The role of hypoxia in canine cancer

Publishing Authors : S. A. Snyder, M. W. Dewhirst and M. L. Hauck

Date Published : Unknown

Abstract
Human oncology has clearly demonstrated the existence of hypoxic tumours and the problematic
nature of those tumours. Hypoxia is a signifi cant problem in the treatment of all types of solid tumours
and a common reason for treatment failure. Hypoxia is a negative prognostic indicator of survival and is
correlated with the development of metastatic disease. Resistance to radiation therapy and chemotherapy
can be because of hypoxia. There are two dominant types of hypoxia recognized in tumours, static
and intermittent. Both types of hypoxia are important in terms of resistance. A variety of physiological
factors cause hypoxia, and in turn, hypoxia can induce genetic and physiological changes. A limited
number of studies have documented that hypoxia exists in spontaneous canine tumours. The knowledge
from the human literature of problematic nature of hypoxic tumours combined with the rapid
growth of veterinary oncology has necessitated a better understanding of hypoxia in canine tumours.
214 S. A. Snyder et al.
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
survival. 14 – 16 Moreover, hypoxia has been demonstrated
in spontaneous canine tumours and correlates
with a worse response to radiation therapy and
a decrease in overall survival. 11
With the advancement of veterinary oncology to
routinely include all standard modalities of treatment,
the role of hypoxia in treatment failure in
veterinary patients necessarily becomes an issue of
importance. A greater understanding of each patient
’ s tumour oxygenation status would improve
application of standard therapy. If a tumour were
well oxygenated, then the patient would benefi t by
potentially not needing additional toxic agents.
Conversely, in patients with tumours that are
found to be hypoxic, chemotherapeutic agents that
target hypoxic cells can be given. Alternatively, the
application of treatments to reverse the effects of
hypoxia may also be applied in suitable patients.
Given the importance of oxygen status in predicting
response to chemotherapy and radiation therapy,
information about the status and nature of
hypoxia in a dog ’ s tumour should improve the clinician
’ s ability to choose appropriate treatment.
Accordingly, the understanding of the biology of
hypoxia and relevance in the clinical setting is of
importance to veterinary oncology.
Hypoxia and cancer therapy
Previously, hypoxia was thought to occur only in
tumours with inappropriate vessels or that outgrew
their blood supply (generally large regions of central
necrosis seen). However, work has demonstrated
that hypoxia can be present in tumours as small as
100 cells – tumours too small to be detected clinically
and unlikely to contain necrosis. 17 – 20
An extensive amount of work has been carried
out to better detect and understand the hypoxic tumour
because radiation therapy is most effective in
a tumour that is well oxygenated. Oxygen is needed
for effective radiation therapy as most cellular
damage is because of free radicals produced during
treatment. Radiation kills cells by inducing multiple
breaks in DNA. These strand breaks can be accomplished
in two ways. Direct damage occurs
when photons cause atoms of the DNA to be ionized
or excited, leading to a chemical or biological
change within the DNA. Indirect damage occurs
when the photon interacts with a secondary molecule
(typically water) and produces a free radical –
this reaction accounts for two-thirds of the damage
caused by radiation. This interaction produces primarily
the hydroxyl radical that in turn damages
the DNA. The primary lesion in DNA that leads to
cell death is the double-stranded break. When oxygen
is present, the damaged DNA is converted
to an organic peroxide, which is not easily repaired
by the cell. If the free radical is not converted to an
organic peroxide, the DNA damage is repairable.
This process is known as the oxygen fi xation hypothesis
( Fig. 1 ). Consequently, radiation therapy
is less effective when cells are hypoxic.
Hypoxia also results in resistance to treatment
with chemotherapy. Some chemotherapeutics depend
on oxygen for greatest effi cacy. Bleomycin is
one example. It is known a
Role of hypoxia in canine cancer 215
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
also causes cell damage by the formation of free
radicals. In addition, hypoxia often increases the
fraction of cells in G 0 . Because many chemotherapeutic
drugs are cell cycle specifi c, they will be less
effective on G 0 cells. 21 Vincristine and methotrexate
are two cell-cycle-specifi c drugs that are less effective
in tumours that are hypoxic. 22 – 24 Interestingly,
chemotherapeutics that are cell cycle independent
are also less effi cacious in hypoxic cells most likely
because of the limitations on diffusion. 25
Physiology and consequences of
tumour hypoxia
Tumour hypoxia is best understood as a problem
of supply and demand. Until recently, it was generally
accepted that tumour hypoxia was caused by
one of two different mechanisms and either one or
both may be the underlying cause of hypoxia in individual
tumours. Classically, perfusion-limited
hypoxia was defi ned as cells that are limited in oxygen
by vascular stasis, while diffusion-limited hypoxia
was lack of oxygen in cells as a consequence
of their distance from the vasculature. It is now realized
that these two mechanisms are not independent
of each other ( Fig. 2 ). The diffusion distance of
oxygen from the microvasculature is dependent on
the oxygen content of the blood, which is controlled
by red cell fl ux. It is now established that
the red cell fl ux is constantly changing; therefore,
tumour cells are constantly experiencing hypoxia –
re-oxygenation injury. 26 Another important contributor
to hypoxia is the longitudinal oxygen
gradient. The vascular pO 2 drops as blood traverses
afferently from the arterial supply. 27,28 Blood oxygen
content in tumours can be quite low, depending
on how far the vasculature is from the feeding
arterioles. In such regions, the diffusion distance of
oxygen may be very short. Although much research
has focused on oxygen delivery as being responsible
for hypoxia, another important factor, the oxygen
consumption rate must be examined.
Oxygen consumption in proliferating cells
Proliferating cells consume oxygen at a rate of approximately
fi ve times that of quiescent cells. 29 This
high consumption rate is one of the most signifi cant
causes underlying tumour hypoxia. Cells consume
and utilize oxygen through mitochondrial respiration.
The high level of oxygen consumption in tumours
is primarily because of a large percentage of
metabolically active cells versus quiescent cells, unlike
the situation in most non-malignant tissue.
