blood supply in hypoxic regions by
in vivo 15O-labelled gases and 18F-FMISO PET using the
rat xenograft model of C6 glioma, focusing on the oxygen
Preparation of 18F-FMISO and 15O-labelled gases
18F-FMISO was produced by the method as described
previously with minor modifications [7, 8]. In brief,
18F-fluoride was produced with a cyclotron by the
18O(p, n)18F nuclear reaction using 18O–H2O. 18F-fluoride
was trapped with an ion-exchange resin QMA sep-pak
light cartridge and eluted from the resin by 210 mmol/L
K2CO3 solution (0.2 mL) and 31 mg/mL Kryptofix 2.2.2 in
acetonitrile solution (K222, 0.7 mL). Water was removed
by azeotropic distillation. To the residue, repeated addition
of acetonitrile solution (0.2 mL) and azeotropic
distillation was performed. The dried 18F-KF/K222 complex
mentioned above was added to the precursor
(3-(2-nitroimidazol-1-yl)-2-O-tetrahydropyranyl-1-O-toluenesulfonylpropanediol 5 mg) in acetonitrile solution (0.7 mL) and
reacted at 110 °C for 10 min. After cooling, the reaction
mixture was added to 0.5 M HCl (0.7 mL). The mixture
was hydrolyzed by heating for 5 min at 100 °C. After
cooling, the mixture was neutralized with 1.5 M sodium
acetate and transferred to the high-pressure liquid
chromatography (HPLC) injector. Crude product was purified
with semi-preparative HPLC (column: YMC-ODS-AM
10 × 250 mm, mobile phase: 3% EtOH/H2O, flow rate:
5.0 mL/min). The purified fraction was evaporated to
dryness, and the residue was dissolved in saline containing
benzyl alcohol (24 μL). The specific activity ranged from
30 to 300 GBq/μmol at the end of synthesis.
Radiochemical purity was higher than 98%.
15O-labelled gases were produced by a 14N(p, n)15O
nuclear reaction with 2.0% O2 (for 15O–CO and 15O–O2) or
2.0% CO2 (for 15O–CO2) added N2 gas target at a 12-MeV
proton 7 μA current accelerated by the CYPRISR HM 12S
in-house cyclotron (Sumitomo Heavy Industry).
Animal preparation and implantation of C6 glioma cells
Male Fischer rats from Charles River Japan, Inc. were
housed under a 12-h light/12-h dark cycle and had free
access to food and water. A rat glioma C6 cell line, which
was derived from gliomas induced by
N-nitrosomethylurea, was provided by the RIKEN BRC. The cells were
cultured in MEM medium (Sigma-Aldrich) with 10% fetal
bovine serum (Sigma-Aldrich) at 37 °C in a humidified
incubator containing 5% CO2. Cultured cells were collected
after washing in PBS and harvested with trypsin. Tumor
xenograft models were established by the subcutaneous
injection of 1 × 10^6 tumor cells suspended in 0.2 mL of
culture medium and Matrigel (1:1; BD Biosciences) into
the bilateral shoulder region of F344 rats (a total of two
sites in the left and right shoulder per one rat). PET
experiments were performed 2 to 3 weeks after the
implantation of C6 glioma cells, when the tumor diameter had
reached 1–2 cm.
Ten rats with tumor xenografts (8 weeks age, body
weight = 220 ± 15 g) were anesthetized with 2%
isoflurane with room air, and a Terumo 24-G indwelling
cannula was inserted into the tail vein. PET/CT data were
acquired with a small-animal PET system (Inveon PET/
CT system, Siemens Medical Solutions) . The animals
were placed in a feet-first supine position in the PET
scanner. 18F-FMISO (69.9 ± 10.1 MBq) was
administered intravenously via the catheter cannula, and
dynamic PET scans (60 min) were started at the same
time (n = 4). Delayed static scans (10 min) were performed
at 3 h post injection in all rats (n = 10). CT acquisition was
performed before or after the PET acquisition.
