NMB-08272; No of Pages 8

Nuclear Medicine and Biology 110 (2022) xxx

Contents lists available at ScienceDirect

Nuclear Medicine and Biology

journal homepage: www.elsevier.com/locate/nucmedbio

Correlation of hypoxia PET tracer uptake with hypoxic radioresistance in

cancer cells: PET biomarkers of resistance to stereotactic
radiation therapy?
Kazumi Chia a,⁎, Rowena L. Paul a, Amanda J. Weeks a, Marium Naeem b, Gregory E.D. Mullen a,
David Landau a, Philip J. Blower a
a
King's College London, School of Biomedical Engineering and Imaging Sciences, St. Thomas' Hospital, London SE1 7EH, UK
b
Department of Medical Physics, Guy's and St Thomas' NHS Foundation Trust, London SE1 9RT, UK

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: The pO2 threshold of an ideal PET hypoxia tracer for radiotherapy planning in cancer would match those
Received 25 January 2022 observed in clinically and biologically relevant processes such as radioresistance and HIF1α expression. To iden-
Received in revised form 24 March 2022 tify such tracers, we directly compared uptake in vitro of hypoxia PET tracers ([18F]FMISO, [64Cu]CuATSM, and an-
Accepted 6 April 2022 alogues [ 64Cu]CuATS, [64Cu]CuATSE, [64Cu]CuCTS, [ 64Cu]CuDTS, [64Cu]CuDTSE, [64Cu]CuDTSM) with levels of
Available online xxxx
radioresistance and HIF1α expression in cultured cancer cells under identical hypoxic conditions ranging from
extreme hypoxia to normoxia. Pimonidazole uptake was also compared as a marker of hypoxia.
Methods: A custom-built hypoxia apparatus enabled all experiments to be performed under identical hypoxic
conditions with constant measurement of pO2 in media using an OxyLab pO2™ probe. HCT116 human colonic
carcinoma and MCF-7 human Caucasian breast adenocarcinoma cells were irradiated using a cobalt teletherapy
unit. Clonogenic assays were used to assess survival. HIF1α expression was determined by western blotting,
tracer uptake by gamma counting and pimonidazole binding by flow cytometry.
Results: Radioresistance, pimonidazole binding and HIF1α expression increased gradually as pO2 decreased between
25 mmHg and 0 mmHg. In contrast, all the PET hypoxia tracers showed a sharp increase in uptake only when pO2
levels fell below 1 mmHg. Above this threshold, tracer uptake was not elevated above that in normoxic cells.
Conclusion: This study highlights an important mismatch in pO2 thresholds between these PET tracers and other
markers of hypoxia: tracer uptake only occurred at oxygen levels that were well below levels that induced
radioresistance, pimonidazole uptake and HIF1α expression. Although their pO2 thresholds do not match the
threshold for resistance to conventionally fractionated radiotherapy (pO2 2.5–10 mmHg), their specificity for
extreme hypoxia (pO2 ≪ 1 mmHg) suggests these PET tracers may be of particular use to predict outcomes in ste-
reotactic radiation therapy where these maximally resistant cells play a key role in determining the biological effect.
© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

1. Introduction Non-invasive imaging using positron-emission tomography (PET)

Since 1955 when Thomlinson and Gray described the anatomical
basis of tumor hypoxia using histological sections of fresh lung cancer
specimens [1], scores of studies have demonstrated tumor hypoxia to
be a characteristic feature of many solid tumours [2]. After more than
50 years of research, we know that tumor hypoxia is a key determinant
not only of cancer prognosis [3,4], but also of response to radiotherapy
[5,6], surgery [7] and chemotherapy [8].
⁎ Corresponding author at: Guy's Cancer Centre, Great Maze Pond, London SE1 9RT, UK.
E-mail address: kazumi.chia2@nhs.net (K. Chia).
has been one of many approaches used in identifying tumor hypoxia
[9] but in contrast to other targets for PET imaging where the molecular
target is easily defined (e.g. αvβ3, prostate-specific membrane antigen,
hexokinase, thymidine kinase), hypoxia is not a well-defined entity
[10–12]. In the body, O2 molecules move along a concentration
gradient from the alveolar air in the lungs to the cytosol (Fig. 1) and
O2 tensions vary along this concentration gradient [13]. If hypoxia is
defined as a state of O2 deficiency in tissue, there will be a multitude
of different critical O2 thresholds below which normal physiological
function is disturbed in different ways. In oncology, different critical
O2 thresholds have been demonstrated for a number of anti-cancer
therapies and important cellular functions [14]. In other disease states
such as ischaemic heart disease and ischaemic brain disease, critical O2

