**3.6 Anti-angiogenic agents**

As stated earlier, two anti-angiogenic agents, SU-5416 and ZD-6474, have also been identified as potent inhibitors of oxygen consumption (Ansiaux et al., 2007, 2009). Our major findings regarding those specific anti-angiogenic agents were the following: (a) SU-5416 and ZD-67474 both induce an increase in tumor oxygenation at an early phase of treatment (after 2 days of daily injections); (b) this tumor reoxygenation can be exploited to increase the efficacy of combined radiotherapy; (c) the mechanism of increase in tumor oxygenation does not involve a ''normalization'' of the tumor vasculature as described previously for thalidomide in the same tumor model (Ansiaux et al., 2005) but is consistent with the decrease in the rate of oxygen consumption by the tumor cells. Indeed, at this early stage of the treatment, no apparent remodeling of the tumor vasculature and no changes in tumor perfusion and permeability parameters were observed, using histological and DCE-MRI analysis, respectively. We however demonstrated a reduction in tumor oxygen consumption after those treatments. The reduction factor in oxygen consumption observed was sufficient to abolish tumor hypoxia, as we reported previously using the treatments listed above.

#### **4. Hyperthermia: Combining provascular and oxygen consumption effects in a single treatment**

Hyperthermia is a potent adjuvant therapy with radiotherapy and chemotherapy, and the perfect illustration of a strategy combining transient, local vasodilatation with the inhibition of tumor cell respiration. The heat treatment consists of elevating the temperature of tumors to a supra-physiological range of 40°C to 45°C at which tumor reoxygneation occurs with limited skin toxicity. Hyperthermia induces a graded response in tissues characterized by decreased oxygen consumption at temperatures ≥ 40°C, vasodilatation between 41°C and 41.5°C, and vascular damage above 42°C. Although direct tumor cell killing was

Targeting Tumor Perfusion and Oxygenation Modulates

**5.1.1 Electron Paramagnetic Resonance (EPR) oximetry** 

convenient as they can diffuse in the whole tissue.

(Swartz et al., 2004).

**5.1.2 19F relaxometry** 

2011).

Hypoxia and Cancer Sensitivity to Radiotherapy and Systemic Therapies 299

disorders and in treatment response, the lack of easy-to-use and efficient methods to quantify oxygen deprivation hampers further pathophysiological understanding and restricts the clinical implementation of oxygen mapping techniques resulting in the absence of gold standard for measuring hypoxia in the day-to-day practice (Tatum et al., 2006). Below is a non exhaustive review of the most relevant non invasive methods able to assess tumor hemodynamic parameters. Methods to measure absolute pO2 mostly encompass EPR oximetry and 19F relaxometry whereas indirect methods include Blood Oxygen Level-Dependent (BOLD) MRI, Oxygen enhanced relaxation MRI, Oxygen enhanced longitudinal relaxation MRI and positron emission tomography (PET) tracers retained in hypoxic regions. Of note, our group has also developed innovative methods to assess tumor oxygen consumption *in vitro* and *in vivo*, which have been reviewed elsewhere (Jordan & Gallez,

EPR is a MR method that detects only species containing unpaired electrons (Gallez & Swartz, 2004a). One of the numerous applications of EPR is *in vivo* oximetry. Molecular oxygen is a triplet radical that possesses two unpaired electrons which are responsible for its paramagnetism. However, EPR is not able to detect oxygen itself when dissolved in fluids near room temperature: in biological systems, the output signal lines are so broadened as to be undetectable. Indirect methods exist. Most of these methods rely on the paramagnetic properties of molecular oxygen, which acts as an efficient relaxer for other paramagnetic species (Gallez et al., 2004b). The enhancement of relaxation rates scales linearly with the concentration of oxygen over a wide range of oxygen tensions. The lack of detectable levels of endogenous paramagnetic species makes it necessary to use exogenous paramagnetic materials. Variations in pO2 of less than 1mmHg can be detected using particulate materials. While EPR spectroscopy provides local measurements, EPR imaging techniques provide spatially resolved measurements of these materials. The spatial distribution of free radicals can be performed utilizing magnetic field gradients in a manner similar to that of MRI. Spectral–spatial EPR imaging encodes both the spatial distribution of the spin probe and the spectral information, which allows the mapping of molecular oxygen (Kuppusamy et al., 2003). For this purpose, the use of soluble EPR materials such as trityl radicals is more