15O-labelled gases inhalation PET
The next day after FMISO PET, the tumoral blood flow
(TBF), tumoral metabolic rate of oxygen (TMRO2),
oxygen extraction fraction (OEF), and tumoral blood volume
(TBV) were measured with a steady-state inhalation
method under artificial ventilation of 15O–CO2, 15O–O2,
and 15O–CO gases mixed with room air and oxygen
[10, 11]. Rats were anesthetized with 2% isoflurane with
room air during the arterial cannulation in the femoral
artery, later switched to intramuscular injection of
midazolam (1.2 mg/kg body weight), xylazine (4.8 mg/kg body
weight), and butorphanol (1.6 mg/kg body weight). The
stability of the anesthesia was already established in the
previous study . Tracheotomy was performed, and
artificial ventilation was started (tidal volume 2–3 mL,
Inhalation of the 15O–CO2 (200 MBq/min) or 15O–O2
(400 MBq/min) gas was continued during the PET
measurements for 16 min. The inhalation time of the 15O–CO
gas (400 MBq/min) was 3 min, and the PET
measurements were continued for a total of 13 min. Arterial blood
sampling (0.1 mL) was performed during the steady-state
PET acquisition in the 15O–CO2 and 15O–O2 gases
studies (13 and 16 min after the start of the scanning) and
10 min after the start of the scanning in the 15O–CO gas
study (A total of five blood samplings per rat). The
radioactivity and weight of the whole blood and plasma after
centrifugation (3000 round/min (800g), 3 min) were
measured with a well scintillation counter (BeWell, Molecular
Imaging Labo). CT acquisition was performed after the
PET acquisition for the attenuation and scatter correction.
Systemic blood pressure (BP) and heart rate (HR) were
indirectly measured with a tail-cuff apparatus (BP-98A-L,
Softron). Arterial blood gas (ABG) analysis was performed
from the blood samples (i-STAT system, Abbott Point of
Care Inc). The body temperature was maintained with a
heating sheet system.
Quantitative data calculation
All PET data were reconstructed with 3-dimensional
ordered-subset expectation maximization followed by
maximum a posteriori (OSEM3D-MAP) (16 subsets, 2
OSEM3D, and 18 MAP iterations) with attenuation and
scatter correction. The image matrix was 128 × 128 × 159,
which yielded a voxel size of 0.776 × 0.776 × 0.796 mm.
Quantitative values of TBF, TMRO2, OEF, and TBV
were calculated by the steady-state inhalation method
by Frackowiak et al. [10, 11]. Detailed calculation formulas
were written in our previous study . In brief, TBF was
calculated from the 15O–CO2 PET using the radioactivity
counts of the whole blood during the steady state. TMRO2
and OEF were calculated from the 15O–CO2 and 15O–O2
PET using the radioactivity counts of the whole blood and
plasma during the steady state. TBV was calculated from
the 15O–CO PET using the radioactivity counts of the
whole blood. Functional images of the TBF, TMRO2, OEF,
and TBV were reconstructed using in-house software
from the last 6-min frame of each 15O-labelled gases
study. A tissue–blood partition coefficient for water was
fixed at 0.91 mL/g . The correction value of the
hematocrit between the great vessels and the tissue was
fixed at 0.70 . The TBV data were used to correct for
intravascular hemoglobin-bound 15O2 .
Quantitative PET data analysis
18F-FMISO PET images were co-registered to
15O-labelled gases PET with reference to the CT image using
AMIDE software (Ver. 1.0.1) and PMOD software (Ver.
3.404). To compare the distribution of the two PET
images, multiple small volumes of interest (VOIs, 1-mm
sphere) were manually placed over the tumor of
18FFMISO PET images (static images 3 h post injection) and
functional images of 15O-labelled gases PET according to
a previous study . In setting VOIs, co-registered
coronal images of 18F-FMISO (window level 0–3) and TBF
(window level 0–80) were displayed using view tool of
PMOD and 1-mm sphere VOIs were paved on PET
images over the entire tumor where there is an uptake of
18F-FMISO or TBF (Additional file 1: Figure S1).
In addition, 8-mm sphere VOIs were placed on the
forelimb muscle and used for the evaluation of
background tissue activity to determine the threshold level of
significant FMISO uptake. The quantitative values of
TBF, TMRO2, OEF, and TBV were plotted against the
18F-FMISO SUV for correlation analysis. Next, VOIs
were divided into three groups according to the
18FFMISO SUV: group Low, low 18F-FMISO uptake region
(less than 1.0 in SUV); group Medium, moderate
18FFMISO uptake region (between 1.0 and 2.0 in SUV); and
group High, high 18F-FMISO uptake region (more than
2.0 in SUV). The threshold value between group Low
and Medium was determined by the background tissue
activity, using the forelimb muscle uptake of 18F-FMISO
SUV (the value obtained by adding the average and
twice the standard deviation). The threshold value
between group Medium and High was determined
according to the previous study which reported the 18F-FMISO
SUV of 2.0 corresponded to the pO2 measurement of
2.5 mmHg .