https://doi.org/10.1016/j.nucmedbio.2022.04.004
0969-8051/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: K. Chia, R.L. Paul, A.J. Weeks, et al., Correlation of hypoxia PET tracer uptake with hypoxic radioresistance in cancer cells:
PET biomarkers of resis..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2022.04.004
K. Chia, R.L. Paul, A.J. Weeks et al.
Oxygen
Air Lung Artery Capillary Tissue
Fig. 1. Diagrammatic representation of the pO2 gradient from atmospheric air to the cell.
(Based on data from ref. 13).
thresholds are different again [15]. Therefore it is unlikely that a single
hypoxia imaging agent will have universal application, even within
one clinical speciality such as oncology. The pO2 threshold of an ideal
PET hypoxia tracer for radiotherapy planning in cancer would match
those observed in clinically and biologically relevant processes such as
radioresistance and HIF1α expression.
The aims of this study were as follows: 1) For the first time, to deter-
mine the extent to which radiosensitivity and HIF1α expression corre-
late with hypoxic PET tracer uptake in the same in vitro cell system
under controlled conditions; 2) To do so using rigorous, validated,
continuous measurement of pO2 in the cell suspension rather than
relying on the oxygen content of the gas cylinder used; 3) To apply
the resulting understanding to better clinical application and
interpretation of PET hypoxia tracers, particularly in the field of radio-
therapy where newer techniques such as stereotactic radiation therapy
are opening up fresh opportunities for hypoxia targeting/modification
strategies [16]. [ 18F]FMISO is the “prototypical” clinical nitroimidazole-
based PET hypoxia tracer and remains the most well-validated [9]. Cop-
per bis(thiosemicarbazone) complexes in which the diimine backbone
carries two alkyl groups (among which [64Cu]CuATSM is the prototype)
are well-validated in vitro as having selective uptake in hypoxic cells
[17–19], but in vivo, some studies show correlation with hypoxia [18,
20] and others do not; analogues with improved pharmacokinetics
have been evaluated recently [18,21]. Therefore, [ 18F]FMISO, [ 64Cu]
CuATSM and a selection of analogous copper complexes with subtle
changes to the bis(thiosemicarbazone) side chains, known to exhibit se-
lective uptake in hypoxic cells but with potentially different hypoxia se-
lectivity and pharmacokinetics, were included in the evaluation.
2. Materials and methods
Production and quality control of copper-64 was performed as de-
scribed previously and is outlined in Supplemental Data [22]. Synthesis
of copper-64 bis(thiosemicarbazone) complexes was performed as pre-
viously described except for the DTSE complex, synthesis of which is de-
scribed in Supplemental Data. Production and quality control of [ 18F]
FMISO was performed as described in Supplemental Data [17,19].
Threshold oxygen levels for radioresistance, PET radiotracer uptake,
pimonidazole uptake and HIF1α expression were defined as the pO2
at which the value of each of the parameters was the mean of the
maximum and minimum plateau levels. This value was determined
objectively by fitting (least squares) the data to the sigmoid function
(unless otherwise indicated) chosen from GraphPad Prism (version 5.
03 for Windows; GraphPad Software, San Diego, CA) giving the best fit
(measured as R2) and interpolating the resulting equation.
Nuclear Medicine and Biology 110 (2022) xxx
2.1. Cell culture
HCT116 human colonic carcinoma and MCF-7 human Caucasian
breast adenocarcinoma cells authenticated by microsatellite genotyping
were obtained directly from the European Collection of Cell Cultures
(Health Protection Agency Cell Culture Collections, Salisbury, UK), cul-
tured (37 °C, 95% air and 5% CO2) as monolayers and used in the
exponential phase of growth. For all experiments, cells were
trypsinised immediately prior to use to form a cell suspension. Both
cell lines were regularly assessed for characteristic morphology and
absence of mycoplasma infection (LookOut® Mycoplasma PCR Detec-
tion Kit, Sigma-Aldrich, Poole, Dorset, UK). Culture media and additives
were obtained from PAA Laboratories (Yeovil, Somerset, UK). HCT116
cells were maintained in McCoy's 5a medium supplemented with 10%
FBS, 2 mM glutamine, 100 units/mL penicillin and 100 μg/mL strepto-
Cell mycin. MCF-7 cells were maintained in DMEM (low glucose, 1 g/L) sup-
plemented with 10% FBS, 2 mM glutamine, 100 units/mL penicillin and
100 μg/mL streptomycin. An additional 10% FBS was added to the com-
plete medium used in MCF-7 clonogenic assays. Cell concentration and
viability were determined using a CASY® DT cell counter (Roche Diag-
nostics Ltd., Burgess Hill, West Sussex, UK).
2.2. Hypoxia
Custom-made glass vessels (LEL Glassblowing, March, Cambs., UK)
were designed to contain the hypoxic cell suspension allowing all ex-
periments to be carried out using the same set-up (Supplementary
Fig. 1a). In all experiments, cells were trypsinised and the 40 mL suspen-
sion placed into the vessel and equilibrated with the chosen atmosphere
for 30 min to achieve stable temperature and pO2 (Supplementary Fig.
1b). Certified mixtures of O2 with 5% carbon dioxide and balance
nitrogen were obtained to cover the range of severe and moderate
hypoxia (BOC Special Products, Guildford, Surrey, UK). The gas mixes
supplied were defined in units of percent O2. Assuming atmospheric
pressure of 760 mmHg a factor of 7.6 converts the units to mmHg (0%
= 0 mmHg, 0.1% = 0.76 mmHg, 0.8% = 6.08 mmHg, 1.5% = 11.4
mmHg, 2.7% = 20.5 mmHg, 3.5% = 26.6 mmHg, 4.5% = 34.2 mmHg,
7.3% = 55.5 mmHg and 21% = 159.6 mmHg). Dissolved O2 and
temperature were monitored throughout all experiments with an
OxyLab pO2™ O2 probe (Oxford Optronix, Oxford, Oxfordshire, UK)
with a built-in thermocouple allowing automatic temperature correc-
tion of pO2 measurements. The probes were individually calibrated by
the manufacturer at 37 °C at six different O2 levels created by bubbling
certified gases (0, 1, 2, 5, 14 and 21%). Due to small statistical
fluctuations, readings <0 mmHg were recorded during some
experiments performed at 0% O2 and are directly recorded as such
without correction. To counteract regional O2 depletion in the medium
caused by cellular respiration, suspensions were continuously agitated
either by a plate shaker (SSM5 set at 1080 rpm) in cell irradiation
studies or a rocker (SSM4 set at 65 oscillations/min) (Stuart, Bibby
Scientific Ltd., Stone, Staffordshire, UK) in all other experiments, to aid
O2 diffusion into the medium from the overlying gas. A stable pH of 7.
5 was maintained in the cell suspension, monitored using Hydrion™
Brilliant pH dip sticks (Sigma-Aldrich).
2.3. Cell irradiation
Cells were trypsinised and the suspension (1 × 10 6/mL for HCT116
and 2 × 105/mL for MCF-7) in 40 mL medium was placed in a custom-
built perspex rig attached to a T1000 Cobalt 60 teletherapy unit (Supple-
mentary Fig. 1c). The pO2 in the suspension was measured continuously
for 2 min immediately prior to γ-irradiation to ensure stability. The ra-
diation dose administered in all experiments was 2 Gy as this is a stan-