EPR oximetry was compared with other methods that provide direct or indirect measurements of tumor oxygenation: with polarographic electrodes, the distribution of nitroimidazoles, the BOLD effect in MRI, and pO2 recordings using OxyLite (reviewed in Gallez et al., 2004b). Two major challenges are now considered for moving this technology into the clinic: (i) assuring biocompatibility of the oxygen sensors in humans and (ii) modifying the instruments so that they can be used for humans instead of small animals

19F NMR spectroscopy and imaging of perfluorocarbon (PFC) emulsions (hydrocarbons with protons having been replaced with fluorine nuclei) has been extensively exploited to measure the oxygen tension of biological systems in preclinical studies. The 19F MR signal of the PFC is sensitive to the pO2 of the surrounding tumor tissue, and acts as an oximeter. The principle behind 19F MR oximetry relies on the linear increase of the NMR spin-lattice relaxation rate R1 (=1/T1) of PFC emulsions with increasing oxygen tension (Mason et al.

demonstrated *in vitro* at higher temperatures, long-term tumor control has never been demonstrated using hyperthermia as the sole treatment modality. Vasodilation only modestly contributes to tumor reoxygenation at the low thermal doses. Increased pO2 rather primarily results from changes in oxygen consumption in the target cells, and at least two different processes have been identified to contribute to this response. It is now well demonstrated that an important target of heat is proteins among which enzymes of the respiratory chain are more sensitive to heat inactivation/denaturation than glycolytic enzymes (Lepock et al., 1987; Kelleher et al., 1995). But the inhibition of mitochondrial respiration by heat lasts longer than the turnover time of respiratory enzymes, suggesting the existence of an additional mechanism. Dewhirst in collaboration with our group (Moon et al., 2010) recently demonstrated that mild hyperthermia activates the transcription factor hypoxia-inducible factor 1 (HIF-1) through an hypoxia-independent mechanism involving the sequential activation of Extracellular signal-Regulated Kinases (ERK) by heat shock, ERK-induced upregulation of the expression of the Nox1 subunit of NAD(P)H oxidase, increased ROS production by NAD(P)H oxidase, ROS-induced HIF-1 protein stabilization, and, ultimately, HIF-1 activation. HIF-1 target genes include most glycolytic enzymes and transporters as well as major pro-angiogenic molecules such as VEGF. Among these genes, we showed that pyruvate dehydrogenase kinase 1 (PDK1) largely mediates the inhibition of mitochondrial respiration by heat in tumor cells through inhibiting pyruvate dehydrogenase (PDH), i.e., the enzyme coupling glycolysis to the tricarboxylic acid (TCA) cycle (Moon et al., 2010). Furthermore, consistent with the increase in VEGF expression that we also observed in heat-treated tumors, we documented an increased vascular density in perfused tumor areas where oxygen is extracted from the blood. Tumor reoxygenation by mild hyperthermia is thus a multifaceted process involving the combination of decreased O2 consumption by tumor cells and increased O2 delivery by blood vessels. This and the fact that reoxygenation occurs at thermal doses lower than those inducing vascular damage justifies the use of mild hyperthermia as a combination treatment notably with radiotherapy. Although several clinical trials have confirmed that combining heat and radiotherapy is indeed associated with better patient treatment outcome (Brizel et al., 1996; Jones et al., 2003, 2005; Vujaskovic et al., 2003), the future clinical development of hyperthermia strongly relies on designing tools allowing for homogeneous thermal dose distribution and improving imaging techniques able to correlate thermal maps of tumors to the clinical outcome of patients (Dewhirst et al., 2010).

#### **5. Non invasive imaging of tumor oxygenation and perfusion**

The study of magnetic resonance (MR) markers over the past decade has provided evidence that the tumor microenvironment and hemodynamics play a major role in determining therapy outcome. Therefore, the identification of relevant non-invasive imaging endpoints is of crucial importance in the management of cancer patients. Improvement of the therapeutic index was evidenced in numerous preclinical studies that used multimodal imaging. The impact of non-invasive imaging in oncology extends from guiding preclinical development of targeted biomarkers and therapeutic agents, to assisting in the diagnosis and staging of tumors in the clinic, as well as monitoring the therapeutic response.

