**1. Introduction**

288 Advances in Cancer Therapy

Tsihlias, J.; Kapusta, L. & Slingerland, J. (1999). The prognostic significance of altered cyclindependent kinase inhibitors in human cancer. *Annu Rev Med,* Vol.50, pp. 401-23 VanderWel, S. N.; Harvey, P. J.; McNamara, D. J.; Repine, J. T.; Keller, P. R.; Quin, J., 3rd;

Wang, W. L.; Conley, A.; Reynoso, D.; Nolden, L.; Lazar, A. J.; George, S. & Trent, J. C.

Wesierska-Gadek, J.; Maurer, M.; Zulehner, N. & Komina, O. (2011). Whether to target

Wolfel, T.; Hauer, M.; Schneider, J.; Serrano, M.; Wolfel, C.; Klehmann-Hieb, E.; De Plaen, E.;

Wyatt, P. G.; Woodhead, A. J.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D. M.;

stromal tumor. *Cancer Chemother Pharmacol,* Vol.67 Suppl. 1, pp. S15-24 Weinberg, R. A. (2007). *The biology of cancer*, Garland Science, ISBN 0-8153-4078-8, New York,

abstract 3531

USA

No.2, pp. 341-9

No.16, pp. 4986-99

*Med Chem,* Vol.48, No.7, pp. 2371-87

melanoma. *Science,* Vol.269, No.5228, pp. 1281-4

of the oral multi-CDK inhibitor PHA-848125. *J Clin Oncol,* Vol.26, (May 20 Suppl.),

Booth, R. J.; Elliott, W. L.; Dobrusin, E. M.; Fry, D. W. & Toogood, P. L. (2005). Pyrido[2,3-d]pyrimidin-7-ones as specific inhibitors of cyclin-dependent kinase 4. *J* 

(2011). Mechanisms of resistance to imatinib and sunitinib in gastrointestinal

single or multiple CDKs for therapy? That is the question. *J Cell Physiol,* Vol.226,

Hankeln, T.; Meyer zum Buschenfelde, K. H. & Beach, D. (1995). A p16INK4ainsensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human

Davis, D. J.; Devine, L. A.; Early, T. R.; Feltell, R. E.; Lewis, E. J.; McMenamin, R. L.; Navarro, E. F.; O'Brien, M. A.; O'Reilly, M.; Reule, M.; Saxty, G.; Seavers, L. C.; Smith, D. M.; Squires, M. S.; Trewartha, G.; Walker, M. T. & Woolford, A. J. (2008). Identification of N-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3 carboxamide (AT7519), a novel cyclin dependent kinase inhibitor using fragmentbased X-ray crystallography and structure based drug design. *J Med Chem,* Vol.51, Hypoxia, a partial pressure of oxygen (pO2) below physiological needs, is a limiting factor affecting the efficiency of radiotherapy. Indeed, the reaction of reactive oxygen species (ROS, produced by water radiolysis) with DNA is readily reversible unless oxygen stabilizes the DNA lesion. While normal tissue oxygenation is around 40 mm Hg, both rodent and human tumors possess regions of tissue oxygenation below 10 mm Hg, at which tumor cells become increasingly resistant to radiation damage (radiobiological hypoxia) (Gray, 1953). Because of this so-called "oxygen enhancement effect", the radiation dose required to achieve the same biologic effect is about three times higher in the absence of oxygen than in the presence of normal levels of oxygen (Gray et al., 1953; Horsman & van der Kogel, 2009). Hypoxic tumor cells, which are therefore more resistant to radiotherapy than well oxygenated ones, remain clonogenic and contribute to the therapeutic outcome of fractionated radiotherapy (Rojas et al., 1992).