dard dose per fraction used clinically, and the time to deliver this dose
was approximately 3 min [23]. The syringe used to remove the sample
immediately following irradiation was pre-flushed with the gas mix
2
K. Chia, R.L. Paul, A.J. Weeks et al.
used in the experiment and samples were placed on ice for immediate
plating for the clonogenic assay (see Supplemental Data for details).
For each atmosphere, experiments were performed in triplicate and un-
irradiated control samples were taken. The following equation was used
to fit the data: y = (1 + axc) / (1 + bx c), where x = pO2 (mmHg), y =
relative radiosensitivity (defined as and constrained to be unity at pO2
= 0), and a, b and c are constants optimised separately for each cell line.
2.4. PET tracer retention
The radiotracer stock was diluted with PBS so that 100 kBq in 100 μL
was added to MCF-7 and HCT116 cell suspensions in 40 mL medium
(both cell lines at 1 × 10 6/mL) that had been equilibrated with the cho-
sen atmosphere. Three 200 μL samples were removed with a glass, gas-
tight syringe (Hamilton Company, Reno, NV) pre-flushed with the rele-
vant gas mix, at 1, 5, 15, 30, and 60 min and placed on ice in gas-flushed
crimp-top glass vials (Chromacol, Welwyn Garden City, UK) before
centrifuging at 400g for 30 s. The supernatants (180 μL) and cell pellets
(including 20 μL residual supernatant) were counted in a gamma coun-
ter (Wallac 1282-001 Compugamma CS, Turku, Finland) using
Ultroterm software. Percent radioactivity retained in cells, correcting
for the 20 μL of supernatant left with the cell pellet, was calculated as
[cell pellet counts − (supernatant counts / 9)] / (cell pellet counts + su-
pernatant counts) × 100%. In [ 18F]FMISO experiments, because % uptake
in cells was very low, additional samples were taken at 120 min and a
wash step was added using culture medium to remove unbound [ 18F]
FMISO prior to counting. The following sigmoidal equation was used
to fit the data: y = a + [24] / (1 + 10 logc-x) where x = pO2
(controlled variable), y = % radiotracer uptake (measured variable)
and a, b and c are fitted constants, a representing maximum plateau %
uptake, b representing minimum plateau % tracer uptake, and c
representing half maximal uptake (threshold pO2). The optimised
value of c was taken as the objective threshold.
2.5. Pimonidazole binding
Pimonidazole (HPI, Inc., Burlington, MA) was added to HCT116 cell
suspensions (1 × 10 6/mL in 40 mL) that had been equilibrated with
the chosen atmosphere for 30 min to give a final concentration of 200
μM. After 2 h incubation, 2 × 10 5 cells in 200 μL medium were removed,
centrifuged and resuspended in cold PBS, fixed in 70% ethanol and
stored at −20 °C until they could be processed (by a published method
[22]). Fixed and frozen samples were incubated with fluorescein iso-
thiocyanate (FITC)-conjugated mouse monoclonal IgG1 anti-
pimonidazole antibody (HPI, Inc., Burlington, MA) or FITC-conjugated
mouse monoclonal IgG1 isotype antibody (Caltag-MedSystems Ltd.,
Buckingham, UK) at room temperature. Flow cytometry of
pimonidazole binding was carried out on a FACSCalibur flow
cytometer using BD Cellquest Pro software (BD, Oxford, UK), acquiring
approximately 10,000 events for each analysis. To fit the data, a general-
ised (i.e. constraint-free) variant of the sigmoid equation used above for
radiosensitivity data was used: y = (ax c) / (bc + xc) + d where x = pO2
(controlled variable), y = fluorescence intensity (measured variable,
assumed to be proportional to pimonidazole uptake) and a, b, c and d
are constants optimised (least squares) for the data.
2.6. HIF1α determination
Suspensions of MCF-7 cells (2 × 105 cells in 40 mL) were equilibrated
with the chosen atmosphere for 1.5 h. Samples were transferred using
chilled pipette tips into chilled centrifuge tubes before pelleting cells by
centrifugation at 4 °C. Nuclear protein extracts were prepared using NE-
PER® Nuclear and Cytoplasmic Extraction kit (Pierce Biotechnology,
Rockford, IL) according to the manufacturer's instructions and stored at
−80 °C until required. HIF1α protein was determined using western blot-
ting (see Supplemental Data for details). Since there was no indication of
3
Nuclear Medicine and Biology 110 (2022) xxx
sigmoid form in the data, an exponential function was used to fit the data
using GraphPad Prism version 5.03 for Windows (GraphPad Software, San
Diego, CA): y = [24]exp(−cx) + a, where x = pO2 (controlled variable),
y = normalised optical density of western blot (measured variable,
assumed to be proportional to HIF1α expression), and a, b and c are
constants optimised (least squares) separately for the data: a represents
value of y when x = 0, b represents the minimum plateau value of y
and was constrained to zero since this is the smallest possible value for
x (and this was consistent with the real data). Threshold pO2 was
objectively taken as the value of x when y = (a − b) / 2.
3. Results
3.1. Dissolved oxygen concentration
Despite use of the same cell density, cell line, rocker rate, tempera-
ture, growth phase and certified gas mixtures for each corresponding
experiment, pO2 readings in the cell suspension often varied between
experiments (Supplementary Table 1). Despite continuous agitation of
the cell suspension to aid O2 diffusion into the medium from the
overlying gas, the pO2 measured in the cell suspension was
consistently lower than expected from the gas phase composition (as
compared with cell-free medium). The difference was most pronounced
when using a gas mix with <1 mmHg O2 where the expected (based on
cell-free measurements) pO2 was threefold higher than the real value
measured in the cell suspension (Fig. 2). The reason for this is that
despite agitating the medium with a rocker, limiting the cell
concentration to 1 million per mL, and the use of a large surface area
vessel (78.5 cm 2), gas exchange at the surface of the medium was
unable to keep up with the O2 consumption by cells and became
limiting, especially at very low O2 levels (<1 mmHg O2). This
reinforced the importance of directly measuring and reporting the pO2
of the cell suspension rather than assuming equilibration with the O2
content of the gas mixture to which the cells were exposed (which is
frequent practice in literature in this field). Cell viability was assessed
under these experimental conditions and was found to be >99%
(Supplementary Fig. 2).
3.2. PET tracer retention
PET tracer uptake, radiosensitivity, HIF1α expression and
pimonidazole binding data were plotted on identical pO2 axes in order
Ratio to expected/observed
5
4
pO2 in medium
3
2
1
0
0 10 20 30 40
pO2 in gas mix (mmHg)
Fig. 2. Difference in pO2 between expected level based on gas mix (○) and observed level
in cell free medium (■) and cell containing (HCT116 cells at 1 × 106/mL) medium (Δ) as
measured by OxyLab oxygen sensor. The difference was most pronounced when using a
gas mix with <1 mmHg O2 where the expected (based on cell-free measurements) pO2
was threefold higher than the real value measured in the cell suspension.
K. Chia, R.L. Paul, A.J. Weeks et al. Nuclear Medicine and Biology 110 (2022) xxx