Figure 2. There is a limited diffusion distance of oxygen of approximately 150 microns out from the vasculature. Therefore,
cells at greater distances are hypoxic and eventually anoxic. This is an example of diffusion-limited hypoxia (white arrow).
Tumour vasculature is often abnormal, and there can be a high degree of red cell fl ux. The dramatic change in red cell fl ux
in these vessels often leaves even those cells close to a vessel in a hypoxic or transient anoxic state (black arrow). This is an
example of perfusion-limited hypoxia. Both types of hypoxia occur simultaneously.
216 S. A. Snyder et al.
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Previous work has shown that either a four-fold increase
in tumour blood fl ow or an 11-fold increase
in arterial pO 2 is necessary to abolish tumour hypoxia.
Alternatively, only a modest decrease of 30%
in the oxygen consumption rate is needed to achieve
the same outcome. 30 Oxygen consumption is also
infl uenced by host infl ammatory macrophages.
Macrophages are documented to contribute to hypoxia
through increased oxygen consumption. 31 In
addition to inducing hypoxia, the high oxygenation
consumption rate within the tumour microenvironment
can also causes large amounts of CO 2 to be
excreted into the extracellular space, thereby contributing
to acidifi cation of the tumour. 32,33
Tumour acidosis and hypoxia
Tumours are often found to have pH values around
6.8. 34 Tumour acidosis has signifi cant negative impact
on response to therapy as well. Although several
mechanisms can contribute to tumour acidosis,
high cellular respiration is one of the most important
causes of a lower intratumoural pH. Another
cause of tumour acidosis is the tumour ’ s ability to
survive and proliferate using glycolysis (anaerobic
metabolism). 35 Hypoxic tumours tend to have an
increased rate of glycolysis rather than mitochondrial
respiration due in part to the lack of oxygen
required for aerobic metabolism. Glycolysis results
in the sustained production of lactic acid, thereby
further acidifying the extracellular space of the tumour.
Tumours found to be acidic are more resistant
to chemotherapy and radiation therapy and
are generally more aggressive with a greater potential
for metastasis. 35,36
Abnormal tumour vasculature and hypoxia
One hallmark of tumours is their abnormal and
tortuous blood vessels. Abnormal vessels are often
the result of an increase in levels of vascular endothelial
growth factor (VEGF), which is robustly
induced by hypoxia. 37 Many tumour vessels are dilated,
have blind ends and are leaky because of loose
endothelial junctions. 38 Tortuous and abnormally
branching vessels lead to changes including deformation
of red cells, turbulent and unpredictable
blood fl ow and high sheer stress. 39 All these factors
contribute to areas of tumour vasculature devoid of
red blood cells and other areas with complete lack
of fl ow – both resulting in large regions of hypoxic
cells. Also, as the tumour expands rapidly, there are
regions of cells beyond the diffusion distance of oxygen
from the vessels. The outer limit of oxygen diffusion
from a blood vessel is approximately 200  m,
with diffusion routinely only reaching 150  m. 2
The hypoxic cells are stimulated to produce more
VEGF, setting up a loop promoting the formation
of more abnormal vessels and leading to increasing
microscopic regions of hypoxia. 40
Once the abnormal vasculature infrastructure is
in place, many regions of tumour may not be able
to be re-oxygenated. For example, if a region of tumour
is hypoxic because it contains blind-ended
vessels, the tumour cells may produce large
amounts of VEGF in order to stimulate angiogenesis,
but the additional abnormal vessels do not relieve
the hypoxia. Another confounding factor with
increased angiogenesis is the potential to induce
areas of intermittent hypoxia in the tumour cells.
The constant vascular remodelling happening
within the tumour is the most likely cause for intermittent
hypoxia. 41,42 Cells that experience hypoxia
induce VEGF production bringing in new
vessels, relieving the hypoxia and allowing the cells
to survive and proliferate. As the tumour grows
and vascular remodelling continues, cells again become
hypoxic because of increased distance from
the vessels and/or increased oxygen consumption.
The cells may now be experiencing their second,
third or more exposure to hypoxia, that is intermittent
hypoxia. Intermittent hypoxia may also be
a consequence of instabilities in red cell fl ux within
the tumour vasculature. The cells may not be at a
great distance from the vasculature, but because of
the fl ux and even occasional backwards fl ow, the
tumour cells experience multiple cycles of hypoxia
and re-oxygenation. 43,44 The consequence of hypoxia
and re-oxygenation includes the expression
of a whole host of genes and proteins promoting
growth, survival and metastasis. 45
Static versus intermittent hypoxia
Independent of the mechanism of hypoxia, there are
two dominant resulting types of hypoxia currently
Role of hypoxia in canine cancer 217
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
recognized in tumours, static and intermittent.
These two types of hypoxia do not occur independently
although. Static hypoxia is defi ned as ongoing
hypoxia within a cell, while intermittent
hypoxia is defi ned as a cell experiencing fl uctuations
between hypoxia and normoxia. Static hypoxia
has been well documented as a problem for
cancer therapy, and much ongoing work is focused
on understanding how to overcome this type of hypoxia.
Cells that experience static hypoxia often
change both their physical and their genetic make
up to survive the hostile environment.
Until recently, intermittent hypoxia has received
little attention. Intermittent hypoxia is documented
in the literature as early as 1979 by Brown. 46
Several reports have shown that intermittently hypoxic
cells are less responsive than normoxic and
statically hypoxic cells to both radiation and chemotherapy.
47 – 49 Intermittent hypoxia not only induces
a different cellular phenotype than static
hypoxia, but the cells undergoing intermittent hypoxia
may also be subjected to changes associated
with re-oxygenation, such as increased amounts of
ROS, induction of stress-response genes, additional
stabilization of hypoxia inducible factor 1-
alpha (HIF-1 ) and subsequent activation of the
unfolded protein response. 50,51 ROS are of particular
interest as they can be both benefi cial and
problematic. As discussed, ROS are needed to
cause strand breaks and free radical fi xation during
radiation therapy. However, ROS can cause
stabilization of HIF even during times of normoxia.
