**2. Improving oxygen delivery to the tumor: The provascular approach**

Tumors are highly heterogeneous and this heterogeneity extends to the tumor vasculature (see introduction). Beside neovessels that are the target of anti-angiogenic agents, human and rodent tumors also contain blood vessels that are structurally mature (Mattson et al., 1978; Peterson & Mattson, 1984). These vessels possess the minimal contractile features (such as pericytes or vascular smooth muscle cells) endowing them with vasocontractile properties. The intrinsic reactivity of tumor-feeding vessels modulates oxygen delivery and the accessibility of circulating drugs to the tumor. A selective and transient dilation of these vessels should thus improve the tumor response to radiotherapy (which depends on tumor oxygenation) and chemotherapy (which depends on perfusion and on the vascular exchange area). We termed this approach 'provascular' to contrast with antivascular and antiangiogenic approaches that are destructive by nature (Sonveaux, 2008). The net effect of systemic vasodilation on tumor pO2 is unpredictable because it primarily depends on the arrangement of vessels (in series or in parallel) between the tumor and surrounding host tissues (Zlotecki et al., 1995). The key issue to resolve is thus to identify tumor-selective vasodilators. Treatment optimization would also require to monitor on individual bases the tumor response to treatment, preferentially using early surrogate, predictive and noninvasive markers.

#### **2.1 Nitric Oxide (NO) and endothelin-1 (ET1) related strategies**

Physiologically, the vascular tone is determined by the balance between nitric oxide (NO, a potent vasodilator) and endothelin-1 (ET1, a potent vasoconstrictor) (Sonveaux & Feron, 2005a). A number of studies have explored the functionality and the provascular exploitability of these 2 systems, as described below.

#### **2.1.1 Exogenous and endogenous NO**

NO was initially investigated for its vasodilatory activity and NO-donors were anticipated to improve the therapeutic efficacy of chemo- and radiotherapy upon combinational delivery (Sonveaux et al., 2009). We first considered the effect of the application of exogenous NO on tumor hemodynamic parameters and radiation response. This was performed by systemic administration of NO donor compounds, including isosorbide dinitrate (Jordan et al., 2000), Xanthinol Nicotinate (Segers et al., 2010), and S-nitrosocaptopril (Jordan et al., 2010a). The stimulation of the production of endogenous NO was also achieved by administration of insulin (Jordan et al., 2002). All treatments resulted in a transient acute improvement of experimental tumor oxygenation with a consecutive increase in tumor radiosensitivity upon sequential administration of X-rays during the

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

**2. Improving oxygen delivery to the tumor: The provascular approach** 

**2.1 Nitric Oxide (NO) and endothelin-1 (ET1) related strategies** 

exploitability of these 2 systems, as described below.

**2.1.1 Exogenous and endogenous NO** 

Tumors are highly heterogeneous and this heterogeneity extends to the tumor vasculature (see introduction). Beside neovessels that are the target of anti-angiogenic agents, human and rodent tumors also contain blood vessels that are structurally mature (Mattson et al., 1978; Peterson & Mattson, 1984). These vessels possess the minimal contractile features (such as pericytes or vascular smooth muscle cells) endowing them with vasocontractile properties. The intrinsic reactivity of tumor-feeding vessels modulates oxygen delivery and the accessibility of circulating drugs to the tumor. A selective and transient dilation of these vessels should thus improve the tumor response to radiotherapy (which depends on tumor oxygenation) and chemotherapy (which depends on perfusion and on the vascular exchange area). We termed this approach 'provascular' to contrast with antivascular and antiangiogenic approaches that are destructive by nature (Sonveaux, 2008). The net effect of systemic vasodilation on tumor pO2 is unpredictable because it primarily depends on the arrangement of vessels (in series or in parallel) between the tumor and surrounding host tissues (Zlotecki et al., 1995). The key issue to resolve is thus to identify tumor-selective vasodilators. Treatment optimization would also require to monitor on individual bases the tumor response to treatment, preferentially using early surrogate, predictive and non-