a Radiotracer uptake b Radiosensitivity

80 2.8

Relative Radiosensitivity
[64Cu]CuATSM (HCT116)
% Radiotracer uptake 2.4
60 [64Cu]CuATSM (MCF7)
18
[ F]FMISO (HCT116)
[18F]FMISO (MCF7) 2.0
40

1.6
20 HCT116

1.2 MCF-7
0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Oxygen (mmHg) Oxygen (mmHg)

c Pimonidazole binding d HIF1

Median Fluorescence Intensity

50 0.6

Normalized Densitometry
40 0.5

0.4
30
0.3
20
0.2

10
0.1

0 0.0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Oxygen (mmHg) Oxygen (mmHg)

Fig. 3. (a) pO2 dependence of tracer uptake after incubation for 30 min ([64Cu]CuATSM) and 120 min ([18F]FMISO) (samples taken in triplicate, mean and SEM shown. Unseen bars are
hidden within data points); (b) the pO2 dependence of cellular radiosensitivity (for each atmosphere, experiments were performed in triplicate and unirradiated control samples were
taken; mean and SEM shown. Unseen bars are hidden within data points); (c) pO2 dependence of pimonidazole binding in HCT116 cells (each data point represents an individual
experiment performed at a specific pO2); (d) pO2 dependence of HIF1α expression in MCF-7 cells (see Fig. 6 for the corresponding western blot “A”). This graph is representative of
two sets of experiments covering the range of pO2 levels. The second western blot and graph can be found in Supplementary Fig. 4. Horizontal axes are identical in (a) to (d) for easy
comparison, which shows that threshold pO2 for onset of PET tracer uptake is much lower than that for radioresistance, pimonidazole uptake or HIF1α expression.