After 15O-labelled gases PET/CT, pimonidazole (hypoxia
marker, HypoxyprobeTM-1, 60 mg/kg) was injected
60 min before euthanasia. The tumor tissue samples were
resected and fixed overnight with 4% paraformaldehyde
and later cryoprotected in 30% sucrose in PBS before
storage at −80 °C. Frozen sections (10–14 μm thick) were
obtained with a cryostat (CryoStar NX70, Thermo
Scientific) and mounted on glass slides. After blocking
with blocking agent (Dako REAL Peroxidase-Blocking
Solution) for 5 min at room temperature, incubations with
the primary antibody against pimonidazole (FITC-MAb1,
1/100 dilution) for 30 min and the second antibody (HRP
linked to rabbit anti-FITC, 1/100 dilution with Dako
REAL Antibody Diluent) for 30 min were performed in
order. Mounted sections were coverslipped after
dehydration in ethanol and clearance with xylene, followed by
3,3′Diaminobenzidine (DAB) (Dako Envision) and hematoxylin
Correlation analyses of TBF, TMRO2, OEF, and TBV
against the 18F-FMISO SUV were performed with
Spearman’s correlation coefficient. The quantitative values of
TBF, TMRO2, OEF, and TBV were compared among the
three groups with multiple comparison tests (Games–
Howell test). Probability values of less than 0.05 were
considered to denote statistical significance using SPSS
version 19.0 (SPSS).
The number of animals and tumor regions which were
included in the analysis are summarized in Table 1. The
time activity curves of 18F-FMISO PET showed increased
accumulation in the tumor and wash out from
background tissue over time (Fig. 1a). Hypoxic regions in the
tumor were clearly visualized at 3 h post injection of
18F-FMISO (Fig. 1b).
The mean systolic and diastolic BP, HR, PaO2, PaCO2,
hemoglobin concentration (Hb), hematocrit (Ht), and
oxygen saturation (SaO2) during 15O-labelled gases PET
are summarized in Table 2. The time activity curves of
15O-labelled gases PET reached a steady state
approximately 10 min after continuous inhalation of 15O–CO2 or
15O–O2 gas (Fig. 2). Figure 3a shows mild but almost the
same distribution pattern in TMRO2 as compared to TBF,
partial elevation of OEF in the area close to the center,
Static 18F-FMISO PET (3 h post injection)
15O–O2 PET (TMRO2 and OEF)
In one rat, TMRO2 and OEF images were not generated and excluded from the
evaluation due to the technical reason
and a high TBV spot in the marginal zone of the tumor.
Co-registered images of 18F-FMISO and TBF showed that
intratumoral hypoxic regions with FMISO uptakes were
located as lining the inside of the high TBF area and there
was no accumulation in the central area inside the FMISO
uptake, suggesting a necrotic region (Fig. 3b–d).
Relationships between 18F-FMISO uptake and
quantitative values of 15O-labelled gases PET are shown in
Fig. 4a. There were moderate negative correlations
between 18F-FMISO SUV and TBF (r = −0.56 and p < 0.01),
and weak negative correlations between 18F-FMISO SUV
and TMRO2 (r = −0.38 and p < 0.01) and 18F-FMISO SUV
and TBV (r = −0.38 and p < 0.01). Quantitative values in
the Low (L), Medium (M), and High (H) groups were as
follows: TBF, (L) 55 ± 30, (M) 32 ± 17, and (H) 30 ±
15 mL/100 mL/min; OEF, (L) 33 ± 14, (M) 36 ± 17, and
(H) 41 ± 16%; TMRO2, (L) 2.8 ± 1.3, (M) 1.9 ± 1.0, and (H)
2.1 ± 1.1 mL/100 mL/min; and TBV, (L) 5.7 ± 2.1, (M)
4.3 ± 1.9, and (H) 3.9 ± 1.2 mL/100 mL, respectively
(Fig. 4b). Intratumoral hypoxic regions (group Medium
and High) showed significantly lower TBF, TMRO2, and
TBV values than non-hypoxic regions (group Low). OEF
showed significant increase in the severe hypoxic region
compared to non-hypoxic and mild hypoxic regions.
These findings suggested that oxygen demand was
relatively maintained by increased oxygen extraction
in the hypoxic region.