Tumor hypoxia results from the imbalance between oxygen delivery by poorly efficient blood vessels and oxygen consumption by tumor cells with high metabolic activities. On the one hand, oxygen delivery is impaired by structural abnormalities present in the tumor vasculature (Munn, 2003). They include caliber variations with dilated and narrowed single branches of tumor vessels, non-hierarchical vascular networks, disturbed precapillary architecture, and incomplete vascular walls. These structural abnormalities cause numerous functional impairments, i.e. increased transcapillary permeability, increased vascular permeability, interstitial hypertension, and increased flow resistance (Boucher et al., 1996; McDonald & Baluk, 2002). It is however important to note that, although hastily formed immature tumor microvessels lack smooth muscle layer(s) and are therefore unable to provide autoregulation, it is not uncommon to find mature blood vessels with smooth muscle layers and neural junctions inside slow-growing tumors (e.g. most human tumors) (Feron, 2004). On the other hand, the altered tumor cell metabolism with elevated metabolic rates also contributes to the occurrence of hypoxic regions in tumors and further causes extracellular acidification. Tumor hypoxia occurs in two ways: chronic hypoxia (or diffusion-limited hypoxia), and acute hypoxia (or perfusion-limited or fluctuating hypoxia). Chronic hypoxia has classically been thought to result from long diffusion distances

Targeting Tumor Perfusion and Oxygenation Modulates

which can have dramatic consequences on outcome (Ang, 2010).

monitor tumor perfusion and oxygenation.

This chapter will explore 2 strategies to radiosensitize tumors to X-rays: to increase oxygen delivery by exploiting the reactivity of mature tumor vessels (the so-called 'provascular' approach) and to decrease oxygen consumption by tumor cells. The goal of the provascular approach is to temporarily increase tumor perfusion and oxygenation through pharmacological interventions. Accordingly, radiotherapy could benefit from tumor reoxygenation whereas a decrease in interstitial pressure could facilitate tumor accessibility to circulating drugs. Alternatively, a second approach is to decrease the oxygen consumption by tumor cells since theoretical modeling studies demonstrated that reducing O2 consumption was far more efficient at reducing tumor hypoxia than increasing blood pO2 or flow (Secomb et al., 1995). We will also describe attempts to combine both approaches that can be considered as complementary strategies. The evaluation and validation of these adjuvant therapies require imaging techniques capable to accurately

From the clinical analyses cited above, it also appeared that the variation in the results among the trials reflects a considerable heterogeneity among tumors and that patient individualization would be mandatory for the success of such therapeutic approach. There is therefore an essential need to predict individually the presence of hypoxic regions in tumors. Based on individual tumor characteristics and/or the ability to alleviate tumor hypoxia, it will become possible to adapt the individual treatment either by delivering optimal radiation doses into the resistant areas or by delivering an associated treatment for

Hypoxia and Cancer Sensitivity to Radiotherapy and Systemic Therapies 291

effect of radiotherapeutic management in head and neck cancer (Overgaard et al., 1998). Since these results, nimorazole administration became part of the standard irradiation protocol for Head and Neck cancer in Denmark. However, these findings have had no impact on general clinical practice except for Denmark because earlier generations of these agents induced rather severe peripheral neuropathy and because nearly all of the individual phase III trials yielded negative or inconclusive results (Ang, 2010). The ARCON protocol consists in the combination of accelerated radiotherapy to overcome tumor cell proliferation, carbogen breathing to overcome diffusion-limited hypoxia, and nicotinamide to minimize capillary bed shutdown and thereby reduce perfusion-related acute hypoxia. Phase I and II clinical trials have shown the feasibility and tolerability of the treatment and have produced promising results in term of tumor control, in particular in cancer of the head and neck and bladder (Kaanders et al., 2002). A large improvement in survival was demonstrated using this approach during radiotherapy for bladder cancer. The randomized multicentre phase III trial, designed and coordinated at Mount Vernon Cancer Centre, demonstrated a 13% benefit in overall survival when radiotherapy was combined with carbogen and nicotinamide compared to radiotherapy alone (ASTRO, 2009). Results from a large phase III trial launched to test this regimen will become available within the next 2 years. Finally, Tirapazamine (TPZ) has attracted interest after preclinical studies showing that in addition to augmenting the cytotoxicity of both radiation and cisplatin, this compound selectively kills hypoxic cells (Rischin et al., 2001; 2005). Phase I and II studies of the combination of TPZ with radiation and cisplatin were performed on patients with locally advanced head and neck carcinoma. On the basis of the phase II data, a large international phase III trial was launched (Peters et al., 2010). Surprisingly, the combination did not show any evidence of improvement in overall survival. Nevertheless, patients were not previously screened for tumor hypoxia, the study was multicentric with some centers enrolling fewer than five patients and a consequent decrease in the quality of radiotherapy planning and delivery