to easily compare the different O2 thresholds. Hypoxia-specific reten-
tion of [ 64Cu]CuATSM was seen in both cell lines with similar maximal
percentage retention levels (Fig. 3a). [ 18F]FMISO also showed hypoxia-
selective retention but with a much lower maximal percentage reten-
tion level in HCT116 cells than in MCF-7 cells (Fig. 3a). Fig. 4 shows
[ 64Cu]CuATSM and [ 18F]FMISO retention curves over time for HCT116
and MCF-7 cells under anoxic conditions. [ 64Cu]CuATSM retention
Radiotracer uptake
80
% Radiotracer uptake
plateaued past the 15 min time point (hence the subsequent uptake
measurements at all pO2 levels were curtailed at 60 min) but [ 18F]
FMISO retention was much lower, increased more slowly and
continued to plateau beyond 120 min (Fig. 4). Supplementary Fig. 3
shows [ 64Cu]CuATSM and [ 18F]FMISO retention curves for different
pO2 levels and at all time points. The uptake data were also plotted
with pO2 values on a log scale in order to better estimate the half-
maximal values, which occurred at very low pO2 levels not easily
represented on a linear scale. While the bis(thiosemicarbazone)
complexes showed wide variation in % uptake in cells (as expected
since they were designed to have a range of lipophilicity values in
order to control pharmacokinetics), all of them, as well as [ 18F]FMISO,
[64Cu]CuATSM (HCT116) showed a sharp increase in uptake only when pO2 levels fell below

[64Cu]CuATSM (MCF-7) 1 mmHg (Fig. 5). The pO2 levels producing a half-maximal cellular
60 [18F]FMISO (HCT116)
[ 64Cu]CuATSM retention at 30 min were approximately 0.60 mmHg
(HCT116) and 0.75 mmHg (MCF-7). The pO2 values for half-maximal
[18F]FMISO (MCF-7)
40 [ 18F]FMISO binding at 120 min were approximately 0.03 mmHg (MCF-
7) and 0.02 mmHg (HCT116) (Fig. 5). The additional copper tracers
(complexes of ATS, CTS, ATSE, DTS, DTSM, and DTSE), were assessed in
20
the HCT116 cell line only and all showed similar hypoxia-selective re-
tention with half-maximal retention values in the range 0.1–1.
0 1 mmHg (Fig. 5, Supplementary Table 2). The steepness of the uptake
0 30 60 90 120
curves near the threshold pO2 value meant that few data points could
Time (min)
be collected in this region (as is generally the case with steep sigmoid
curves), limiting the precision with which the thresholds could be
Fig. 4. Percent [64Cu]CuATSM and [18F]FMISO uptake in HCT116 and MCF-7 cells under
measured. Nevertheless, no calculated threshold values exceeded 1.
anoxic conditions at 1, 5, 15, 30, 60, 120 min, showing slower uptake kinetics for [18F]
FMISO (samples taken in triplicate, mean and SEM shown. Unseen bars are hidden 1 mmHg and none of the tracers gave % uptake levels significantly
within data points). higher than baseline normoxic levels until pO2 fell below 1 mmHg.

4
K. Chia, R.L. Paul, A.J. Weeks et al. Nuclear Medicine and Biology 110 (2022) xxx

HCT116 cells [64Cu]CuCTS maximal response of HIF1α in MCF-7 cells was in the range 10–13
80 [64Cu]CuATS mmHg (Supplementary Table 2).
[64Cu]CuATSM
% Radiotracer uptake

60
40
20
0
10-4 10-3 10-2 10-1 100 101
Oxygen (mmHg)
Fig. 5. Percentage uptake of hypoxia tracers plotted against directly measured dissolved O2
levels using a log scale at 120 and 30 min time points [18F]FMISO and 64Cu-labeled bis
(thiosemicarbazones) respectively. Data were derived from HCT116 cells unless
otherwise stated (samples taken in triplicate, mean and SEM shown. Unseen bars are
hidden within data points).
3.3. Radiosensitivity
Relative radiosensitivity, defined as (1 − surviving fraction) / (1 −
surviving fraction at 0 mmHg O2), of both cell lines showed very little
decline as pO2 fell from 140 to 25 mmHg, but decreased rapidly
between 25 and 0 mmHg (Fig. 3b). The maximum increase in relative
radiosensitivity (RR) due to increasing pO2 was 2.5 for HCT116 cells
and 1.9 for MCF7 cells (the RR under anoxic conditions is defined as 1.
0). The pO2 for half-maximal radiation sensitivity was around 7 mmHg
for both cell lines (Supplementary Table 2). Supplementary Table 3
lists the relative radiosensitivity data for both cell lines.
3.4. Pimonidazole binding
Pimonidazole adduct formation in HCT116 cells increased sharply as
pO2 dropped below 25 mmHg, reaching a plateau below 10 mmHg O2
(Fig. 3c). The pO2 producing half-maximal pimonidazole binding was
17 mmHg (Supplementary Table 2). Cell samples from pimonidazole
binding experiments using MCF-7 cells could not be analysed due to se-
vere clumping of cells despite the use of a cell detachment solution and a
cell strainer.
3.5. HIF1α protein expression
In total,18 individual hypoxia experiments were carried out at vari-
ous different pO2 levels. The nuclear HIF1α extracts were analysed on
two separate western blots. As the two blots were not exposed at the
same time, the data were kept separate and two individual graphs
were generated. Levels of nuclear HIF1α protein increased markedly
as pO2 dropped below 50 mmHg, reaching a maximum at an O2
sensor reading of −0.1and 0.2 mmHg for blots A (Figs. 3d and 6) and
B (Supplementary Fig. 4a and b) respectively. The pO2 for half-
Oxygen (mmHg)
6 .1 7 5
15 -0 0.1 3.8 8.2
HIF1
TBP
Fig. 6. Western blot “A” analysis of HIF1α protein levels as a function of pO2 in MCF-7 cells
(see Fig. 3d for graphical representation of the data).
[64Cu]CuATSM (MCF7)
4. Discussion
[64Cu]CuDTS
[64Cu]CuDTSM
To our knowledge, this is the first study to compare directly the pO2
[64Cu]CuATSE
dependence of radiotracer retention with that of cellular
[64Cu]CuDTSE
[18F]FMISO (MCF7)
radiosensitivity, pimonidazole binding and HIF1α expression under
[18F]FMISO
identical controlled conditions. It confirms previous reports that
significant hypoxia-selective cellular retention of [ 18F]FMISO and
102
[ 64Cu]CuATSM (and its analogues) occurs in vitro, but it also shows
that these tracers are only able to identify maximally hypoxic cells and
not cells that are at intermediate levels of hypoxia. We also highlight
an important mismatch between these PET tracers and other markers
of hypoxia: tracer uptake only occurred at oxygen levels that were one
or two orders of magnitude below levels that induced pimonidazole up-
take, HIF1α expression and radioresistance. In these respects, the PET
tracer evaluated do not meet the criteria of an ideal PET hypoxia tracer
for radiotherapy planning in cancer, which should have a pO2
threshold for increased uptake that matches those observed in
clinically and biologically relevant processes such as radioresistance
and HIF1α expression.
Despite differences in absolute % uptake and rate of uptake between
tracers and between cell lines (attributable to different lipophilicity and
cell permeability of the tracers and possibly different intracellular re-
ducing agents and their concentrations in the different cell lines), all
the PET tracers in both cell lines examined shared a similar pattern of
uptake in response to decreasing pO2 in the medium: a steep sigmoid
curve, making them essentially binary, with a “switch” between high
and low uptake (Fig. 5). The extreme steepness of the sigmoid curves
permitted only limited sampling around the inflexion of the curves
which made accurate determination of half-maximal values difficult
and necessarily approximate. However, the sigmoid curves provided
unambiguous qualitative indication of low pO2 thresholds (Fig. 5 and
Supplementary Table 2). [ 64Cu]CuCTS also showed a higher pO2
threshold than other bisthiosemicarbazone complex analogues in the
isolated perfused rat heart model [18,21]. In contrast to PET tracer up-
take, the response curves for radiosensitivity, pimonidazole binding
and HIF1α expression showed much higher half-maximal pO2 values
(7–17 mmHg) and dramatically less steep slopes (Fig. 3).
The threshold values in the present work are comparable to those re-
ported by us previously but significantly lower (more extremely hyp-
oxic) than some previously reported by others. The threshold pO2 of 0.
02 and 0.03 mmHg for [ 18F]FMISO in HCT116 and MCF-7 cells respec-
tively is lower than previously published values of 720–2300 ppm (0.
55–1.75 mmHg) in V-79, EMT6 (UW), RIF-1, and CaOs-1 cells [25]. Sim-
ilarly, our finding of 0.6 mmHg for [64Cu]CuATSM in HCT116 cells and 0.
75 mmHg in MCF-7 cells is lower than the 2500 ppm (1.9 mmHg) re-
ported by Lewis et al. in EMT6 cells [20]. [ 18F]FMISO demonstrated
greater maximal percentage retention level in HCT116 cells than in
MCF-7 cells, a finding similar to that of Rasey et al. where a 10-fold dif-
ference was seen between EMT6 and V79 cells [25]. It is to be expected
that the different cell lines used give rise to different threshold pO2
4 .2 .7 .4 .4 values, but more important may be the different experimental
17 22 31 51 methods, in particular those for pO2 measurement. The previously
published [ 18F]FMISO data referred to above were acquired by gassing
of cell monolayers in a very thin (100 μm) layer of overlying liquid.
This technique reduces the equilibration time between gas and liquid
to 6 s but does not involve direct monitoring of the pO2 in the liquid
overlying the cells [26,27], therefore the O2 concentrations reported
must be regarded as less reliable. The [ 64Cu]CuATSM data from Lewis
et al. were obtained by plotting tracer retention against O2 content of
the gas rather than pO2 measured in the cell suspension [20]. Our
preliminary measurements (Fig. 2) confirmed that when cells were
present, oxygen consumption led to significantly lower pO2 in the cell
suspension than that predicted from the gas phase composition based