The pimonidazole-staining immunohistochemistry
revealed a heterogeneous intratumoral distribution of the
hypoxic regions, and high intensity staining was
observed mainly at the edge of the viable region of the
tumor section (Fig. 5a–c). The central area of the tumor
showed necrotic regions partially accompanied by hypoxic
cells (Fig. 5d). Pimonidazole staining corresponded to
18FFMISO uptake regions, surrounded by blood flow
preserved regions (Fig. 6).
We have reported herein on the relationship between
blood supply and oxygen consumption in intratumoral
hypoxic regions according to the 18F-FMISO SUV. Our
results indicated that tumor cells in the hypoxic
environment try to maintain the oxygen metabolism against
decreased blood supply similar to the misery perfusion
which has been observed in steno-occlusive disease in
the brain [10, 16]. As shown in Fig. 4, we observed wide
range of TBF or TMRO2 values in the tumors. This
variability is considered to result from intertumoral
heterogeneity. In some tumors, we observed decreased trend
of TBF and TBV, increased trend of OEF, and stable
TMRO2 against the increase of 18F-FMISO SUV
(Additional file 1: Figure S2). However, weak correlation
was observed between OEF and 18F-FMISO SUV as a
whole, possibly due to the intertumoral heterogeneity of
Fig. 1 a The time activity curves of maximum SUV in the tumor and background tissue of 18F-FMISO PET. b Coronal images of 18F-FMISO PET
and PET/CT fusion (3 h post injection). Tumors are located in the bilateral shoulder region of rats (red arrows)
blood flow and oxygen utility level. Figure 4b shows
significant increase of OEF along with increased 18F-FMISO
SUV, suggesting that high 18F-FMISO uptake regions
could survive the hypoxic conditions by the mechanism of
Table 2 Mean ± standard deviation values of BP, HR, and arterial
blood gas data during PET measurement
Systolic/diastolic BP (mmHg)
Previous studies have reported perfusion and oxygen
metabolism in patients with cerebral gliomas using
15Olabelled gases PET [17–20]. The TBF and TBV values
varied widely depending on the vascularity of the tumor,
whereas significantly decreased TMRO2 and OEF values
were observed in the tumor region compared with
normal gray matter or white matter in the brain. Compared
to the normal brain tissue, glioma cells demanded less
oxygen and received excessive TBF, which has been
defined as the condition of luxury perfusion. The authors
concluded that the mechanism of this luxury perfusion
is unknown, whether it was due to decreased oxygen
consumption as a result of adaptation to hypoxic
conditions or was inherent in the metabolism of the gliomas.
In our study, relatively increased OEF was observed in
Fig. 2 The time activity curves (mean ± standard deviation) of whole tumor of 15O-labelled gases PET (15O–CO2 and 15O–O2 gases, radioactivity
count without decay correction; 15O–CO gas, radioactivity count with decay correction)
Fig. 3 a Functional images of 15O-labelled gases PET using 6-min data during steady-state acquisition (coronal slices of PET/CT fusion). TBF and
TMRO2 images showed marginal dominant distribution (arrow). OEF images showed partial elevation in the area close to the center (arrow head)
and TBV images showed a high spot in the marginal zone of the tumor (arrow head). b Axial, c coronal, and d sagittal images of 18F-FMISO PET
(rainbow color scale), TBF PET (fire color scale), and co-registered fusion images. The upper figures are tumors in the right side and the lower figures
are tumors in the left side
Fig. 4 a Relationships between the 18F-FMISO SUV and quantitative values of TBF, TMRO2, OEF, and TBV. b Quantitative values (mean ± standard
deviation) of group Low with less than 1.0 in 18F-FMISO SUV, group Medium with between 1.0 and 2.0 in 18F-FMISO SUV, and group High with
more than 2 in 18F-FMISO SUV (**p < 0.01 with the multiple comparison test)
Fig. 5 Immunohistochemistry of the intratumoral hypoxic region
(pimonidazole staining). a Overall image of the tumor (summed
image of ×20 magnification), b boundary zone: red color square, c
hypoxic region: yellow color square, and d necrotic region: blue color
square (×200 magnification)
hypoxic regions against non-hypoxic regions, but OEF
was decreased compared to the cerebral OEF (65 ± 9.1%)
in the normal rat brain according to our previous study
. This result was consistent with the previous study
in human by Mineura et al., which reported significantly
decreased OEF in the tumor compared to the normal
brain. The new findings of our study was that
intratumoral hypoxic regions showed relatively increased
oxygen extraction as well as decreased blood flow compared
to the non-hypoxic regions.