between tumor vessels as the consequence of the more rapid expansion of tumors cells than that of the supporting vasculature (Vaupel et al., 1989). It is now well established that steep longitudinal gradients of pO2 along the vascular tree, as opposed to radial diffusion of oxygen, can largely contribute to deficiencies in tumor oxygen supply (Dewhirst et al., 1999). The origin of acute hypoxia in tumors is not firmly established. The commonly held view has been that acute hypoxia results primarily from vascular stasis, which stems from one of three causes: 1) vascular collapse in regions of high tumor interstitial pressure, 2) vessel plugging by leukocytes, and 3) impingement of tumor cells on the vascular lumen. It has been demonstrated that temporal instability in tumor red blood cell flux could lead to transient hypoxia (Kimura et al., 1996), and Dewhirst linked temporal changes in microvessel red blood cell flux to changes in the oxygen content in the same vessel (Dewhirst et al., 1996). Factors that may contribute to flow fluctuations include arteriolar vasomotion and rapid vascular modeling (Baudelet et al., 2004, 2006; Dewhirst et al., 1996; Patan et al., 1996). More recent studies indicate a widespread presence of fluctuating hypoxia in solid tumors (Cardena-Navia et al., 2008).

The effect of tumor hypoxia on the response to treatment by ionizing radiation has been demonstrated in a multitude of experimental studies. In a series of clinical studies in the early nineties, Vaupel and others showed definitively that measurements of pO2 by polarographic microelectrodes provided useful criteria for predicting the response of tumors to radiation therapy (Gatenby et al., 1988; Hockel et al., 1993; Okunieff et al., 1993; Stone et al., 1993; Thomas et al., 1994). These results stimulated considerable efforts in defining and evaluating therapeutic approaches designed to overcome tumor hypoxia as source of resistance (Horsman & van der Kogel, 2009). A particular area under focus is thus to combine radiotherapy with treatments that increase tumor pO2. Other approaches consist to chemically radiosensitize hypoxic cells or alternatively to exploit hypoxia as a mean to selectively kill the resistant population of hypoxic cells. Before the advent of imaging methods able to provide non-invasively oxygen estimation, animal and clinical studies were generally designed to evaluate the effect of a given treatment on tumor pO2 as measured by Eppendorf or histological markers of tumor hypoxia. The clinical end points were generally locoregional control and survival. Modifiers of oxygen delivery tested in clinical trials included hyperbaric oxygen therapy (HBO), oxygen and carbogen breathing. Hypoxic cell radiosensitizers (possessing a selective toxicity for the radioresistant hypoxic cells) tested in clinical trials included metronidazole, misonidazole, nimorazole, and tirapazamine. In a systematic review, J. Overgaard (2007) identified 10,108 patients in 86 randomized trials designed to modify tumor hypoxia in patients treated with curative attempted primary radiation therapy alone. Overall modification of tumor hypoxia significantly improved the effect of radiotherapy for the outcome of locoregional control and with an associated significant overall survival benefit. No significant influence was found on the incidence of distant metastases or on the risk of radiation-related complications. From this meta-analysis, the authors concluded in 2007 that "Ample data exist to support a high level of evidence for the benefit of hypoxic modification. However, hypoxic modification still has no impact on general clinical practice".

