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Keywords:

  • canine;
  • hypoxia;
  • resistance;
  • tumour

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

Human oncology has clearly demonstrated the existence of hypoxic tumours and the problematic nature of those tumours. Hypoxia is a significant 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

Hypoxia has been a suspected cause for treatment failure in human oncology since the 1950s. The study of hypoxia in human medicine over the past 50 years has revealed that it is, in fact, a cause of treatment resistance for both radiation and chemotherapy. Pivotal work by Gray in 1953 and Thomlinson and Gray in 1955 lead to the subsequent in-depth study of the role of hypoxia in radiation therapy.1,2 These investigators found large regions of necrosis in human lung tumours and discovered that a thin ring of viable tumour cells consistently surrounded the necrosis. They calculated the diffusion distance of oxygen and concluded that lack of oxygen was the cause of the necrosis. Additionally, they concluded that the decreasing oxygen concentration from the vessel wall out through the tissue would leave cells at different oxygen tensions depending on their distance from the vessel. This gradient implied that near the necrotic edge, there might be residual hypoxic, but viable cells that could give rise to tumour regrowth after radiation.

Reasons for hypoxia-induced radioresistance are lack of reactive oxygen species (ROS), low proliferation rate of hypoxic cells compared with aerobic cells and upregulation of hypoxia responsive genes that promote growth and survival of cells, metastasis and a more aggressive phenotype.1, 3–6 Hypoxic cells are also more resistant to chemotherapy. One reason for chemotherapy resistance is that hypoxic cells are farther from the vasculature and may not be exposed to lethal drug concentrations.7 Low rates of cell proliferation also have fewer cells in the stage of the cell cycle most susceptible to these drugs. Even drugs that are classified as hypoxic cytotoxins are less efficacious because of diffusion limitations.8

Since Thomlinson and Gray’s seminal work, tumour hypoxia has been demonstrated to exist in numerous tumour types and has been well documented as a significant cause of treatment failure.9–13 A variety of clinical studies have shown that hypoxic human tumours are: (1) more likely to fail locally after radiotherapy; (2) more invasive and likely to develop metastases; and (3) a negative prognostic indicator of disease free and overall 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 benefit 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

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 fixation hypothesis (Fig. 1). Consequently, radiation therapy is less effective when cells are hypoxic.

image

Figure 1. Mechanisms of radiation cell kill. Radiation causes a single-stranded or double-stranded break directly and also interacts with water creating a ROS for direct DNA damage. If molecular oxygen is present during radiation treatment, 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. Image courtesy of Genetics Home Reference (Internet) Bethesda (MD), National Library of Medicine (USA): 2003 (updated 12 October 2007; cited 16 October 2007). Available from http://ghr.nlm.nih.gov/handbook/illustrations/dnastructure. Image was altered to show DNA strand breaks and the attachment of oxygen at the location of the breaks.

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Hypoxia also results in resistance to treatment with chemotherapy. Some chemotherapeutics depend on oxygen for greatest efficacy. Bleomycin is one example. It is known as a radiomimetic, an iron-containing compound and works by the formation of free radicals and DNA fixation. Oxygen is required for the formation of an iron–oxygen complex; this complex acts as an oxygen free radical by inducing strand breaks. This complex also attaches to the ends of the damaged DNA for fixation. More importantly, the widely used drug, doxorubicin, also causes cell damage by the formation of free radicals. In addition, hypoxia often increases the fraction of cells in G0. Because many chemotherapeutic drugs are cell cycle specific, they will be less effective on G0 cells.21 Vincristine and methotrexate are two cell-cycle-specific drugs that are less effective in tumours that are hypoxic.22–24 Interestingly, chemotherapeutics that are cell cycle independent are also less efficacious in hypoxic cells most likely because of the limitations on diffusion.25

Physiology and consequences of tumour hypoxia

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

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 defined 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 flux. It is now established that the red cell flux 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 pO2 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.

image

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 flux. The dramatic change in red cell flux 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.

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Oxygen consumption in proliferating cells

Proliferating cells consume oxygen at a rate of approximately five times that of quiescent cells.29 This high consumption rate is one of the most significant 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. Previous work has shown that either a four-fold increase in tumour blood flow or an 11-fold increase in arterial pO2 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 influenced by host inflammatory 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 CO2 to be excreted into the extracellular space, thereby contributing to acidification of the tumour.32,33

Tumour acidosis and hypoxia

Tumours are often found to have pH values around 6.8.34 Tumour acidosis has significant 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 flow 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 flow – 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 μm, with diffusion routinely only reaching 150 μm.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 flux within the tumour vasculature. The cells may not be at a great distance from the vasculature, but because of the flux and even occasional backwards flow, 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

Independent of the mechanism of hypoxia, there are two dominant resulting types of hypoxia currently recognized in tumours, static and intermittent. These two types of hypoxia do not occur independently although. Static hypoxia is defined as ongoing hypoxia within a cell, while intermittent hypoxia is defined as a cell experiencing fluctuations 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-1α) and subsequent activation of the unfolded protein response.50,51 ROS are of particular interest as they can be both beneficial and problematic. As discussed, ROS are needed to cause strand breaks and free radical fixation 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 anti-apoptotic, metastasis and cell survival genes both when the cells are hypoxic and when they have been re-oxygenated.

Hypoxia and gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

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, α and β, that is constitutively expressed in the cytoplasm of cells.55,56 During normoxic conditions, prolyl hydroxylases contained in the cell cytoplasm modifies the α-subunit by adding hydroxy residues to two prolines. The hydroxylation allows the von Hippel Lindau (VHL) tumour suppressor complex protein to bind with HIF-1α. Once the VHL complex binds the α-subunit, it is shuttled to the proteosome for degradation. Induction of hypoxia prevents the prolyl hydroxylases from hydroxylating the α-subunit because oxygen is required for this reaction. If levels of HIF-1α increase, it dimerizes with the β-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 meserchymal-epithelial transition factor (cMET)].62,66–68

Hypoxia and veterinary oncology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

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 field 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 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 first direct evidence of the presence of hypoxia with spontaneous canine tumours.

The first 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 pO2 value of less than 2.5 mmHg.13 These results showed that canine tumours contain a significant amount of radiobiologically hypoxic cells that could lead to treatment resistance. This study also verified that spontaneous canine soft tissue sarcomas are a reliable model for the study of tumour hypoxia. Following the verification of hypoxia in canine patients, the same group examined the changes in tumour pO2 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 fixed points in time. An additional study observed the changes in pO2 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 first documenting direct evidence of intermittent hypoxia in a spontaneous canine tumour. Even though tumour pO2 varied widely during radiation therapy, a subsequent study showed that baseline pO2 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References

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 sarcoma15,81,82 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-iodo-benzylguanidine.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 flow or arterial pO2 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 flow or arterial pO2 is difficult to attain. However, a significant decrease in oxygen consumption can be accomplished by the use of such compounds as glucose, glucocorticoids, non-steroidal anti-inflammatory 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 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 flow, vascular permeability and re-oxygenation. The first 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 specified temperature water bath. Since then, studies have shown that hyperthermia improves tumour oxygenation in canine tumours,95,96 especially when the baseline pO2 values were low.12

This review serves to summarize the small, but rapidly expanding field 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 beneficial 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. Top of page
  2. Abstract
  3. Introduction
  4. Hypoxia and cancer therapy
  5. Physiology and consequences of tumour hypoxia
  6. Static versus intermittent hypoxia
  7. Hypoxia and gene expression
  8. Hypoxia and veterinary oncology
  9. Overcoming hypoxia
  10. References
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