We selected 18F-FMISO for a hypoxic tracer as it had
been used most extensively in the previous study .
18F-FMISO PET significantly correlated with the
pimonidazole immunohistochemical staining and the
endogenous hypoxia related marker CA IX . 18F-FMISO has
no protein binding, diffuses freely, and accumulates in
those regions with a low oxygen concentration, which
enables a delayed scan (2–3 h post injection) to achieve
the best contrast [23, 24]. We compared the two PET
scans in this study, and multiple VOI-based comparison
was a feasible method as reported in the previous study
to prevent high variability of data in the voxel based
analysis . VOI size of 1 mm was selected because
intratumoral distribution of 18F-FMISO was heterogeneous,
and larger VOIs led to ambiguous evaluation between
the hypoxic and edge regions. In 15O-labelled gases
experiments, we need at least 2 h for total anesthesia time
from the preparation of an arterial sampling line to the
end of 15O-labelled gases PET. Performing 15O-labelled
gases and FMISO studies in 1 day is an extreme
overload for rats due to the prolonged anesthesia. We
performed less invasive FMISO PET at first and set the
interval of 1 day between FMISO and 15O-labelled gases
PET in this study. The anesthesia methods were
different between FMISO PET and 15O-labelled gases PET.
We monitored the systemic blood pressure, heart rate,
and arterial blood gas data during 15O-labelled gases PET
and confirmed that these factors were within physiological
range and stable (Table 2). In addition, pimonidazole
staining after 15O-labelled gases PET matched with the
18F-FMISO PET. It was considered that the difference
about anesthesia had a minor effect for the evaluation of
tumor perfusion and oxygen metabolism.
As for the arterial measurement during 15O-labelled
gases PET, two blood samples were taken per each scan
during steady state (13 and 16 min after the start of the
scanning) and the average value was used for the
quantitative calculation. Percent differences of the radioactivity
Fig. 6 Comparison among pimonidazole staining, 18F-FMISO PET, TBF, and fusion image. Co-registered 18F-FMISO and TBF images were resliced
to the same section as pimonidazole staining for comparison using the PMOD software
between the two samples were 4.5 ± 3.3% for the whole
blood and 6.0 ± 5.6% for the plasma. The measurements
of arterial blood were considered to be sufficiently
accurate and stable.
There are two types of tumor hypoxia, acute hypoxia
(perfusion limited hypoxia) and chronic hypoxia
(diffusion limited hypoxia) [6, 21]. Acute hypoxia is a
transient hypoxic condition which can change from day to
day, whereas chronic hypoxia is a rather stable
condition. There is a possibility that hypoxic regions could be
different between 18F-FMISO PET and 15O-labelled
gases PET. Okamoto reported the high reproducibility of
tumor hypoxia by comparing two 18F-FMISO PET scans
within a 48-h interval in patients with head and neck
cancer . They concluded that the values for the
18FFMISO PET uptake (SUV and uptake ratio of tumor to
muscle) and the hypoxic volume between the two scans
were highly reproducible. Furthermore, we performed
immunohistochemical staining with pimonidazole after
15O-labelled gases PET, which corresponded to the
18FFMISO PET findings seen on the previous day. These
results demonstrated that the main hypoxia in the tumor
was chronic hypoxia. However, acute component cannot
be excluded in the low TBF regions as we evaluated one
time point evaluation of 15O-labelled gases PET. Further
study is necessary to evaluate the involvement of acute
ischemia by investigating the temporal change, such as
repetitive 15O-labelled gases PET studies.
As shown in Figs. 3 and 5, the central regions in the
tumors were deficient in 18F-FMISO uptake and blood
flow, which corresponded to the necrotic regions. These
regions were excluded from the VOIs analysis in this
study as multiple small sphere VOIs were placed on the
tumor with 18F-FMISO uptake or blood flow uptake.
However, since some necrotic regions were mixed
with viable hypoxic cells as shown in the
pimonidazole staining images, it is not always possible to
clearly separate the hypoxic and necrotic regions in
This study demonstrated that tumor cells were
resistant to the reduction in blood flow through the increased
OEF and it will be an effective treatment strategy to
modulate the blood flow, such as therapy using
bevacizumab with a monoclonal antibody targeting vascular
endothelial growth factor which normalizes vascular
architecture, to deliver anti-tumor drugs more effectively
and enhance the radiosensitizing effect through
oxygenation. Bevacizumab is effective for some cancer patients
in combination with chemotherapy. Recently, treatment
with bevacizumab has been performed in patients with
malignant gliomas, which led to an improved blood flow
to the tumor [26, 27]. However, it was reported that high
doses of bevacizumab might have the risk of increased
hypoxia due to the decreased perfusion in the tumor
. Conversely, low dose could not yield survival
benefit in non-small-cell lung cancer patients treated with 15
or 7.5 mg/kg bevacizumab with chemotherapy .