Currently, the most advanced therapeutic interventions used in the clinic to target tumor hypoxia are either the DAHANCA (Danish head and neck cancer) trial, the application of the ARCON (Accelerated radiotherapy, carbogen and nicotinamide) protocol, and phase III studies with Tirapazamine. In the DAHANCA phase III study, nimorazole has been used as hypoxic radiosensitizer on 422 patients, and it was shown that this compound improves the

between tumor vessels as the consequence of the more rapid expansion of tumors cells than that of the supporting vasculature (Vaupel et al., 1989). It is now well established that steep longitudinal gradients of pO2 along the vascular tree, as opposed to radial diffusion of oxygen, can largely contribute to deficiencies in tumor oxygen supply (Dewhirst et al., 1999). The origin of acute hypoxia in tumors is not firmly established. The commonly held view has been that acute hypoxia results primarily from vascular stasis, which stems from one of three causes: 1) vascular collapse in regions of high tumor interstitial pressure, 2) vessel plugging by leukocytes, and 3) impingement of tumor cells on the vascular lumen. It has been demonstrated that temporal instability in tumor red blood cell flux could lead to transient hypoxia (Kimura et al., 1996), and Dewhirst linked temporal changes in microvessel red blood cell flux to changes in the oxygen content in the same vessel (Dewhirst et al., 1996). Factors that may contribute to flow fluctuations include arteriolar vasomotion and rapid vascular modeling (Baudelet et al., 2004, 2006; Dewhirst et al., 1996; Patan et al., 1996). More recent studies indicate a widespread presence of fluctuating

The effect of tumor hypoxia on the response to treatment by ionizing radiation has been demonstrated in a multitude of experimental studies. In a series of clinical studies in the early nineties, Vaupel and others showed definitively that measurements of pO2 by polarographic microelectrodes provided useful criteria for predicting the response of tumors to radiation therapy (Gatenby et al., 1988; Hockel et al., 1993; Okunieff et al., 1993; Stone et al., 1993; Thomas et al., 1994). These results stimulated considerable efforts in defining and evaluating therapeutic approaches designed to overcome tumor hypoxia as source of resistance (Horsman & van der Kogel, 2009). A particular area under focus is thus to combine radiotherapy with treatments that increase tumor pO2. Other approaches consist to chemically radiosensitize hypoxic cells or alternatively to exploit hypoxia as a mean to selectively kill the resistant population of hypoxic cells. Before the advent of imaging methods able to provide non-invasively oxygen estimation, animal and clinical studies were generally designed to evaluate the effect of a given treatment on tumor pO2 as measured by Eppendorf or histological markers of tumor hypoxia. The clinical end points were generally locoregional control and survival. Modifiers of oxygen delivery tested in clinical trials included hyperbaric oxygen therapy (HBO), oxygen and carbogen breathing. Hypoxic cell radiosensitizers (possessing a selective toxicity for the radioresistant hypoxic cells) tested in clinical trials included metronidazole, misonidazole, nimorazole, and tirapazamine. In a systematic review, J. Overgaard (2007) identified 10,108 patients in 86 randomized trials designed to modify tumor hypoxia in patients treated with curative attempted primary radiation therapy alone. Overall modification of tumor hypoxia significantly improved the effect of radiotherapy for the outcome of locoregional control and with an associated significant overall survival benefit. No significant influence was found on the incidence of distant metastases or on the risk of radiation-related complications. From this meta-analysis, the authors concluded in 2007 that "Ample data exist to support a high level of evidence for the benefit of hypoxic modification. However, hypoxic modification still has no impact on

Currently, the most advanced therapeutic interventions used in the clinic to target tumor hypoxia are either the DAHANCA (Danish head and neck cancer) trial, the application of the ARCON (Accelerated radiotherapy, carbogen and nicotinamide) protocol, and phase III studies with Tirapazamine. In the DAHANCA phase III study, nimorazole has been used as hypoxic radiosensitizer on 422 patients, and it was shown that this compound improves the

hypoxia in solid tumors (Cardena-Navia et al., 2008).