5
K. Chia, R.L. Paul, A.J. Weeks et al.
on cell-free measurements (particularly at the low end of the pO2
range). Therefore it is not unexpected that our measured threshold
values of pO2 for [ 64Cu]CuATSM, and indeed all the other tracers, are
lower and probably more realistic than many reported in literature
that were not directly measured.
Pimonidazole immunohistochemistry is often used to characterise
tissue hypoxia from an immunohistochemical perspective and as a
“gold–standard” hypoxia marker against which to validate PET hypoxia
tracer accumulation [27–31]. The binding characteristic of pimonidazole
has been described by some as almost binary, showing strong binding at
≤10 mmHg O2 but none above 10 mmHg [32]. However, others have
demonstrated measureable pimonidazole binding at much higher
levels of O2 than 10 mmHg [33]. In this study, we found that
pimonidazole binding increased sharply below ~25 mmHg O2, with a
half-maximal binding threshold of 17 mmHg which is much higher
than that of all the PET tracers used in this study.
At first glance, this mismatch is surprising, especially since, in the
case of [ 18F]FMISO, the mechanism of intracellular trapping is purport-
edly the same as that of pimonidazole, as both are nitroimidazole deriv-
atives. However, the difference in the half-maximal binding threshold
may lie in the much lower concentration of the PET tracer (by at least
a million-fold) compared to pimonidazole in the experiments. The
widely accepted mechanism of hypoxia selectivity of both the
nitroimidazole and copper bis(thiosemicarbazone) derivatives is that
after entry into the cell, intracellular reduction converts the probe into
an intermediate that can either undergo further reaction which leads
to trapping, or can be re-oxidised by molecular oxygen allowing escape
from the cell; hypoxia favours the former pathway. According to our hy-
pothesis, at the PET tracer concentration, even very low levels of molec-
ular oxygen would be sufficient to reoxidise the probe and allow its re-
lease, whereas at the higher concentrations required for immunohisto-
chemical detection of pimonidazole binding, much higher levels of mo-
lecular oxygen would be required to reoxidise a significant fraction of
the probe molecules. Consequently, the threshold pO2 for trapping of
the pimonidazole is much higher than that for the PET tracers, and the
slope of the response curve at this threshold is much less steep, despite
their similar trapping mechanisms. If this behaviour is extrapolated to
the in vivo context, tumor regions with intermediate hypoxia (e.g.
2–20 mmHg), harbouring few extremely hypoxic cells, would show
very little [ 18F]FMISO or [ 64Cu]CuATSM retention but significant
pimonidazole binding. Moreover, in some studies using pimonidazole
in vivo as a comparator for PET tracers, the pimonidazole is injected
with or before the PET tracer; under such circumstances the PET tracer
distribution is likely to be quite different from that in the absence of
pimonidazole (i.e. the manner in which PET tracers would normally be
used clinically). Indeed, while some studies show a spatial correlation
between PET tracer uptake and pimonidazole staining, many others do
not [27–31]. This rationale may explain the findings of a pre-clinical re-
port of an in vivo study involving [18F]EF5, another nitroimidazole derived
hypoxia PET tracer, which showed that the [18F]EF5 was taken up in oxy-
genated tumor regions when administered concurrently with unlabelled
EF5 at immunohistochemistry doses (30 mg/kg), but not when adminis-
tered at tracer level without unlabelled EF5 [34]. We suggest, therefore,
that because of the orders-of magnitude concentration difference be-
tween the contexts of PET imaging and immunohistochemical hypoxia
measurement, the two methods provide different information and non-
concordance does not imply that either is incorrect. Moreover, caution is
required in the design of clinical and preclinical in vivo experiments in
which one is used as a control or gold standard against which to compare
the other.
HIF1α is the key transcription factor in the cellular adaptation to a
hypoxic microenvironment and increased expression of the protein
has been strongly associated with tumorigenesis, poor patient outcomes
and treatment resistance [35]. Novel strategies are currently being de-
veloped to target this crucial survival pathway that has been detected
in a wide range of solid tumours [35,36]. In this study, the expression
6
Nuclear Medicine and Biology 110 (2022) xxx
of nuclear HIF1α protein rapidly increased below pO2 of ~20 mmHg,
reaching a maximum at a pO2 sensor reading of −0.1 mmHg. The pO2
for the half-maximal response of HIF1α was 10–13 mmHg, similar to
values of 1.5–2% (11.4–15.2 mmHg) reported by Jiang et al. using HeLa
cells [37]. Similar to the mismatch between PET tracers and
pimonidazole measurement discussed above, the mismatch between
the pO2 dependency of HIF1α and the hypoxia PET tracers examined
in this study suggest that the PET tracers may be unsuitable as surrogate
markers of hypoxia-dependent HIF1α activation. Due to their very low
pO2 threshold, a negative [ 18F]FMISO or [ 64Cu]CuATSM PET scan
would fail to rule out HIF1α over-expression in a tumor, an important
component of HIF1α directed trials.
The shape of the radiosensitivity response curve (Fig. 3b) is consis-
tent with previous in vitro studies showing that the relative sensitivity
of cells to killing by radiation starts to fall at pO2 below 25 mmHg and
that cells with pO2 < 0.5 mmHg represent the most radioresistant
population [38]. In the clinic, tumours present with varying degrees of
hypoxia and studies using Eppendorf polarographic needle electrodes
have been used to plot the distribution of pO2 directly measured in
tumours (Fig. 7) [39]. These studies also recorded clinical outcomes
following courses of conventionally fractionated (2 Gy per fraction
administered daily over 6–7 weeks) or hyperfractionated radiotherapy
(1.25 Gy per fraction administered twice daily over 5.5–6 weeks) and
showed that hypoxic tumours were strongly linked with worse patient
outcomes [4,5,40]. The pO2 thresholds associated with poorer clinical
outcomes in these studies were in the range 2.5–10 mmHg. Thus, in
the context of conventionally fractionated radiotherapy regimens, an
ideal PET tracer would exhibit a pO2-dependent uptake with a threshold
in the range 2.5–10 mmHg, whereas the pO2 thresholds for all the PET
tracers evaluated in this work (0.02–1.10 mmHg) fall one or two order
of magnitude below these ideal values; their cellular retention is signif-
icantly increased only in extreme hypoxia. This mismatch may weaken
the sought-after correlation between PET tracer retention and resistance
to conventionally fractionated radiotherapy, and could lead to the incor-
rect classification of tumor regions as “normoxic” despite being moder-
ately hypoxic and hence relatively radioresistant and amenable to hyp-
oxia modification therapies.
Although many clinical radiotherapy treatments are delivered using
small individual doses (1.25–3 Gy) administered daily over many weeks
[23], technical advances in image guidance and treatment delivery
methods have led in recent years to the rapid development of
20 Threshold of radioresistance: Stereotactic radiation therapy
Threshold of [64Cu]CuATSM/[18F]FMISO uptake
Threshold of radioresistance: conventionally
fractionationated radiation therapy
Frequency (%)
10
0
0 10 20 30 40 50 60 70 80
Median pO2 (mmHg)
Fig. 7. Chart showing frequency of different median pO2 readings measured in human
tumours compared with threshold pO2 levels for radioresistance in stereotactic radiation
therapy, radioresistance in conventional fractionated radiotherapy and PET tracer