Another study also reported that decreased perfusion also
resulted in the reduced drug delivery after administration of
bevacizumab and scheduling of anti-angiogenic drugs
should be optimized . It was suggested that optimizing
the individual dose could improve patient outcome by the
pre-treatment evaluation with imaging techniques, such
as FMISO and 15O-labelled gases or water PET, as
perfusion and hypoxic conditions are heterogeneous in each
cancer patient. In addition, there is no enough evidence
about temporal change of perfusion and oxygen
metabolism after bevacizumab therapy. The next step of our study
is to elucidate how the oxygen metabolism could be
changed after the vascular normalization with bevacizumab.
There are some limitations in this study. In
smallanimal PET systems, the spatial resolution (full width at
half maximum, FWHM) of 15O is approximately
doubled compared to 18F or 11C due to the longer positron
range . The spatial resolution is about 1.5 mm
FWHM in this study, and the partial volume effect
(PVE) might be larger on 15O-labelled gases PET images
than 18F-FMISO PET (9). PVE is a principle problem of
PET that has been long discussed. We must pay
attention to the underestimation by PVE in the evaluation of
intratumoral distribution with PET. However, in our
previous study, elevated OEF with decreased cerebral blood
flow was detected by 15O-labelled gases PET in the rat
stroke model, suggesting the feasibility to detect the
subtle changes in tumor perfusion or oxygen consumption.
Next, we fixed the tissue–blood partition coefficient as
0.91 mL/g and the correction value of the hematocrit as
0.70 according to previous studies of the brain [12, 13].
If the partition coefficient was substantially different in
some area, it has some influence on TBF values.
However, these effects were relatively small in low TBF area,
such as necrotic area. TBF was increased from 20 to 22
or 25 mL/100 mL/min when the partition coefficient
was changed from 0.91 to 0.8 or 0.7, respectively. In
addition, previous study reported tissue heterogeneity
always resulted in underestimations of mean values of
oxygen extraction fraction by the simulation . It may
be necessary to develop a new tracer kinetic model
considering tumor heterogeneity for more accurate
evaluation. Third, this study was conducted on a single tumor
model without using anti-tumor drugs. It is the next
challenge to investigate other types of tumor model
including the relationship between glycolysis and oxidative
phosphorylation and to evaluate the changes in
perfusion and oxygen metabolism after the anti-tumor
therapy or modulation of perfusion and hypoxia.
This study demonstrated the in vivo relationships among
18F-FMISO SUV, TBF, TMRO2, OEF, and TBV values in
the rat xenograft model of C6 glioma with an
immunohistochemical evaluation. Intratumoral hypoxic regions
showed decreased blood flow with increased oxygen
extraction compared to the non-hypoxic regions. These
findings suggested that a treatment strategy to normalize
the blood flow would be a reasonable approach for
oxygen-avid active tumor cells in hypoxic regions to
achieve more effective drug delivery in chemotherapy
and an oxygen sensitizing effect in radiation therapy.
Additional file 1: Figure S1. Multiple VOI settings on coronal fusion
image of 18F-FMISO PET (window level 0–3 in SUV) and TBF PET (window
level 0–80 ml/100 mL/min). VOIs (1-mm sphere) were manually placed over
the entire tumor where there is an uptake of 18F-FMISO or TBF on the
fusion PET images. Figure S2. Relationships between the 18F-FMISO SUV
and quantitative values of TBF, TMRO2, OEF, and TBV of each tumor.
Decreased trend of TBF and TBV, increased trend of OEF, and stable TMRO2
against the increase of 18F-FMISO SUV were observed. (DOC 673 kb)
We would like to thank all the member of the Department of Nuclear
Medicine, Molecular Imaging in Medicine, especially Hitoshi Iimori, Hiroko
Yamato, and Sotaro Momosaki for providing the 18F-FMISO and Rumi Saika
for the excellent technical assistance.
