**3. Decreasing oxygen consumption by tumor cells**

Tumor oxygenation is a matter of supply and demand. Whereas the provascular strategy intends to improve oxygen supply, several strategies are aimed at decreasing oxygen consumption by tumor cells rendering molecular O2 available for the stabilization of radiation-induced DNA damage. Indeed, theoretical modeling studies demonstrated that reducing O2 consumption could be more efficient at reducing tumor hypoxia than increasing blood pO2 or flow (Secomb, 1995). Two main targets can be considered for inhibiting oxygen consumption: (i) direct interference with the mitochondrial respiratory chain (at different levels), and (ii) modulation of the redox status to change the mitochondrial membrane potential (Pilkington et al., 2008); the final aim being a subsequent increase in tumor pO2 and enhancement of the efficacy of radiotherapy.

In contrast to provascular strategies, Laser Doppler flowmetry, DCE-MRI and electron paramagnetic resonance (EPR) oximetry have revealed that the radiosensitizing effects of these treatments are primarily caused by a decrease in the rate of oxygen consumption by tumor cells, thus allowing oxygen to be redirected from a metabolic fate to the stabilization of DNA lesions. Indeed, apart from NO donors, all the treatments described below did not show any significant increase in tumor blood flow concomitant to the increase in tumor oxygenation. Some of them even showed a decrease in tumor blood flow that was

Targeting Tumor Perfusion and Oxygenation Modulates

in the oxygen consumption rate of tumors (Jordan et al., 2007).

**3.5 NO donors** 

**treatment** 

alone (Jordan et al., 2010a).

**3.6 Anti-angiogenic agents** 

Hypoxia and Cancer Sensitivity to Radiotherapy and Systemic Therapies 297

hypothyroidism slows down the neoplastic process, whereas administration of a thyroid hormone preparation restores tumor growth rates (Mishkin et al., 1981; Shoemaker & Dagher, 1979; Theodossiou et al., 1999). In humans, several case reports have indicated a prolonged survival in the presence of hypothyroidism (Cristofanilli et al., 2005; Hercbergs & Leith, 1993). Moreover, a decrease in the thyroid function may also serve to favorably influence the response to treatment. Finally, patients presented an enhanced response rate to chemotherapy and survived significantly longer under hypothyroidism (Hercbergs et al., 2003). Our group recently demonstrated that the thyroid status is associated with a significant change in tumor radiosensitivity since the regrowth delay was increased in hypothyroid mice compared to euthyroid mice. Mechanistically, we demonstrated that the higher level of tumor oxygenation in hypothyroid mice results from a significant reduction

We recently tested whether *S*-nitrosocaptopril, a molecule combining a NO donor and an angiotensin converting enzyme inhibitor (ACE inhibitor), could temporarily improve the hemodynamic status of experimental tumors. We identified a time window during which tumor oxygenation was improved, as a result of a combined effect on tumor blood flow and oxygen consumption. Consequently, the administration of *S*-nitrosocaptopril contributed to the increase in efficacy of radiation therapy, an effect that was not observed with captopril

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** 

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

counteracted by the dramatic decrease in oxygen consumption by tumor cells (i.e. insulin and NS-398). In addition, regarding NO-mediated treatments, our models showed that the radiotherapeutic response not only depended on the tumor pO2 but also on the net level of NO achieved at the time of irradiation, NO itself being able to stabilize irradiation-induced DNA lesions *in vivo* (Jordan et al., 2004).

The first drug that was described to inhibit oxygen consumption in tumors was metaiodobenzylguanidine (MIBG), which causes an inhibition of the mitochondrial site I electron transfer, inhibition of NAD(P)H oxidation, and is described to alter tumor glycolysis by inhibiting oxygen consumption (Biaglow et al., 1998). We consecutively focused on different innovative treatments that may alter oxygen consumption by tumor cells, as listed below.

### **3.1 Insulin**

This hormone was known to increase blood flow in human skeletal muscle and was postulated to be an important modulator of tumor oxygenation (Jordan et al., 2002). Indeed, we showed that insulin had a profound effect on tumor oxygenation that was not due to an increase in tumor blood flow but to a decrease in tumor cell oxygen consumption. The increase in tumor oxygenation resulted in an important enhancement in the sensitivity of tumors to irradiation. The likely scenario involves a stimulation of eNOS and a consequent increase in NO release. As NO regulates mitochondrial respiration by virtue of reversible interactions with cytochrome c oxidase (complex IV), an increase in NO release consequently decreased cell respiration (Jordan et al., 2002). A preclinical study confirmed the dose-dependant increase in tumor oxygenation and radiation sensitivity by insulin, without any increase in the radiation toxicity for normal tissues (Jordan et al., 2006a).

#### **3.2 Glucocorticoids**

Earlier work had demonstrated that the administration of cortisone to rats resulted in both the inhibition of oxygen consumption and the uncoupling of oxidative phosphorylation in liver mitochondria (Kimberg et al., 1968). Glucocorticoids seemed to decrease the cytochrome c oxidase (complex IV) activity of isolated rat kidney mitochondria by a direct mechanism (Simon et al., 1998). Our group showed an important increase in tumor oxygenation induced by an effect on oxygen consumption. Decreased oxygen consumption could be explained by the capacity of glucocorticoids to inhibit cytochrome c oxidase of the mitochondrial respiratory chain. The result of this increase in tumor oxygenation was an improvement of the radiation efficacy by a factor of 1.7 (Crokart et al., 2007).

#### **3.3 Anti-inflammatory drugs**

Several reports indicated that many non-steroidal anti-inflammatory drugs (NSAIDs) uncouple mitochondrial oxidative phosphorylation with important consequences on cell oxygen consumption. However, it was suggested that the response was dependent on the dose as well as on the type of NSAIDs. For the first time, our group reported that the administration of NSAIDs induced a dramatic increase in tumor oxygenation explained by reduced oxygen consumption. An increase in the tumor response was observed when the irradiation was applied at the time of maximal reoxygenation (Crokart et al., 2005).

#### **3.4 Thyroid hormones**

Chronic alteration in the thyroid status has been shown to affect mitochondrial oxygen consumption in skeletal muscle (Gredilla et al., 2001). Also, studies have demonstrated that hypothyroidism slows down the neoplastic process, whereas administration of a thyroid hormone preparation restores tumor growth rates (Mishkin et al., 1981; Shoemaker & Dagher, 1979; Theodossiou et al., 1999). In humans, several case reports have indicated a prolonged survival in the presence of hypothyroidism (Cristofanilli et al., 2005; Hercbergs & Leith, 1993). Moreover, a decrease in the thyroid function may also serve to favorably influence the response to treatment. Finally, patients presented an enhanced response rate to chemotherapy and survived significantly longer under hypothyroidism (Hercbergs et al., 2003). Our group recently demonstrated that the thyroid status is associated with a significant change in tumor radiosensitivity since the regrowth delay was increased in hypothyroid mice compared to euthyroid mice. Mechanistically, we demonstrated that the higher level of tumor oxygenation in hypothyroid mice results from a significant reduction in the oxygen consumption rate of tumors (Jordan et al., 2007).