51 – 54 This allows activation of HIF target
genes responsible for a large number of antiapoptotic,
metastasis and cell survival genes both
when the cells are hypoxic and when they have
been re-oxygenated.
Hypoxia and gene expression
Not only can hypoxia be a direct cause of treatment
resistance in solid tumours but also changes
in gene expression under hypoxic conditions may
lead to a more malignant phenotype. The most
profound change in hypoxic cells is the upregulation
of HIF-1. HIF-1 is a transcription factor made
up of two subunits, and , that is constitutively
expressed in the cytoplasm of cells. 55,56 During
normoxic conditions, prolyl hydroxylases contained
in the cell cytoplasm modifi es the -subunit
by adding hydroxy residues to two prolines. The
hydroxylation allows the von Hippel Lindau
(VHL) tumour suppressor complex protein to
bind with HIF-1 . Once the VHL complex binds
the -subunit, it is shuttled to the proteosome for
degradation. Induction of hypoxia prevents the
prolyl hydroxylases from hydroxylating the -subunit
because oxygen is required for this reaction. If
levels of HIF-1 increase, it dimerizes with the –
subunit. The dimerized HIF-1 initiates transcription
of multiple genes. Genes that are induced by HIF-1
contain hypoxia response elements (HREs). HREs
are binding sites in the promoter regions of genes
that allow the transcription of HIF-regulated genes.
A large percentage of genes upregulated in hypoxic
tumours have been found to contain HREs.
To date, more than 70 genes contain HREs and
more are being constantly being discovered. HREs
have been found in genes involved in metabolic adaptation
(glucose transporter 1 and 3, GAPDH,
carbonic anhydrase IX and lactate dehydrogenase),
57 – 61 apoptosis resistance (erythropoietin and
insulin-like growth factor 2), 62,63 angiogenesis
(VEGF, vascular endothelial growth factor receptor
and transforming growth factor beta) 64,65 and
metastasis [matrix metalloproteinase 2, uroplasminogen
activator receptor and cellular meserchymalepithelial
transition factor (cMET)]. 62,66 – 68
Hypoxia and veterinary oncology
The past 20 years have had a marked increase in the
study of hypoxia in canine tumours. The establishment
and growth of the veterinary radiation oncology
fi eld during the 1970s and 1980s spurred the
exploration of tumour oxygenation in canine patients.
The human literature clearly demonstrated
that hypoxia was a cause for radioresistance, and so,
it made sense that veterinary patient ’ s tumours may
express the same phenotype. Also, an interest in
spontaneous canine tumours as an experimental
model for human tumours began to emerge. Dog
patients were seen as a good model for human disease
as they more closely paralleled humans in terms
of size, environmental exposure, natural development
of disease and genetics. These similarities
218 S. A. Snyder et al.
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
spurred an interest in the underlying mechanisms of
radioresistance in the veterinary patient.
Initially, most studies of canine tumour oxygenation
involved the use of biochemical markers of
hypoxia. CCI-103F and pimonidazole are two exogenous
markers administered to patients for later
determination of hypoxia in routine clinical biopsies.
Both drugs are bound to tissues only when
they are present in a cell at or below a certain
threshold level of oxygen. 69 – 71 These studies provided
the fi rst direct evidence of the presence of
hypoxia with spontaneous canine tumours.
The fi rst direct measurement of tumour hypoxia
in a patient was carried out in dogs with spontaneous
soft tissue sarcomas. Tumour oxygenation was
measured in 11 companion dogs. Almost half the
patients measured had a median pO 2 value of less
than 2.5 mmHg. 13 These results showed that canine
tumours contain a signifi cant amount of radiobiologically
hypoxic cells that could lead to treatment
resistance. This study also verifi ed that spontaneous
canine soft tissue sarcomas are a reliable model
for the study of tumour hypoxia. Following the
verifi cation of hypoxia in canine patients, the same
group examined the changes in tumour pO 2 following
fractionated radiation therapy. Unfortunately,
dogs that were normoxic to begin with
had tumours that became hypoxic after radiation
therapy and dogs that were hypoxic to begin with
remained hypoxic. 72 Both of these studies only observed
the tumour oxygenation at one or two fi xed
points in time. An additional study observed the
changes in pO 2 during fractionated therapy to see if
there was a temporal pattern of oxygenation. They
found that the pO2 of tumours varied widely
throughout treatment. Variations in pO2 were independent
of whether the tumours were well oxygenated
or not at the baseline measurement. 9
This
study is the fi rst documenting direct evidence of
intermittent hypoxia in a spontaneous canine tumour.
Even though tumour pO 2 varied widely during
radiation therapy, a subsequent study showed
that baseline pO 2 was independently prognostic for
overall survival independent of tumour size or histology.
Tumours in this study that contained a
higher number of cells at or below the threshold of
radiobiological hypoxia (10, 5 or 2.5 mmHg oxygen
tension) had the worst overall survival. 11
Overcoming hypoxia
Previous studies have shown some human and rodent
tumours to be comprised almost 50% hypoxic
cells. 73 In clinical human tumours, the average
number of hypoxic cells is approximately 12 –
20%. 74 Studies have also demonstrated that hypoxia
is found in a variety of tumour types,
including breast, 75 – 78 prostate, 79,80 soft tissue sarcoma
15,81,82 and carcinoma of the cervix. 16,83,84 This
knowledge has helped to develop new therapies
and techniques to overcome the effects of hypoxia
or re-oxygenate hypoxic tumours. Some common
ways to improve tumour oxygenation are the use
of hyperthermia, hyperbaric oxygen and oxygen
consumption inhibitors, for example meta-iodobenzylguanidine.