This study was supported by KAKENHI Grant (Number 25861095) from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
No other potential conflict of interest relevant to this article was reported.
TW conducted the experiments, performed the analysis, and wrote the
manuscript. YK, HI, GH, KI, and KA assisted in the experiments. KM and HK
assisted in the data analysis. ES and JH were the supervisors of the study. All
authors contributed to the writing of the article and approved the final version.
All the animal experiments were performed in compliance with the guidelines
of the Institute of Experimental Animal Sciences. The protocol was approved by
the Animal Care and Use Committee of the Osaka University Graduate School
of Medicine (Approval Number 20-144-008).
1. Vaupel P , Thews O , Hoeckel M. Treatment resistance of solid tumors: role of hypoxia and anemia . Med Oncol . 2001 ; 18 ( 4 ): 243 - 59 .
2. Hicks RJ , Rischin D , Fisher R , Binns D , Scott AM , Peters LJ . Utility of FMISO PET in advanced head and neck cancer treated with chemoradiation incorporating a hypoxia-targeting chemotherapy agent . Eur J Nucl Med Mol Imaging . 2005 ; 32 ( 12 ): 1384 - 91 .
3. Arteel GE , Thurman RG , Raleigh JA . Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state . Eur J Biochem . 1998 ; 253 ( 3 ): 743 - 50 .
4. Krohn KA , Link JM , Mason RP . Molecular imaging of hypoxia . J Nucl Med . 2008 ; 49 Suppl 2 : 129S - 48S .
5. Bowen SR , van der Kogel AJ , Nordsmark M , Bentzen SM , Jeraj R. Characterization of positron emission tomography hypoxia tracer uptake and tissue oxygenation via electrochemical modeling . Nucl Med Biol . 2011 ; 38 ( 6 ): 771 - 80 .
6. Chaudary N , Hill RP . Hypoxia and metastasis . Clin Cancer Res . 2007 ; 13 ( 7 ): 1947 - 9 .
7. Tang G , Wang M , Tang X , Gan M , Luo L. Fully automated one-pot synthesis of [18F]fluoromisonidazole . Nucl Med Biol . 2005 ; 32 ( 5 ): 553 - 8 .
8. Oh SJ , Chi DY , Mosdzianowski C , Kim JY , Gil HS , Kang SH , et al. Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module . Nucl Med Biol . 2005 ; 32 ( 8 ): 899 - 905 .
9. Bao Q , Newport D , Chen M , Stout DB , Chatziioannou AF . Performance evaluation of the inveon dedicated PET preclinical tomograph based on the NEMA NU-4 standards . J Nucl Med . 2009 ; 50 ( 3 ): 401 - 8 .
10. Watabe T , Shimosegawa E , Watabe H , Kanai Y , Hanaoka K , Ueguchi T , et al. Quantitative evaluation of cerebral blood flow and oxygen metabolism in normal anesthetized rats: 15O-labeled gas inhalation PET with MRI Fusion . J Nucl Med . 2013 ; 54 ( 2 ): 283 - 90 .
11. Frackowiak RS , Lenzi GL , Jones T , Heather JD . Quantitative measurement of regional cerebral blood flow and oxygen metabolism in man using 15O and positron emission tomography: theory, procedure, and normal values . J Comput Assist Tomogr . 1980 ; 4 ( 6 ): 727 - 36 .
12. Herscovitch P , Raichle ME . What is the correct value for the brain-blood partition coefficient for water ? J Cereb Blood Flow Metab . 1985 ; 5 ( 1 ): 65 - 9 .
13. Cremer JE , Seville MP . Regional brain blood flow, blood volume, and haematocrit values in the adult rat . J Cereb Blood Flow Metab . 1983 ; 3 ( 2 ): 254 - 6 .
14. Lammertsma AA , Jones T. Correction for the presence of intravascular oxygen-15 in the steady-state technique for measuring regional oxygen extraction ratio in the brain: 1. Description of the method . J Cereb Blood Flow Metab . 1983 ; 3 ( 4 ): 416 - 24 .
15. Lohith TG , Kudo T , Demura Y , Umeda Y , Kiyono Y , Fujibayashi Y , et al. Pathophysiologic correlation between 62Cu-ATSM and 18F-FDG in lung cancer . J Nucl Med . 2009 ; 50 ( 12 ): 1948 - 53 .
16. Yamauchi H , Higashi T , Kagawa S , Nishii R , Kudo T , Sugimoto K , et al. Is misery perfusion still a predictor of stroke in symptomatic major cerebral artery disease? Brain . 2012 ; 135 (Pt 8): 2515 - 26 .