Physiologically, the vascular tone is determined by the balance between nitric oxide (NO, a potent vasodilator) and endothelin-1 (ET1, a potent vasoconstrictor) (Sonveaux & Feron, 2005a). A number of studies have explored the functionality and the provascular

NO was initially investigated for its vasodilatory activity and NO-donors were anticipated to improve the therapeutic efficacy of chemo- and radiotherapy upon combinational delivery (Sonveaux et al., 2009). We first considered the effect of the application of exogenous NO on tumor hemodynamic parameters and radiation response. This was performed by systemic administration of NO donor compounds, including isosorbide dinitrate (Jordan et al., 2000), Xanthinol Nicotinate (Segers et al., 2010), and S-nitrosocaptopril (Jordan et al., 2010a). The stimulation of the production of endogenous NO was also achieved by administration of insulin (Jordan et al., 2002). All treatments resulted in a transient acute improvement of experimental tumor oxygenation with a consecutive increase in tumor radiosensitivity upon sequential administration of X-rays during the

chapter.

invasive markers.

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 tumor reoxygenation (see below).

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 clinical use of radiotherapy in its fractionated mode.

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

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

Targeting Tumor Perfusion and Oxygenation Modulates

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

increase in tumor pO2 and enhancement of the efficacy of radiotherapy.

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

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

al., 2008).

Hypoxia and Cancer Sensitivity to Radiotherapy and Systemic Therapies 295

optimal time points for the delivery of radiation. Our group previously studied the modifications in the tumor environment early after treatment with the anti-angiogenic agent thalidomide, with a special focus on a possible normalization of the tumor vasculature (Jain, 2001; Tong et al., 2004) that could be beneficial for radiotherapy. Our results showed an increase in tumor pO2 during the first 2 days of thalidomide treatment, which was likely the result of the ability of thalidomide to modify tumor microenvironmental parameters such as the vascular supply and tumor perfusion, as shown by DCE-MRI (see 5.2.1) and histological analysis using the endothelial marker CD31 (Ansiaux et al., 2005). Indeed, the histological analysis revealed profound modifications in the vascular supply: a reduction in the number of tumor microvessels after thalidomide treatment together with a dilation of the remaining vessels with no decrease in the tumor vascular density. The perfusion measured by DCE-MRI showed an increased plasma volume fraction (see 5.2.1), which could be explained by the shift to larger blood vessel diameters as observed in histology analysis, perhaps due to compensation for the loss of small vessels. Interestingly, similar observations were not obtained using more specific anti-angiogenic agents such as SU-5416 or ZD-6474 (Ansiaux et al., 2006; 2009). For these compounds, tumor reoxygenation was rather due to a decrease in the rate of oxygen consumption by tumor cell and no normalization effect was observed in the tumor models under study (see below). We hypothesized that specific inhibition of vascular endothelial growth factor (VEGF) signaling via VEGFR2 by SU-5416 or ZD-6474 may have been compensated by another angiogenic pathway such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, or Tie-2 signaling (Folkman et al., 2001; Stratmann et al., 1998); contrary to thalidomide which acts on different angiogenic pathways. It is nevertheless important to note that these findings are specific to the tumor models under study, since ZD-6474 was described earlier to be able to decrease both flow and permeability in human colon tumors (Bradley et al., 2008) and was able to induce transient normalization of the vasculature in gliomas (Claes et

deoxygenate and release NO at the site of delivery. While increased tumor perfusion at low dose SNO-Hb is primarily attributable to central effects (i.e., baroreceptor inhibition), SNO-Hb at higher doses could act as a tumor-selective vasodilator and could therefore be used as a radiosensitizing treatment. Inhalation of the NO-donor gas ethyl nitrite, which promotes the S-nitrosylation of intra-erythrocytic Hb in the lungs, could have the same effects while minimizing toxic side effects associated with the administration of naked Hb (Moya et al., 2002; Sonveaux et al., 2007b).