33,85,86 As an alternative to overcoming
hypoxia, some agents take advantage of the
hypoxic cell. One hypoxic cell cytotoxin, topotecan,
a clinically used compound, is a topoisomerase
I inhibitor that needs to be reduced in a hypoxic
environment in order to be activated. 87
As mentioned, a massive increase in tumour
blood fl ow or arterial pO 2 is needed to abolish tumour
hypoxia, but only a minor decrease in the
oxygen consumption rate is needed to achieve the
same outcome. 30 The large increase in fl ow or arterial
pO 2 is diffi cult to attain. However, a signifi cant
decrease in oxygen consumption can be accomplished
by the use of such compounds as glucose,
glucocorticoids, non-steroidal anti-infl ammatory
and insulin. These compounds act by a variety of
mechanisms to decrease the oxygen consumption
of tumours. For example, when glucose is administered
to a patient, the Crabtree effect occurs – a
shift in tumour metabolism towards anaerobic glycolysis.
88 This shift causes a decrease in mitochondrial
respiration resulting in a higher concentration
of oxygen present. The combination of these compounds
with radiation therapy has been shown to
effectively increase the response of patients to
treatment. 33,89 – 93
Hyperthermia is another adjuvant used in the
hope of improving outcome in radiation therapy
patients. Hyperthermia treatments have been
studied extensively in the canine patient. An
interest in tumour oxygenation is rooted in the
work being carried out to elucidate the mechanism
Role of hypoxia in canine cancer 219
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
underlying the effectiveness of hyperthermia used
for human and canine tumours. There are many
theories as to why hyperthermia improves tumour
oxygenation, including changes in blood fl ow,
vascular permeability and re-oxygenation. The
fi rst documented case of hyperthermia used to
treat canine spontaneous tumours was in 1962.
The study author used companion dogs with the
consent of their owners to better translate to human
medicine the results seen in experimental tumours
in mice. 94 All the hyperthermia applications
were carried out with local heating of the tumours
by submersion in a specifi ed temperature water
bath. Since then, studies have shown that hyperthermia
improves tumour oxygenation in canine
tumours, 95,96 especially when the baseline pO 2
values were low. 12
This review serves to summarize the small, but
rapidly expanding fi eld of study of canine tumour
hypoxia. An extensive amount of work in human
oncology has clearly demonstrated the existence of
hypoxic tumours and the problematic nature of
those tumours. The growth of veterinary oncology
during the past 20 years has brought about a need
for a better understanding of the individual patient
’ s tumour. Completed studies have documented
that hypoxia exists in canine tumours, and
this warrants a need for a better understanding of
the degree of hypoxia in each patient ’ s tumour.
The majority of canine tumour hypoxia studies are
carried out using patients with spontaneous tumours.
This is benefi cial for both human and veterinary
oncology. Knowledge that has been gained
in human medicine can guide development of individualized
therapy for the canine patient with
hypoxia, and conversely, studies of spontaneous
canine tumours can help further the study of
hypoxia in human cancer.
References
1. Gray LH , Conger AD , Ebert M , Hornsey S , Scott OC .
The concentration of oxygen dissolved in tissues at
the time of irradiation as a factor in radiotherapy .
British Journal of Radiology 1953 ; 26 : 638 – 648 .
2. Thomlinson RH and Gray LH . The histological
structure of some human lung cancers and the
possible implications for radiotherapy . British
Journal of Cancer 1955 ; 9 : 539 – 549 .
3. Alper T and Howard-Flanders P . Role of oxygen in
modifying the radiosensitivity of E. coli B . Nature
1956 ; 178 : 978 – 979 .
4. Chi J-T , Wang Z , Nuyten DSA , Rodriguez EH ,
Schaner ME , Salim A , Wang Y , Kristensen GB ,
Helland Å , Børresen-Dale A-L , Giaccia A , Longaker
MT , Hastie T , Yang GP , van de Vijver MJ , Brown BO .
Gene expression programs in response to hypoxia:
cell type specifi city and prognostic signifi cance in
human cancers . PLoS Medicine 2006 ; 3 : e47 .
5. Le QT , Denko N and Giaccia AJ . Hypoxic gene
expression and metastasis . Cancer and Metastasis
Reviews 2004 ; 23 : 293 – 310 .
6. Rofstad EK , Mathiesen B , Henriksen K , Kindem K ,
Galappathi K . The tumor bed effect: increased
metastatic dissemination from hypoxia-induced upregulation
of metastasis-promoting gene products .
Cancer Research 2005 ; 65 : 2387 – 2396 .
7. Brown JM and Wilson WR . Exploiting tumour
hypoxia in cancer treatment . Nature Reviews Cancer
2004 ; 4 : 437 – 447 .
8. Cardenas-Navia LI , Secomb TW and Dewhirst
MW . Effects of fl uctuating oxygenation on
tirapazamine effi cacy: theoretical predictions .
International Journal of Radiation Oncology, Biology,
Physics 2007 ; 67 : 581 – 586 .
9. Brurberg KG , Skogmo HK , Graff BA , Olsen DR ,
Rofstad EK . Fluctuations in pO2 in poorly and welloxygenated
spontaneous canine tumors before and
during fractionated radiation therapy . Radiotherapy
and Oncology 2005 ; 77 : 220 .
10. Rohrer Bley C , Wergin M , Roos M , Grenacher B ,
Kaser-Hotz B . Interrelation of directly measured
oxygenation levels, erythropoietin and erythropoietin
receptor expression in spontaneous canine tumours .
European Journal of Cancer 2007 ; 43 : 963 .
11. Rohrer-Bley C , Ohlerth S , Roos M , Wergin M ,
Achermann R , Kaser-Hotz B . Infl uence of
pretreatment polarographically measured
oxygenation levels in spontaneous canine tumors
treated with radiation therapy . Strahlentherapie und
Onkologie 2006 ; 182 : 518 .
12. Thrall DE , LaRue SM , Pruitt AF , Case B , Dewhirst
MW . Changes in tumour oxygenation during
fractionated hyperthermia and radiation therapy in
spontaneous canine sarcomas . International Journal
of Hyperthermia 2006 ; 22 : 365 .