general clinical practice".

effect of radiotherapeutic management in head and neck cancer (Overgaard et al., 1998). Since these results, nimorazole administration became part of the standard irradiation protocol for Head and Neck cancer in Denmark. However, these findings have had no impact on general clinical practice except for Denmark because earlier generations of these agents induced rather severe peripheral neuropathy and because nearly all of the individual phase III trials yielded negative or inconclusive results (Ang, 2010). The ARCON protocol consists in the combination of accelerated radiotherapy to overcome tumor cell proliferation, carbogen breathing to overcome diffusion-limited hypoxia, and nicotinamide to minimize capillary bed shutdown and thereby reduce perfusion-related acute hypoxia. Phase I and II clinical trials have shown the feasibility and tolerability of the treatment and have produced promising results in term of tumor control, in particular in cancer of the head and neck and bladder (Kaanders et al., 2002). A large improvement in survival was demonstrated using this approach during radiotherapy for bladder cancer. The randomized multicentre phase III trial, designed and coordinated at Mount Vernon Cancer Centre, demonstrated a 13% benefit in overall survival when radiotherapy was combined with carbogen and nicotinamide compared to radiotherapy alone (ASTRO, 2009). Results from a large phase III trial launched to test this regimen will become available within the next 2 years. Finally, Tirapazamine (TPZ) has attracted interest after preclinical studies showing that in addition to augmenting the cytotoxicity of both radiation and cisplatin, this compound selectively kills hypoxic cells (Rischin et al., 2001; 2005). Phase I and II studies of the combination of TPZ with radiation and cisplatin were performed on patients with locally advanced head and neck carcinoma. On the basis of the phase II data, a large international phase III trial was launched (Peters et al., 2010). Surprisingly, the combination did not show any evidence of improvement in overall survival. Nevertheless, patients were not previously screened for tumor hypoxia, the study was multicentric with some centers enrolling fewer than five patients and a consequent decrease in the quality of radiotherapy planning and delivery which can have dramatic consequences on outcome (Ang, 2010).

This chapter will explore 2 strategies to radiosensitize tumors to X-rays: to increase oxygen delivery by exploiting the reactivity of mature tumor vessels (the so-called 'provascular' approach) and to decrease oxygen consumption by tumor cells. The goal of the provascular approach is to temporarily increase tumor perfusion and oxygenation through pharmacological interventions. Accordingly, radiotherapy could benefit from tumor reoxygenation whereas a decrease in interstitial pressure could facilitate tumor accessibility to circulating drugs. Alternatively, a second approach is to decrease the oxygen consumption by tumor cells since theoretical modeling studies demonstrated that reducing O2 consumption was far more efficient at reducing tumor hypoxia than increasing blood pO2 or flow (Secomb et al., 1995). We will also describe attempts to combine both approaches that can be considered as complementary strategies. The evaluation and validation of these adjuvant therapies require imaging techniques capable to accurately monitor tumor perfusion and oxygenation.

From the clinical analyses cited above, it also appeared that the variation in the results among the trials reflects a considerable heterogeneity among tumors and that patient individualization would be mandatory for the success of such therapeutic approach. There is therefore an essential need to predict individually the presence of hypoxic regions in tumors. Based on individual tumor characteristics and/or the ability to alleviate tumor hypoxia, it will become possible to adapt the individual treatment either by delivering optimal radiation doses into the resistant areas or by delivering an associated treatment for

Targeting Tumor Perfusion and Oxygenation Modulates

clinical use of radiotherapy in its fractionated mode.

**2.1.2** *S***-nitrosylated hemoglobin and nitrites** 

tumor reoxygenation (see below).