While the use of SNO-Hb exploits hypoxia as a mean to selectively deliver NO to tumors, one can also take advantage of the low pH coupled to the high metabolic activities of many solid tumors. Nitrites for example can be reduced to NO either by enzymatic catalysis (nitrite reductase activities of xanthine oxidase, eNOS and Hb) or by non enzymatic disproportionation, and these processes are facilitated in an acidic microenvironment (Angelo et al., 2006; Godber et al., 2000; Modin et al., 2001; Vanin et al., 2007; Zweier et al., 1999). They have been used clinically as an antidote for cyanide poisoning (Holland & Kozlowski, 1986), which also indicates that they can be safely administrated to humans. We therefore tested whether the low pH of tumors (on average pH 6.7) could be exploited to generate NO from nitrites selectively in tumors. We observed *ex vivo* that nitrite-induced vasodilation was more pronounced at pH 6.7 compared to pH 7.4 (Frerart et al., 2008). We also found that the bioactivity of nitrites at low pH encompassed NO-mediated inhibition of tumor cell respiration, which indicates that the robust and transient increase in tumor pO2 after nitrite delivery to mice is the result of the combination of vasoactive and metabolic responses. When administered to reach a plasma concentration of 100 µM in mice, nitrites sensitized tumors to radiotherapy (Frerart et al., 2008). Further clinical applications are however confronted to financial issues: clinical trials are now warranted whereas nitrites or their use in cancer therapy can not be patented.

#### **2.1.3 Endothelin-1 inhibitors**

Endothelin-1 (ET-1) is a strong vasoconstrictor and an autocrine growth factor produced by tumor cells (Haynes & Webb, 1994; Shichiri et al., 1991). It has a key role in the accommodation of vasoactive blood vessels to variations in intraluminal pressure: ET-1 mediates the myogenic tone, a vasoconstriction that buffers perfusion changes when the blood pressure increases (Huang & Koller, 1997). In tumors, the constant exposure of arterioles to ET-1 *in vivo* results in an increased myogenic tone that can be detected *ex vivo* (Sonveaux et al., 2004). We reasoned that it constituted a reserve for vasorelaxation that could be exploited to sensitize tumors to radio- and chemotherapy. ET-1 induces vasoconstriction when binding to ETA receptors expressed by contractile vascular cells (Maguire & Davenport, 1995). Using the ETA antagonist BQ123, we observed *ex vivo* a vasodilation selectively in tumor vessels (compared to size-matched vessels from nonmalignant tissues) that translated *in vivo* into increased tumor perfusion and oxygenation (Sonveaux et al., 2004). Both responses were tumor-selective and transient. BQ123 as a pretreatment therefore improved the antitumor effects of X-ray radiotherapy and cyclophosphamide (after systemic delivery) (Martinive et al., 2006; Sonveaux et al., 2004).

#### **2.2 Normalization effect of anti-angiogenic agents**

Given that anti-angiogenic agents will likely be combined with radiation therapy, it is critical to understand alterations in tumor oxygenation and perfusion, as well as to define

deoxygenate and release NO at the site of delivery. While increased tumor perfusion at low dose SNO-Hb is primarily attributable to central effects (i.e., baroreceptor inhibition), SNO-Hb at higher doses could act as a tumor-selective vasodilator and could therefore be used as a radiosensitizing treatment. Inhalation of the NO-donor gas ethyl nitrite, which promotes the S-nitrosylation of intra-erythrocytic Hb in the lungs, could have the same effects while minimizing toxic side effects associated with the administration of naked Hb (Moya et al.,

While the use of SNO-Hb exploits hypoxia as a mean to selectively deliver NO to tumors, one can also take advantage of the low pH coupled to the high metabolic activities of many solid tumors. Nitrites for example can be reduced to NO either by enzymatic catalysis (nitrite reductase activities of xanthine oxidase, eNOS and Hb) or by non enzymatic disproportionation, and these processes are facilitated in an acidic microenvironment (Angelo et al., 2006; Godber et al., 2000; Modin et al., 2001; Vanin et al., 2007; Zweier et al., 1999). They have been used clinically as an antidote for cyanide poisoning (Holland & Kozlowski, 1986), which also indicates that they can be safely administrated to humans. We therefore tested whether the low pH of tumors (on average pH 6.7) could be exploited to generate NO from nitrites selectively in tumors. We observed *ex vivo* that nitrite-induced vasodilation was more pronounced at pH 6.7 compared to pH 7.4 (Frerart et al., 2008). We also found that the bioactivity of nitrites at low pH encompassed NO-mediated inhibition of tumor cell respiration, which indicates that the robust and transient increase in tumor pO2 after nitrite delivery to mice is the result of the combination of vasoactive and metabolic responses. When administered to reach a plasma concentration of 100 µM in mice, nitrites sensitized tumors to radiotherapy (Frerart et al., 2008). Further clinical applications are however confronted to financial issues: clinical trials are now warranted whereas nitrites or