13. Achermann R , Ohlerth S , Fidel J , Gardelle O ,
Gassmann M , Roos M , Saunders HM , Scheid A ,
Wergin M , Kaser-Hotz B . Ultrasound guided, preradiation
oxygen measurements using
polarographic oxygen needle electrodes in
spontaneous canine soft tissue sarcomas . In Vivo
2002 ; 16 : 431 – 437 .
220 S. A. Snyder et al.
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
14. Brizel DM , Scully SP , Harrelson JM , Layfi eld LJ ,
Bean JM , Prosnitz LR , Dewhirst MW . Tumor
oxygenation predicts for the likelihood of distant
metastases in human soft tissue sarcoma . Cancer
Research 1996 ; 56 : 941 – 943 .
15. Nordsmark M , Bentzen SM , Rudat V , Brizel D ,
Lartigau E , Stadler P , Becker A , Adam M , Molls M ,
Dunst J , Terris DJ , Overgaard J . Prognostic value of
tumor oxygenation in 397 head and neck tumors
after primary radiation therapy. An international
multi-center study . Radiotherapy and Oncology
2005 ; 77 : 18 – 24 .
16. 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 Research 1996 ; 56 :
4509 – 4515 .
17. Li C-Y , Shan S , Huang Q , Braun RD , Lanzen J , Hu
K , Lin P , Dewhirst MW . Initial stages of tumor
cell-induced angiogenesis: evaluation via skin
window chambers in rodent models . Journal of
the National Cancer Institute 2000 ; 92 : 143 – 147 .
18. Rockwell SC , Kallman R and Fajardo L .
Characteristics of a serially transplanted mouse
mammary tumor and its tissue-culture-adapted
derivative . Journal of the National Cancer Institute
1972 ; 49 : 735 – 749 .
19. Suit HD and Maeda M . Oxygen effect factor and
tumor volume in the C3H mouse mammary
carcinoma. A preliminary report . The American
Journal of Roentgenology, Radium Therapy and
Nuclear Medicine 1966 ; 96 : 177 – 182 .
20. Suit HD and Shalek RJ . Response of anoxic C3H
mouse mammary carcinoma isotransplants (1-25
mm 3 ) to X irradiation . Journal of the National
Cancer Institute 1963 ; 31 : 479 – 495 .
21. Koch S , Mayer F , Honecker F , Schittenhelm M and
Bokemeyer C . Effi cacy of cytotoxic agents used in
the treatment of testicular germ cell tumours under
normoxic and hypoxic conditions in vitro . British
Journal of Cancer 2003 ; 89 : 2133 – 2139 .
22. Hussein D , Estlin EJ , Dive C , Makin GWJ . Chronic
hypoxia promotes hypoxia-inducible factor1{alpha}-dependent
resistance to etoposide and
vincristine in neuroblastoma cells . Molecular Cancer
Therapeutics 2006 ; 5 : 2241 – 2250 .
23. Generali D , Berruti A , Brizzi MP , Campo L ,
Bonardi S , Wigfi eld S , Bersiga A , Allevi G , Milani
M , Aguggini S , Gandolfi V , Pogliotti L , Bottini A ,
Harris AL , Fox SB . Hypoxia-inducible factor-1alpha
expression predicts a poor response to primary
chemoendocrine therapy and disease-free survival
in primary human breast cancer . Clinical Cancer
Research 2006 ; 12 : 4562 – 4568 .
24. Sanna K and Rofstad EK . Hypoxia-induced
resistance to doxorubicin and methotrexate in
human melanoma cell lines in vitro . International
Journal of Cancer 1994 ; 58 : 258 – 262 .
25. Song X , Liu X , Chi W , Liu Y , Wei L , Wang X , Yu J.
Hypoxia-induced resistance to cisplatin and
doxorubicin in non-small cell lung cancer is
inhibited by silencing of HIF-1alpha gene . Cancer
Chemotherapy and Pharmacology 2006 ; 58 : 776 – 784 .
26. Lanzen J , Braun RD , Klitzman B , Brizel D ,
Secomb TW , Dewhirst MW . Direct demonstration
of instabilities in oxygen concentrations within the
extravascular compartment of an experimental
tumor . Cancer Research 2006 ; 66 : 2219 – 2223 .
27. Erickson K , Braun RD , Yu D , Lanzen J , Wilson D ,
Brizel DM , Secomb TW , Biaglow JE , Dewhirst MW .
Effect of longitudinal oxygen gradients on
effectiveness of manipulation of tumor
oxygenation . Cancer Research 2003 ; 63 : 4705 – 4712 .
28. Dewhirst MW , Ong ET , Braun RD , Smith B ,
Klitzman B , Evans SM , Wilson D . Quantifi cation of
longitudinal tissue pO2 gradients in window
chamber tumours: impact on tumour hypoxia .
British Journal of Cancer 1999 ; 79 : 1717 – 1722 .
29. Freyer J . Rates of oxygen consumption for
proliferating and quiescent cells isolated from
multicellular tumor spheroids . Advances in
Experimental Medicine and Biology 1994 ; 345 :
335 – 342 .
30. Secomb T , Hsu R , Ong ET , Gross JF and Dewhirst
MW . Analysis of the effects of oxygen supply and
demand on hypoxic fraction in tumors . Acta
Oncologica 1995 ; 34 : 313 – 316 .
31. James PE , Grinberg OY , Michaels G , Swartz HM .
Intraphagosomal oxygen in stimulated
macrophages . Journal of Cellular Physiology 1995 ;
163 : 241 – 247 .
32. Herst PM and Berridge MV . Cell surface oxygen
consumption: a major contributor to cellular
oxygen consumption in glycolytic cancer cell lines .
Biochimica et Biophysica Acta (BBA) – Bioenergetics
2007 ; 1767 : 170 – 177 .
33. Burd R , Lavorgna SN , Daskalakis C , Wachsberger PR ,
Wahl ML , Biaglow JE , Stevens CW , Leeper DB .