uptake. The latter is a close match for radioresistance in stereotactic radiation therapy
but not conventional fractionated radiotherapy. Data are based on individual patients
and were pooled from across nine separate studies (pO2 data adapted from reference 39).
K. Chia, R.L. Paul, A.J. Weeks et al.
stereotactic radiation therapy (SRT) techniques which involve large
doses of radiation (8–30 Gy) per fraction delivered in 1–5 sessions
[41]. SRT treatments are commonly referred to as stereotactic ablative
radiotherapy (SABR), stereotactic body radiotherapy (SBRT) or stereo-
tactic radiosurgery (SRS) when treating the brain. In constrast to con-
ventionally fractionated radiotherapy, SRT delivers a larger biological ef-
fective dose of radiation by using small margins and ensuring steep dose
drop-off outside the tumor, minimising dose to surrounding normal tis-
sues [42]. It is known from in vivo assays using single, high doses of ra-
diation (10–30 Gy) that the biological effect at these doses is deter-
mined by the maximally resistant cells in the tumor (pO2 < 0.5
mmHg), originally termed the “radiobiological hypoxic fraction” [43].
This pO2 threshold for SRT, unlike the threshold for conventionally frac-
tionated radiotherapy, closely matches the pO2 thresholds for all the PET
tracers evaluated in this work (0.02–1.10 mmHg) which suggests that
these tracers may be more useful in identifying clinically relevant hyp-
oxia in tumours treated with this radiotherapy technique than with con-
ventional fractionation. Furthermore, hypoxia may be of more impor-
tance to SRT than to conventionally fractionated radiotherapy due to
the absence of reoxygenation with single fraction treatments [16].
Early preclinical studies have shown that fractionating radiotherapy
mitigates hypoxic radioresistance by allowing reoxygenation of hypoxic
tumor cells in between treatments, thereby increasing their susceptibil-
ity to radiation damage [44]. Tumours suitable for treatment with SRT
have been shown to harbour hypoxic regions [45,46] and both preclini-
cal and clinical studies have started looking at hypoxia modification
strategies in conjunction with SRT [47,48]; PET hypoxia tracers could
be used to assess such strategies in this context.
5. Conclusion
The in vitro approach taken in this work cannot fully predict the be-
haviour of PET hypoxia tracers in vivo because it does not take into ac-
count pharmacokinetic properties such as hepatobiliary or urinary
clearance, interaction with blood proteins etc. which are critical to the
delivery of the tracers to their target tissues and clearance from non-
target tissues. However, by adopting the in vitro approach we have
been able to achieve a level of control and measurement of pO2 that is
not feasible in the in vivo setting and hence to elucidate fundamental
aspects of tracer behaviour at cellular level. This approach is essential
to understand the mode of action of the tracers and what they can tell
us about hypoxia. Using it, we have shown that hypoxia PET tracers
[ 18F]FMISO and [ 64Cu]CuATSM, and other hypoxia-targeting copper bis
(thiosemicarbazone) complexes evaluated in this work, are significantly
taken up only in cells in extreme hypoxic environments and show a
steep slope at the threshold making them essentially binary tracers.
This behaviour contrasts with the response of pimonidazole binding,
HIF1α expression and relative radiosensitivity, all of which show a less
steep response with a pO2 threshold at intermediate hypoxia. This
contrast highlights risks of using PET scans to predict response to con-
ventionally fractionated radiotherapy or stratifying patients in trials of
hypoxia modification strategies and novel HIF1α targeting agents. Fur-
thermore, the use of pimonidazole binding as a gold standard against
which to validate hypoxia PET tracers should be reconsidered or used
with caution. PET tracers with the ability to identify intermediate
hypoxia (2.5–10 mmHg) and whose cellular uptake response to
pO2 correlates better with HIF1α expression may be more clinically
useful but it is not yet clear that such tracers exist. Strategies to
achieve this might include designing molecules that, once reduced
within cells, are re-oxidised more slowly by molecular oxygen, or
by deliberately using tracers with very low molar activity [49,50].
The pO2 thresholds for all the PET tracers evaluated in this work (0.
02–1.10 mmHg), however, closely match the pO2 threshold for SRT
which suggests that these tracers may be of particular use in stratify-
ing patients for hypoxia modification/targeting studies using this ra-
diotherapy technique.
7
Nuclear Medicine and Biology 110 (2022) xxx
Funding
This work was supported by the Centre of Excellence in Medical En-
gineering funded by the Wellcome Trust and EPSRC (WT088641/Z/09/Z,
WT 203148/Z/16/Z), the KCL-UCL Comprehensive Cancer Imaging Cen-
tre funded by Cancer Research UK & EPSRC, in association with the MRC
and the Department of Health (England), EPSRC Programme Grant [EP/
S032789/1, “MITHRAS”], a Wellcome Trust Multi-User Equipment Grant
(WT212885/Z/18/Z) and the National Institute for Health Research
(NIHR) Comprehensive Biomedical Research Centre award to Guy's &
St Thomas' NHS Foundation Trust in partnership with King's College
London and King's College Hospital NHS Foundation Trust. K.C. received
a Cancer Research UK Clinical Training Fellowship and A.J.W. was sup-
ported by a grant from the Guy's and St. Thomas' Charity (Grant to
P.J.B.). The views expressed are those of the authors and not necessarily
those of the NHS, the NIHR or the DoH.
Declaration of competing interest
The authors declare that they have no known competing financial in-
terests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We thank posthumously Kath Martin for synthesis and characteriza-
tion of new bisthiosemicarbazones and copper complexes. We thank
Dr. Judith P. Banath (British Columbia Cancer Research Centre) for the
anti-pimonidazole staining protocol and Dr. Jim Ballinger for helpful
comments on the manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.nucmedbio.2022.04.004.
References
[1] Thomlinson R, Gray L. The histological structure of some human lung cancers and
the possible implications for radiotherapy. Br J Cancer. 1955;IX:539–49.
[2] Vaupel P, Hockel M, Mayer A. Detection and characterization of tumor hypoxia using
pO2 histography. Antioxid Redox Signal. 2007;9:1221–36.
[3] Brizel DM, Scully SP, Harrelson JM, Layfield LJ, Bean JM, Prosnitz LR, et al. Tumor ox-
ygenation predicts for the likelihood of distant metastases in human soft tissue sar-
coma. Cancer Res. 1996;56:941–3.
[4] Brizel DM, Sibley GS, Prosnitz LR, Scher RL, Dewhirst MW. Tumor hypoxia adversely
affects the prognosis of carcinoma of the head and neck. Int J Radiat Oncol Biol Phys.
1997;38:285–9.
[5] Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radia-
tion response in advanced squamous cell carcinoma of the head and neck. Radiother
Oncol. 1996;41:31–9.
[6] Gray LH, Conger AD, Ebert M, Nat R, Hornsey S, Scott OCA. The concentration of ox-
ygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J
Cancer. 1953;26:638–48.
[7] Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P. Association between
tumor hypoxia and malignant progression in advanced cancer of the uterine cervix.
Cancer Res. 1996;56:4509–15.
[8] Teicher BA, Lazo JS, Sartorelli AC. Classification of anti-neoplastic agents by their se-
lective toxicities toward oxygenated and hypoxic tumor-cells. Cancer Res. 1981;41:
73–81.
[9] Chia K, Fleming IN, Blower PJ. Hypoxia imaging with PET: which tracers and why?
Nucl Med Commun. 2012;33:217–22.
[10] Haubner R, Weber WA, Beer AJ, Vabuliene E, Reim D, Sarbia M, et al. Noninvasive vi-
sualization of the activated alphavbeta3 integrin in cancer patients by positron emis-