17. Ito M , Lammertsma AA , Wise RJ , Bernardi S , Frackowiak RS , Heather JD , et al. Measurement of regional cerebral blood flow and oxygen utilisation in patients with cerebral tumours using 15O and positron emission tomography: analytical techniques and preliminary results . Neuroradiology . 1982 ; 23 ( 2 ): 63 - 74 .
18. Mineura K , Sasajima T , Kowada M , Ogawa T , Hatazawa J , Shishido F , et al. Perfusion and metabolism in predicting the survival of patients with cerebral gliomas . Cancer . 1994 ; 73 ( 9 ): 2386 - 94 .
19. Mineura K , Sasajima T , Itoh Y , Sasajima H , Kowada M , Tomura N , et al. Blood flow and metabolism of central neurocytoma: a positron emission tomography study . Cancer . 1995 ; 76 ( 7 ): 1224 - 32 .
20. Mineura K , Shioya H , Kowada M , Ogawa T , Hatazawa J , Uemura K. Blood flow and metabolism of oligodendrogliomas: a positron emission tomography study with kinetic analysis of 18F-fluorodeoxyglucose . J Neurooncol . 1999 ; 43 ( 1 ): 49 - 57 .
21. Wijsman R , Kaanders JH , Oyen WJ , Bussink J. Hypoxia and tumor metabolism in radiation oncology: targets visualized by positron emission tomography . Q J Nucl Med Mol Imaging . 2013 ; 57 ( 3 ): 244 - 56 .
22. Dubois L , Landuyt W , Haustermans K , Dupont P , Bormans G , Vermaelen P , et al. Evaluation of hypoxia in an experimental rat tumour model by [(18)F]fluoromisonidazole PET and immunohistochemistry . Br J Cancer . 2004 ; 91 ( 11 ): 1947 - 54 .
23. Mendichovszky I , Jackson A. Imaging hypoxia in gliomas . Br J Radiol . 2011 ; 84 Spec No 2: S145 - 58 .
24. Carlin S , Humm JL. PET of hypoxia: current and future perspectives . J Nucl Med . 2012 ; 53 ( 8 ): 1171 - 4 .
25. Okamoto S , Shiga T , Yasuda K , Ito YM , Magota K , Kasai K , et al. High reproducibility of tumor hypoxia evaluated by 18F-fluoromisonidazole PET for head and neck cancer . J Nucl Med . 2013 ; 54 ( 2 ): 201 - 7 .
26. Schwarzenberg J , Czernin J , Cloughesy TF , Ellingson BM , Pope WB , Geist C , et al. 3 ′ -deoxy-3′-18F-fluorothymidine PET and MRI for early survival predictions in patients with recurrent malignant glioma treated with bevacizumab . J Nucl Med . 2012 ; 53 ( 1 ): 29 - 36 .
27. Gonzalez J , Kumar AJ , Conrad CA , Levin VA . Effect of bevacizumab on radiation necrosis of the brain . Int J Radiat Oncol Biol Phys . 2007 ; 67 ( 2 ): 323 - 6 .
28. Jain RK . Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia . Cancer Cell . 2014 ; 26 ( 5 ): 605 - 22 .
29. Reck M , von Pawel J , Zatloukal P , Ramlau R , Gorbounova V , Hirsh V , et al. Overall survival with cisplatin-gemcitabine and bevacizumab or placebo as first-line therapy for nonsquamous non-small-cell lung cancer: results from a randomised phase III trial (AVAiL) . Ann Oncol . 2010 ; 21 ( 9 ): 1804 - 9 .
30. Van der Veldt AA , Lubberink M , Bahce I , Walraven M , de Boer MP , Greuter HN , et al. Rapid decrease in delivery of chemotherapy to tumors after antiVEGF therapy: implications for scheduling of anti-angiogenic drugs . Cancer Cell . 2012 ; 21 ( 1 ): 82 - 91 .
31. Levin CS , Hoffman EJ . Calculation of positron range and its effect on the fundamental limit of positron emission tomography system spatial resolution . Phys Med Biol . 1999 ; 44 ( 3 ): 781 - 99 .
32. Lammertsma AA , Jones T. Low oxygen extraction fraction in tumours measured with the oxygen-15 steady state technique: effect of tissue heterogeneity . Br J Radiol . 1992 ; 65 ( 776 ): 697 - 700 .