Tumor oxygenation and acidifi cation are increased
in melanoma xenografts after exposure to
hyperglycemia and meta-iodo-benzylguanidine .
Radiation Research 2003 ; 159 : 328 – 335 .
34. Tannock IF and Rotin D . Acid pH in tumors and its
potential for therapeutic exploitation . Cancer
Research 1989 ; 49 : 4373 – 4384 .
35. Walenta S and Mueller-Klieser WF . Lactate: mirror
and motor of tumor malignancy . Seminars in
Radiation Oncology 2004 ; 14 : 267 – 274 .
Role of hypoxia in canine cancer 221
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
36. Brizel DM , Schroeder T , Scher RL , Walenta S ,
Clough RW , Dewhirst MW , Mueller-Klieser W .
Elevated tumor lactate concentrations predict for an
increased risk of metastases in head-and-neck
cancer . International Journal of Radiation Oncology,
Biology, Physics 2001 ; 51 : 349 – 353 .
37. Carmeliet P . VEGF as a key mediator of
angiogenesis in cancer . Oncology 2005 ; 69 : 4 – 10 .
38. Dvorak H . Tumors: wounds that do not heal.
Similarities between tumor stroma generation and
wound healing . The New England Journal of
Medicine 1986 ; 315 : 1650 – 1659 .
39. Pettersson A , Nagy JA , Brown LF , Sundberg C ,
Morgan E , Jungles S , Carter R , Krieger JE , Manseau
EJ , Harvey VS , Eckelhoefer IA , Feng D , Dvorak AM ,
Mulligan RC and Dvorak HF . Heterogeneity of the
angiogenic response induced in different normal
adult tissues by vascular permeability factor/
vascular endothelial growth factor . Laboratory
Investigation 2000 ; 80 : 99 – 115 .
40. Cao Y , Li CY , Moeller BJ , Yu D , Zhao Y , Dreher MR ,
Shan S , Dewhirst MW . Observation of incipient
tumor angiogenesis that is independent of hypoxia
and hypoxia inducible factor-1 activation . Cancer
Research 2005 ; 65 : 5498 – 5505 .
41. Patan S , Munn LL , Tanda S , Roberge S , Jain RK ,
Jones RC . Vascular morphogenesis and remodeling
in a model of tissue repair: blood vessel formation
and growth in the ovarian pedicle after
ovariectomy . Circulation Research 2001 ; 89 : 723 –
731 .
42. Patan S , Tanda S , Roberge S , Jones RC , Jain RK ,
Munn LL . Vascular morphogenesis and remodeling
in a human tumor xenograft: blood vessel
formation and growth after ovariectomy and tumor
implantation . Circulation Research 2001 ; 89 : 732 –
739 .
43. Kimura H , Braun RD , Ong ET , Hsu R , Secomb TW ,
Papahadjopoulos D , Hong K , Dewhirst MW .
Fluctuations in red cell fl ux in tumor microvessels
can lead to transient hypoxia and reoxygenation in
tumor parenchyma . Cancer Research 1996 ; 56 :
5522 – 5528 .
44. Dewhirst MW , Kimura H , Rehmus SW , Braun RD ,
Papahadjopoulos D , Hong K and Secomb TW .
Microvascular studies on the origins of perfusionlimited
hypoxia . British Journal of Cancer
Supplement 1996 ; 27 : S247 – S251 .
45. Rofstad EK , Galappathi K , Mathiesen B and Ruud
EB . Fluctuating and diffusion-limited hypoxia in
hypoxia-induced metastasis . Clinical Cancer
Research 2007 ; 13 : 1971 – 1978 .
46. Brown J . Evidence for acutely hypoxic cells in
mouse tumours, and a possible mechanism of
reoxygenation . British Journal of Radiology 1979 ; 52 :
650 – 656 .
47. Martinive P , Defresne F , Bouzin C , Saliez J , Lair F ,
Gregoire V , Michiels C , Dessy C , Feron O .
Preconditioning of the tumor vasculature and
tumor cells by intermittent hypoxia: implications
for anticancer therapies . Cancer Research 2006 ; 66 :
11736 – 11744 .
48. Wilson JL , Burchell J and Grimshaw MJ .
Endothelins induce CCR7 expression by breast
tumor cells via endothelin receptor a and hypoxiainducible
factor-1 . Cancer Research 2006 ; 66 :
11802 – 11807 .
49. Dewhirst MW . Intermittent hypoxia furthers the
rationale for hypoxia-inducible factor-1 targeting .
Cancer Research 2007 ; 67 : 854 – 855 .
50. Koumenis C , Wouters BG . “ Translating ” tumor
hypoxia: unfolded protein response (UPR)-
dependent and UPR-independent pathways .
Molecular Cancer Research 2006 ; 4 : 423 – 436 .
51. Chandel NS , McClintock DS , Feliciano CE , Wood TM ,
Melendez JA , Rodriguez AM , Schumacker PT .
Reactive oxygen species generated at mitochondrial
complex III stabilize hypoxia-inducible factor-1alpha
during hypoxia. A mechanism of O2 sensing . Journal
of Biological Chemistry 2000 ; 275 : 25130 – 25138 .
52. Brauchle M , Funk J , Kind P , Werner S . Ultraviolet B
and H 2 O 2 are potent inducers of vascular
endothelial growth factor expression in cultured
keratinocytes . Journal of Biological Chemistry 1996 ;
271 : 21793 – 21797 .
53. Mansfi eld KD , Guzy RD , Pan Y , Young RM , Cash
TP , Schumacker PT , Simon MC . Mitochondrial
dysfunction resulting from loss of cytochrome c
impairs cellular oxygen sensing and hypoxic HIF-
[alpha] activation . Cell Metabolism 2005 ; 1 : 393 – 399 .
54. Pan Y , Mansfi eld KD , Bertozzi CC , Rudenko V ,
Chan DA , Giaccia AJ , Simon MC . Multiple factors
affecting cellular redox status and energy
metabolism modulate hypoxia-inducible factor
prolyl hydroxylase activity in vivo and in vitro .