Hypoxia and Cancer Sensitivity to Radiotherapy and Systemic Therapies 293

reoxygenation window identified for each tumor model (Jordan et al., 2010a). The reoxygenation effect was shown to be due to an increase in tumor blood flow for Isosorbide Dinitrate, Xanthinol Nicotinate and S-nitrosocaptopril, using either dynamic contrastenhanced magnetic resonance imaging (DCE-MRI), where the number of perfused voxels and/or Ktrans, Kep, or Vp parameters was increased (see 5.2.1) , or patent blue staining (Jordan & Gallez, 2010b). Importantly, for some co-treatments, the increase in blood flow occurred concomitantly with a decrease in the rate of oxygen consumption by tumor cells. Inhibition of tumor cell respiration is the main mechanism accounting for insulin-induced

Endogenous NO is produced by a series of enzymes collectively termed NO-synthases (NOS). The endothelial isoform, eNOS, is adapted for the local stimulation of vasodilation following a response to stimuli that release calcium from intracellular stores and promote a calcium-calmodulin-dependent release of eNOS from its inhibitory complex with caveolin-1 (Cav-1) (Arnold et al., 1977; Michel et al., 1997). This mode of activation allows the transient production of micromolar amounts of NO responsible for vasodilatation. Using myography, we showed that this system is insensitive to classical eNOS stimulators (such as acetylcholine) selectively in tumor arterioles, thus suggesting that strategies able to restore eNOS activity would selectively target tumor vessels (Sonveaux et al., 2002). Among different treatments, we have found that ionizing radiations themselves were able to restore the normal vasodilatory properties of tumor vessels. X-rays, through the production of reactive oxygen species (ROS), indeed induce an increase in eNOS expression concomitantly with a decrease in Cav-1 expression, which removes a functional brake promoting eNOS activation (Sonveaux et al., 2002, 2009). Irradiations further stimulate NO production through the ROS-dependent activation of the PI3 kinase pathway, a well described pathway supporting Akt-mediated eNOS phosphorylation (on Ser1177, human sequence) and activation (Sonveaux et al., 2003, 2007a). We documented that radiation-induced vasodilation takes an active part in the antitumor effects of X-rays by showing that eNOS inhibition between the first and second irradiation of a clinical regimen of fractionated radiotherapy results in the total loss of the antitumor efficacy of the second dose, whereas eNOS inhibition before a single dose does not preclude cytotoxic effects (Sonveaux et al., 2002). Active vasodilation after each of the consecutive doses of fractionated radiotherapy is associated with a window of tumor reoxygenation that offers a scientific rationale for the

A smart delivery of exogenous NO would help to resolve the Steal Effect, a process through which systemic vasodilation may in fact reduce tumor perfusion and oxygenation by redirecting blood to normal blood vessels that are generally more sensitive to vasoactive treatments and constitute a denser network (Zlotecki et al., 1995). Using hemoglobin (Hb) is an interesting approach because Hb is a physiological NO carrier (in the form of Snitrosothiol) poised to deliver NO selectively in hypoxic tissues such as tumors (Sonveaux et al., 2005b). NO delivery, indeed, is possible only after the conformational change associated with Hb deoxygenation (Jia et al., 1996; McMahon et al., 2002; Stamler et al., 1997). Using cell-free human S-nitrosylated Hb (SNO-Hb) in rats, we documented a transient increase in tumor perfusion, but only when SNO-Hb was delivered in oxygenated blood (i.e., intraarteriolar injection or intravenous injection concomitantly with carbogen breathing) (Sonveaux et al., 2005b). In deoxygenated blood, SNO-Hb would otherwise readily

potentiating the efficacy of radiation treatments. In the early nineties, invasive techniques such as polarographic electrodes have been used in clinical studies to definitely establish the value of hypoxia as a predictive marker of the response of tumors to irradiation. Although this method was successful in demonstrating the central role played by tumor hypoxia in the clinical response to radiation therapy, it has never been used in standard clinical practice because of its invasiveness and the difficulty to systematically carry out longitudinal studies in individual patients. Fortunately, it is now possible to estimate tumor oxygenation by using minimally or non invasive techniques. This will be the purpose of the last part of this chapter.