Endothelin-1 (ET-1) is a strong vasoconstrictor and an autocrine growth factor produced by tumor cells (Haynes & Webb, 1994; Shichiri et al., 1991). It has a key role in the accommodation of vasoactive blood vessels to variations in intraluminal pressure: ET-1 mediates the myogenic tone, a vasoconstriction that buffers perfusion changes when the blood pressure increases (Huang & Koller, 1997). In tumors, the constant exposure of arterioles to ET-1 *in vivo* results in an increased myogenic tone that can be detected *ex vivo* (Sonveaux et al., 2004). We reasoned that it constituted a reserve for vasorelaxation that could be exploited to sensitize tumors to radio- and chemotherapy. ET-1 induces vasoconstriction when binding to ETA receptors expressed by contractile vascular cells (Maguire & Davenport, 1995). Using the ETA antagonist BQ123, we observed *ex vivo* a vasodilation selectively in tumor vessels (compared to size-matched vessels from nonmalignant tissues) that translated *in vivo* into increased tumor perfusion and oxygenation (Sonveaux et al., 2004). Both responses were tumor-selective and transient. BQ123 as a pretreatment therefore improved the antitumor effects of X-ray radiotherapy and cyclophosphamide (after systemic delivery) (Martinive et al., 2006; Sonveaux et al., 2004).

Given that anti-angiogenic agents will likely be combined with radiation therapy, it is critical to understand alterations in tumor oxygenation and perfusion, as well as to define

2002; Sonveaux et al., 2007b).

their use in cancer therapy can not be patented.

**2.2 Normalization effect of anti-angiogenic agents** 

**2.1.3 Endothelin-1 inhibitors** 

optimal time points for the delivery of radiation. Our group previously studied the modifications in the tumor environment early after treatment with the anti-angiogenic agent thalidomide, with a special focus on a possible normalization of the tumor vasculature (Jain, 2001; Tong et al., 2004) that could be beneficial for radiotherapy. Our results showed an increase in tumor pO2 during the first 2 days of thalidomide treatment, which was likely the result of the ability of thalidomide to modify tumor microenvironmental parameters such as the vascular supply and tumor perfusion, as shown by DCE-MRI (see 5.2.1) and histological analysis using the endothelial marker CD31 (Ansiaux et al., 2005). Indeed, the histological analysis revealed profound modifications in the vascular supply: a reduction in the number of tumor microvessels after thalidomide treatment together with a dilation of the remaining vessels with no decrease in the tumor vascular density. The perfusion measured by DCE-MRI showed an increased plasma volume fraction (see 5.2.1), which could be explained by the shift to larger blood vessel diameters as observed in histology analysis, perhaps due to compensation for the loss of small vessels. Interestingly, similar observations were not obtained using more specific anti-angiogenic agents such as SU-5416 or ZD-6474 (Ansiaux et al., 2006; 2009). For these compounds, tumor reoxygenation was rather due to a decrease in the rate of oxygen consumption by tumor cell and no normalization effect was observed in the tumor models under study (see below). We hypothesized that specific inhibition of vascular endothelial growth factor (VEGF) signaling via VEGFR2 by SU-5416 or ZD-6474 may have been compensated by another angiogenic pathway such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, or Tie-2 signaling (Folkman et al., 2001; Stratmann et al., 1998); contrary to thalidomide which acts on different angiogenic pathways. It is nevertheless important to note that these findings are specific to the tumor models under study, since ZD-6474 was described earlier to be able to decrease both flow and permeability in human colon tumors (Bradley et al., 2008) and was able to induce transient normalization of the vasculature in gliomas (Claes et al., 2008).