Molecular and Cellular Biology 2007 ; 27 : 912 – 925 .
55. Hutchison GJ , Valentine HR , Loncaster JA ,
Davidson JA , Hunter RD , Roberts SA , Harris AL ,
Stratford IJ , Price PM , West CM . Hypoxiainducible
factor 1{alpha} expression as an intrinsic
marker of hypoxia: correlation with tumor oxygen,
pimonidazole measurements, and outcome in
locally advanced carcinoma of the cervix . Clinical
Cancer Research 2004 ; 10 : 8405 – 8412 .
56. Sobhanifar S , Aquino-Parsons C , Stanbridge EJ ,
Olive P . Reduced expression of hypoxia-inducible
factor-1{alpha} in perinecrotic regions of solid
tumors . Cancer Research 2005 ; 65 : 7259 – 7266 .
222 S. A. Snyder et al.
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
57. Chen C , Pore N , Behrooz A , Ismail-Beigi F , Maity A .
Regulation of glut1 mRNA by hypoxia-inducible
factor-1. Interaction between H-ras and hypoxia .
Journal of Biological Chemistry 2001 ; 276 : 9519 – 9525 .
58. Wykoff CC , Beasley NJP , Watson PH , Turner KJ ,
Pastorek J , Sibtain A , Wilson GD , Turley H , Talks KL ,
Maxwell PH , Pugh CW , Ratcliffe PJ , Harris AL .
Hypoxia-inducible expression of tumor-associated
carbonic ahydrases . Cancer Research 2000 ; 60 :
7075 – 7083 .
59. Lu S , Gu X , Hoestje S , Epner DE . Identifi cation of
an additional hypoxia responsive element in the
glyceraldehyde-3-phosphate dehydrogenase gene
promoter . Biochimica et Biophysica Acta (BBA) –
Gene Structure and Expression 2002 ; 1574 : 152 .
60. Firth JD , Ebert BL and Ratcliffe PJ . Hypoxic
regulation of lactate dehydrogenase A . Journal of
Biological Chemistry 1995 ; 270 : 21021 – 21027 .
61. Semenza GL , Jiang B-H , Leung SW , Passantino R ,
Concordet J-P , Maire P , Giallongo A . Hypoxia
response elements in the aldolase A, enolase 1,
and lactate dehydrogenase A gene promoters
contain essential binding sites for hypoxia-inducible
factor 1 . Journal of Biological Chemistry 1996 ; 271 :
32529 – 32537 .
62. Semenza GL and Wang GL . A nuclear factor
induced by hypoxia via de novo protein synthesis
binds to the human erythropoietin gene enhancer at
a site required for transcriptional activation .
Molecular and Cellular Biology 1992 ; 12 : 5447 – 5454 .
63. Burroughs KD , Oh J , Barrett JC , DiAgustine RP .
Phosphatidylinositol 3-kinase and mek1/2 are
necessary for insulin-like growth factor-I-induced
vascular endothelial growth factor synthesis in
prostate epithelial cells: a role for hypoxia-inducible
factor-1? Molecular Cancer Research 2003 ; 1 : 312 – 322 .
64. Levy NS , Chung S , Furneaux H , Levy AP . Hypoxic
stabilization of vascular endothelial growth factor
mRNA by the RNA-binding protein HuR . Journal
of Biological Chemistry 1998 ; 273 : 6417 – 6423 .
65. Jiang B-H , Agani F , Passaniti A , Semenzq GL .
V-SRC induces expression of hypoxia-inducible
factor 1 (HIF-1) and transcription of genes
encoding vascular endothelial growth factor and
enolase 1: involvement of HIF-1 in tumor
progression . Cancer Research 1997 ; 57 : 5328 – 5335 .
66. Eoin PC and Cormac TT . Hypoxia-responsive
transcription factors . Pfl ugers Archiv European
Journal of Physiology 2005 ; 450 : 363 .
67. Semenza GL . Hydroxylation of HIF-1: oxygen
sensing at the molecular level . Physiology 2004 ;
19 : 176 – 182 .
68. Vordermark D , Kaffer A , Riedl S , Katzer A , Flentje M .
Characterization of carbonic anhydrase IX (CA IX)
as an endogenous marker of chronic hypoxia in live
human tumor cells . International Journal of
Radiation Oncology, Biology, Physics 2005 ; 61 : 1197 .
69. Cline JM , Thrall D , Page RL , Franko AJ and Raleigh JA .
Immunohistochemical detection of a hypoxia
marker in spontaneous canine tumours . British
Journal of Cancer 1990 ; 62 : 925 – 931 .
70. Cline JM , Rosner GL , Raleigh JA , Thrall DF .
Quantifi cation of CCI-103F labeling heterogeneity in
canine solid tumors . International Journal of Radiation
Oncology, Biology, Physics 1997 ; 37 : 655 – 662 .
71. Zeman E , Calkins DP , Cline JM , Thrall DE and
Raleigh JA . The relationship between proliferative
and oxygenation status in spontaneous canine
tumors . International Journal of Radiation Oncology,
Biology, Physics 1993 ; 27 : 891 – 898 .
72. Achermann RE , Ohlerth SM , Rohrer-Bley C ,
Gassmann M , Inteeworn N , Roos M , Schärz M ,
Wergin M , Kaser-Holz B . Oxygenation of
spontaneous canine tumors during fractionated
radiation therapy . Strahlentherapie und Onkologie
2004 ; 180 : 297 .
73. Moulder JE and RS . Hypoxic fractions of solid
tumors: experimental techniques, methods of analysis,
and a survey of existing data . International Journal of
Radiation Oncology, Biology, Physics 1984 ; 10 : 695 – 712 .
74. Denekamp J , Fowler J and Dische S . The proportion
of hypoxic cells in a human tumor . International
Journal of Radiation Oncology, Biology, Physics 1977 ;
2 : 1227 – 1228 .