#### **5.1 Tumor oxygenation measurements**

There is a critical need for developing dynamic, non-invasive methods for direct oxygen mapping in the clinical practice. Although hypoxia is recognized as a crucial issue in many

demonstrated *in vitro* at higher temperatures, long-term tumor control has never been demonstrated using hyperthermia as the sole treatment modality. Vasodilation only modestly contributes to tumor reoxygenation at the low thermal doses. Increased pO2 rather primarily results from changes in oxygen consumption in the target cells, and at least two different processes have been identified to contribute to this response. It is now well demonstrated that an important target of heat is proteins among which enzymes of the respiratory chain are more sensitive to heat inactivation/denaturation than glycolytic enzymes (Lepock et al., 1987; Kelleher et al., 1995). But the inhibition of mitochondrial respiration by heat lasts longer than the turnover time of respiratory enzymes, suggesting the existence of an additional mechanism. Dewhirst in collaboration with our group (Moon et al., 2010) recently demonstrated that mild hyperthermia activates the transcription factor hypoxia-inducible factor 1 (HIF-1) through an hypoxia-independent mechanism involving the sequential activation of Extracellular signal-Regulated Kinases (ERK) by heat shock, ERK-induced upregulation of the expression of the Nox1 subunit of NAD(P)H oxidase, increased ROS production by NAD(P)H oxidase, ROS-induced HIF-1 protein stabilization, and, ultimately, HIF-1 activation. HIF-1 target genes include most glycolytic enzymes and transporters as well as major pro-angiogenic molecules such as VEGF. Among these genes, we showed that pyruvate dehydrogenase kinase 1 (PDK1) largely mediates the inhibition of mitochondrial respiration by heat in tumor cells through inhibiting pyruvate dehydrogenase (PDH), i.e., the enzyme coupling glycolysis to the tricarboxylic acid (TCA) cycle (Moon et al., 2010). Furthermore, consistent with the increase in VEGF expression that we also observed in heat-treated tumors, we documented an increased vascular density in perfused tumor areas where oxygen is extracted from the blood. Tumor reoxygenation by mild hyperthermia is thus a multifaceted process involving the combination of decreased O2 consumption by tumor cells and increased O2 delivery by blood vessels. This and the fact that reoxygenation occurs at thermal doses lower than those inducing vascular damage justifies the use of mild hyperthermia as a combination treatment notably with radiotherapy. Although several clinical trials have confirmed that combining heat and radiotherapy is indeed associated with better patient treatment outcome (Brizel et al., 1996; Jones et al., 2003, 2005; Vujaskovic et al., 2003), the future clinical development of hyperthermia strongly relies on designing tools allowing for homogeneous thermal dose distribution and improving imaging techniques able to correlate

thermal maps of tumors to the clinical outcome of patients (Dewhirst et al., 2010).

The study of magnetic resonance (MR) markers over the past decade has provided evidence that the tumor microenvironment and hemodynamics play a major role in determining therapy outcome. Therefore, the identification of relevant non-invasive imaging endpoints is of crucial importance in the management of cancer patients. Improvement of the therapeutic index was evidenced in numerous preclinical studies that used multimodal imaging. The impact of non-invasive imaging in oncology extends from guiding preclinical development of targeted biomarkers and therapeutic agents, to assisting in the diagnosis and staging of

There is a critical need for developing dynamic, non-invasive methods for direct oxygen mapping in the clinical practice. Although hypoxia is recognized as a crucial issue in many

**5. Non invasive imaging of tumor oxygenation and perfusion** 

tumors in the clinic, as well as monitoring the therapeutic response.

**5.1 Tumor oxygenation measurements** 

disorders and in treatment response, the lack of easy-to-use and efficient methods to quantify oxygen deprivation hampers further pathophysiological understanding and restricts the clinical implementation of oxygen mapping techniques resulting in the absence of gold standard for measuring hypoxia in the day-to-day practice (Tatum et al., 2006). Below is a non exhaustive review of the most relevant non invasive methods able to assess tumor hemodynamic parameters. Methods to measure absolute pO2 mostly encompass EPR oximetry and 19F relaxometry whereas indirect methods include Blood Oxygen Level-Dependent (BOLD) MRI, Oxygen enhanced relaxation MRI, Oxygen enhanced longitudinal relaxation MRI and positron emission tomography (PET) tracers retained in hypoxic regions. Of note, our group has also developed innovative methods to assess tumor oxygen consumption *in vitro* and *in vivo*, which have been reviewed elsewhere (Jordan & Gallez, 2011).