sion tomography and [18F]Galacto-RGD. PLoS Med. 2005;2:e70.
[11] Hillier SM, Maresca KP, Femia FJ, Marquis JC, Foss CA, Nguyen N, et al. Preclinical
evaluation of novel glutamate-urea-lysine analogues that target prostate-specific
membrane antigen as molecular imaging pharmaceuticals for prostate cancer. Can-
cer Res. 2009;69:6932–40.
[12] Som P, Atkins HL, Bandoypadhyay D, Fowler JS, MacGregor RR, Matsui K, et al. A fluo-
rinated glucose analog, 2-fluoro-2-deoxy-D-glucose (F-18): nontoxic tracer for rapid
tumor detection. J Nucl Med. 1980;21:670–5.
[13] Ward JP. Oxygen sensors in context. Biochim Biophys Acta. 2008;1777:1–14.
[14] Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and
molecular aspects. J Natl Cancer Inst. 2001;93:266–76.
K. Chia, R.L. Paul, A.J. Weeks et al.
[15] Pell VR, Baark F, Mota F, Clark JE, Southworth R. PET imaging of cardiac hypoxia: hit-
ting hypoxia where it hurts. Curr Cardiovasc Imaging Rep. 2018;11:7.
[16] Brown JM, Carlson DJ, Brenner DJ. The tumor radiobiology of SRS and SBRT: are more
than the 5 rs involved? Int J Radiat Oncol Biol Phys. 2014;88:254–62.
[17] Dearling JLJ, Lewis JS, Muller GED, Welch MJ, Blower PJ. Copper bis
(thiosemicarbazone) complexes as hypoxia imaging agents: structure-activity rela-
tionships. J Biol Inorg Chem. 2002;7:249–59.
[18] Handley MG, Medina RA, Mariotti E, Kenny GD, Shaw KP, Yan R, et al. Cardiac hyp-
oxia imaging: second-generation analogues of 64Cu-ATSM. J Nucl Med. 2014;55:
488–94.
[19] McQuade P, Martin KE, Castle TC, Went MJ, Blower PJ, Welch MJ, et al. Investigation
into cu-64-labeled Bis(selenosemicarbazone) and Bis(thiosemicarbazone) com-
plexes as hypoxia imaging agents. Nucl Med Biol. 2005;32:147–56.
[20] Lewis JS, McCarthy DW, McCarthy TJ, Fujibayashi Y, Welch MJ. Evaluation of 64Cu-
ATSM in vitro and in vivo in a hypoxic tumor model. J Nucl Med. 1999;40:177–83.
[21] Medina RA, Mariotti E, Pavlovic D, Shaw KP, Eykyn TR, Blower PJ, et al. 64Cu-CTS: a
promising radiopharmaceutical for the identification of low-grade cardiac hypoxia
by PET. J Nucl Med. 2015;56:921–6.
[22] Jauregui-Osoro M, De Robertis S, Halsted P, Gould S-M, Yu Z, Paul RL, et al. Produc-
tion of copper-64 using a hospital cyclotron: targetry, purification and quality anal-
ysis. Nucl Med Commun. 2021;42:1024–38.
[23] RCo Radiologists. Radiotherapy dose fractionation. Third Edition Royal College of
Radiologists; 2019..
[24] Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A,
et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Sci-
ence. 2003;300:1155–9.
[25] Rasey JS, Nelson NJ, Chin L, Evans ML, Grunbaum Z. Characteristics of the binding of
labeled fluoromisonidazole in cells-in vitro. Radiat Res. 1990;122:301–8.
[26] Koch JC. A thin-film culturing technique allowing rapid gas-liquid equilibration (6
sec) with no toxicity to mammalian cells. Radiat Res. 1984;97:434–42.
[27] Oh M, Tanaka T, Kobayashi M, Furukawa T, Mori T, Kudo T, et al. Radio-copper-la-
beled cu-ATSM: an indicator of quiescent but clonogenic cells under mild hypoxia
in a Lewis lung carcinoma model. Nucl Med Biol. 2009;36:419–26.
[28] Carlin S, Zhang H, Reese M, Ramos NN, Chen Q, Ricketts SA. A comparison of the im-
aging characteristics and microregional distribution of 4 hypoxia PET tracers. J Nucl
Med. 2014;55:515–21.
[29] KC McCall, Humm JL, Bartlett R, Reese M, Carlin S. Copper-64-diacetyl-bis(N(4)-
methylthiosemicarbazone) pharmacokinetics in FaDu xenograft tumors and correla-
tion with microscopic markers of hypoxia. Int J Radiat Oncol Biol Phys. 2012;84:
e393-e9.
[30] O'Donoghue JA, Zanzonico P, Pugachev A, Wen BX, Smith-Jones P, Cai SD, et al. As-
sessment of regional tumor hypoxia using F-18-fluoromisonidazole and cu-64(II)-
diacetyl-bis(N4-methylthiosemicarbazone) positron emission tomography: com-
parative study featuring microPET imaging, Po-2 probe measurement, autoradio-
graphy, and fluorescent microscopy in the R3327-AT and FaDu rat tumor models.
Int J Radiat Oncol Biol Phys. 2005;61:1493–502.
[31] Yuan H, Schroeder T, Bowsher JE, Hedlund LW, Wong T, Dewhirst MW. Intertumoral
differences in hypoxia selectivity of the PET imaging agent Cu-64(II)-diacetyl-bis(N-
4-methylthiosemicarbazone). J Nucl Med. 2006;47:989–98.
[32] Raleigh JA, Chou SC, Arteel GE, Horsman MR. Comparisons among pimonidazole
binding, oxygen electrode measurements, and radiation response in C3H mouse tu-
mors. Radiat Res. 1999;151:580–9.
Nuclear Medicine and Biology 110 (2022) xxx
[33] Koch CJ. Importance of antibody concentration in the assessment of cellular hypoxia
by flow cytometry: EF5 and pimonidazole. Radiat Res. 2008;169:677–88.
[34] Chitneni SK, Bida GT, Zalutsky MR, Dewhirst MW. Comparison of the hypoxia PET
ttacer 18F-EF5 to immunohistochemical marker EF5 in 3 different human tumor xe-
nograft models. J Nucl Med. 2014;55:1192–7.
[35] Semenza GL. Hypoxia-inducible factors: mediators of cancer progression and targets
for cancer therapy. Trends Pharmacol Sci. 2012;33:207–14.
[36] Talks KL, Turley H, Gatter KC, Maxwell PH, Pugh CW, Ratcliffe PJ, et al. The expression
and distribution of the hypoxia-inducible factors HIF-1α and HIF-2α in Normal
human tissues, cancers, and tumor-associated macrophages. Am J Pathol. 2000;
157:411–21.
[37] Jiang BH, Semenza GL, Bauer C, Marti HH. Hypoxia-inducible factor 1 levels vary ex-
ponentially over a physiologically relevant range of O2 tension. Am J Physiol Cell
Physiol. 1996;271:C1172–80.
[38] Steel GG. Basic Clinical Radiobiology. 3rd ed. London: Arnold; 2002..
[39] Adam MF, Dorie MJ, Brown JM. Oxygen tension measurements of tumors growing in
mice. Int J Radiat Oncol Biol Phys. 1999;45:171–80.
[40] Fyles AW, Milosevic M, Wong R, Kavanagh MC, Pintilie M, Sun A, et al. Oxygenation
predicts radiation response and survival in patients with cervix cancer. Radiother
Oncol. 1998;48:149–56.
[41] Consortium SU. Stereotactic Ablative Body Radiation Therapy: A Resource; 2019..
[42] Eschmann SM, Paulsen F, Bedeshem C, Machulla HJ, Hehr T, Bamberg M, et al.
Hypoxia-imaging with (18)F-misonidazole and PET: changes of kinetics during ra-
diotherapy of head-and-neck cancer. Radiother Oncol. 2007;83:406–10.
[43] Hall EJ, Giaccia AJ. Radiobiology for the radiologist. 6th ed. Lippincott Williams &
Wilkins; 2006..
[44] Fowler JF, Sheldon PW, Denekamp J, Field SB. Optimum fractionation of the C3H
mouse mammary carcinoma using x-rays, the hypoxic-cell radiosensitizer Ro-07-
0582, or fast neutrons. Int J Radiat Oncol Biol Phys. 1976;1:579–92.
[45] Qian Y, Von Eyben R, Liu Y, Chin FT, Miao Z, Apte S, et al. (18)F-EF5 PET-based
imageable hypoxia predicts local recurrence in tumors treated with highly confor-
mal radiation therapy. Int J Radiat Oncol Biol Phys. 2018;102:1183–92.
[46] Kelada OJ, Decker RH, Nath SK, Johung KL, Zheng MQ, Huang Y, et al. High single
doses of radiation may induce elevated levels of hypoxia in early-stage non-small
cell lung cancer tumors. Int J Radiat Oncol Biol Phys. 2018;102:174–83.
[47] Wittenborn TR, Horsman MR. Targeting tumour hypoxia to improve outcome of ste-
reotactic radiotherapy. Acta Oncol. 2015;54:1385–92.
[48] Drzymala RE, Wasserman TH, Won M, Shaw E, Cmelak AJ, Loeffler J, et al. A phase I-B
trial of the radiosensitizer: etanidazole (SR-2508) with radiosurgery for the treat-
ment of recurrent previously irradiated primary brain tumors or brain metastases
(RTOG study 95–02). Radiother Oncol. 2008;87:89–92.
[49] Blower PJ, Castle TC, Cowley AR, Dilworth JR, Donnelly PS, Labisbal E, et al. Structural
trends in copper(II) bis(thiosemicarbazone) radiopharmaceuticals. Dalton Trans.
2003.;4416–25.
[50] Blower PJ, Went MJ, Martin KE, Smith GE. Imaging hypoxia in vivo by controlling the
electrochemistry of copper radionuclide complexes. J Labelled Compd
Radiopharmaceut. 2007;50:354–9.