75. Chaudary N and Hill R . Hypoxia and metastasis in
breast cancer . Breast Disease 2007 ; 26 : 55 – 64 .
76. Knowles HJ and Harris AL . Hypoxia and oxidative
stress in breast cancer: hypoxia and tumourigenesis .
Breast Cancer Research 2001 ; 3 : 318 – 322 .
77. Vaupel P , Mayer A , Briest S and Höckel M . Hypoxia
in breast cancer: role of blood fl ow, oxygen
diffusion distances, and anemia in the development
of oxygen depletion . Advances in Experimental
Medicine and Biology 2005 ; 566 : 333 – 342 .
78. Vaupel P , Schlenger K , Knoop C , Hockel M .
Oxygenation of human tumors: evaluation of tissue
oxygen distribution in breast cancers by
computerized O 2 tension measurements . Cancer
Research 1991 ; 51 : 3316 – 3322 .
79. Milosevic M , Chung P , Parker C , Bristow R , Toi A ,
Panzarella T , Warde P , Catton C , Menard C , Bayley A ,
Gospodarowicz M and Hill R . Androgen withdrawal
in patients reduces prostate cancer hypoxia:
implications for disease progression and radiation
response . Cancer Research 2007 ; 67 : 6022 – 6025 .
80. Movsas B , Chapman J , Hanlon AL , Horwitz EM ,
Pinover WH , Greenberg RE , Stobbe C and Hanks
GE . Hypoxia in human prostate carcinoma: an
Role of hypoxia in canine cancer 223
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 4, 213–223
Eppendorf PO2 study . American Journal of Clinical
Oncology 2001 ; 24 : 458 – 461 .
81. Francis P , Namlos H , Müller C , Edén P , Fernebro J ,
Berner JM , Bjerkehagen B , Akerman M , Bendahl
PO , Isinger A , Rydholm A , Myklebost O and
Nilbert M . Diagnostic and prognostic gene
expression signatures in 177 soft tissue sarcomas:
hypoxia-induced transcription profi le signifi es
metastatic potential . BMC Genomics 2007 ; 8 : 73 .
82. Nordsmark M , Overgaard M and Overgaard J .
Pretreatment oxygenation predicts radiation
response in advanced squamous cell carcinoma of
the head and neck . Radiotherapy and Oncology 1996 ;
41 : 31 – 39 .
83. Lyng H , Sundfor K and Rofstad EK . Oxygen tension
in human tumours measured with polarographic
needle electrodes and its relationship to vascular
density, necrosis and hypoxia . Radiotherapy and
Oncology 1997 ; 44 : 163 – 169 .
84. Fyles AW , Milosevic M , Wong R , Kavanagh M-C ,
Pintilie M , Sun A , Chapman W , Levin W , Manchul L ,
Keane TJ . Oxygenation predicts radiation response
and survival in patients with cervix cancer .
Radiotherapy and Oncology 1998 ; 48 : 149 – 156 .
85. Bennett M , Feldmeier J , Smee R and Milross C .
Hyperbaric oxygenation for tumour sensitisation to
radiotherapy . Cochrane Database of Systematic
Reviews 2005 ; 4 : CD005007 .
86. Song CW , Park HJ , Lee CK , Griffi n R . Implications
of increased tumor blood fl ow and oxygenation
caused by mild temperature hyperthermia in tumor
treatment . International Journal of Hyperthermia
2005 ; 21 : 761 – 767 .
87. Kim JH , Kim SH , Kolozsvary A , Khil MS .
Potentiation of radiation response in human
carcinoma cells in vitro and murine fi brosarcoma
in vivo by topotecan, an inhibitor of DNA
topoisomerase I . International Journal of Radiation
Oncology, Biology, Physics 1992 ; 22 : 515 – 518 .
88. Crabtree H . Observations on the carbohydrate
metabolism of tumours . Biochemical Journal 1929 ;
23 : 536 – 545 .
89. Crokart N , Jordan BF , Baudelet C , Cron GO ,
Hotton J , Radermacher K , Grégoire V , Beghein N ,
Martinire P , Bouzin C , Feron C , Gallez B ,
Dewhirst MW , Navia IC , Brizel DM , Willett C ,
Secomb TW . Glucocorticoids modulate tumor
radiation response through a decrease in tumor
oxygen consumption . Clinical Cancer Research
2007 ; 13 : 630 – 635 .
90. Crokart N , Radermacher K , Jordan BF , Baudelet C ,
Cron GO , Grégoire V , Beghein N , Bouzin C ,
Feron O , Gallez B . Tumor radiosensitization by
anti-infl ammatory drugs: evidence for a new
mechanism involving the oxygen effect . Cancer
Research 2005 ; 65 : 7911 – 7916 .
91. Jordan BF , Grégoire V , Demeure RJ , Sonveaux P ,
Feron O , O’Hara J , Vanhulle VP , Delzenne N , Gallez B .
Insulin increases the sensitivity of tumors to
irradiation: involvement of an increase in tumor
oxygenation mediated by a nitric oxidedependent
decrease of the tumor cells oxygen
consumption . Cancer Research 2002 ; 62 :
3555 – 3561 .
92. Secomb TW , Hsu R and Dewhirst MW . Synergistic
effects of hyperoxic gas breathing and reduced
oxygen consumption on tumor oxygenation: a
theoretical model . International Journal of Radiation
Oncology, Biology, Physics 2004 ; 59 : 572 .
93. Snyder SA , Lanzen JL , Braun RD , Rosner G ,
Secomb TW , Biaglow J , Brizel DM , Dewhirst MW .
Simultaneous administration of glucose and
hyperoxic gas achieves greater improvement in
tumor oxygenation than hyperoxic gas alone .
International Journal of Radiation Oncology, Biology,
Physics 2001 ; 51 : 494 .
94. Crile G . Selective destruction of cancers after
exposure to heat . Annals of Surgery 1962 ; 156 :
404 – 407 .
95. Thrall DE , LaRue SM , Yu

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