Use of Xanthinol Nicotinate as a co-treatment for radio- and chemo-therapy in experimental tumors.

The tumor micro-environment plays a key role in the tumor resistance to cytotoxic treatments. It has been demonstrated that it is possible to modulate the tumor microenvironment to potentiate anti-cancer therapy. Here, we made the hypothesis that the vasoactive agent xanthinol nicotinate (XN) could be an important modulator of the tumor perfusion and oxygenation. Using functional non invasive techniques (in vivo EPR oximetry and dynamic contrast enhanced MRI), we were able to define a time window in which tumor oxygenation, flow and permeability were significantly increased in the TLT tumor model implanted in muscles of mice. As a consequence of the alleviation of tumor hypoxia, we found out that XN was able to radiosensitize the tumors when applying 10 Gy of X-Rays during the reoxygenation of the tumors (enhancement in radiation response of 1.4). Moreover, the administration of cyclosphosphamide (50 mg/kg) used as a chemotherapeutic agent was more efficient when applying the treatment after XN administration (enhancement in response to chemotherapy of 2.7). These results show the importance of the dynamic evolution of the tumor microenvironment on the response to treatments, and that XN is an efficient modulator of the tumor hemodynamics that may potentiate cytotoxic treatments.

Glucocorticoids modulate tumor radiation response through a decrease in tumor oxygen consumption.

PURPOSE: We hypothesized that glucocorticoids may enhance tumor radiosensitivity by increasing tumor oxygenation (pO(2)) through inhibition of mitochondrial respiration. EXPERIMENTAL DESIGN: The effect of three glucocorticoids (hydrocortisone, dexamethasone, and prednisolone) on pO(2) was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO(2) (t(max), 30 min after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO(2) measurements were done using electron paramagnetic resonance. The oxygen consumption rate of tumor cells after in vivo glucocorticoid administration was measured using high-frequency electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging. RESULTS: All glucocorticoids tested caused a rapid increase in pO(2). At t(max), tumor perfusion decreased, indicating that the increase in pO(2) was not caused by an increase in oxygen supply. Also at t(max), global oxygen consumption decreased. When irradiation (25 Gy) was applied at t(max), the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7). CONCLUSION: These results show the potential usefulness of the administration of glucocorticoids before irradiation.

Thyroid status is a key modulator of tumor oxygenation: implication for radiation therapy.

In normal tissues, thyroid hormones play a major role in the metabolic activity and oxygen consumption of cells. Because the rate of oxygen consumption is a key factor in the response of tumors to radiation, we hypothesized that thyroid hormones may affect the metabolic activity of tumor cells and hence modulate the response to cytotoxic treatments. We measured the influence of thyroid status on the tumor microenvironment in experimental tumors. Hypothyroidism and hyperthyroidism were generated in mice by chronic treatment with propyl thiouracil and l-thyroxine. Thyroid status significantly modified tumor pO(2) as measured with EPR oximetry. Mechanistically, this was the result of the profound changes in oxygen consumption rates. Thyroid status was associated with a significant change in tumor radiosensitivity since the regrowth delay was increased in hypothyroid mice compared to euthyroid mice, an effect that was abolished when temporarily clamped tumors were irradiated. This study provides unique insights into the impact of modulating tumor oxygen consumption and could have implications in the management of cancer patients with thyroid disorders.

Tumor radiosensitization by antiinflammatory drugs: evidence for a new mechanism involving the oxygen effect.

We hypothesized that nonsteroidal antiinflammatory drugs (NSAIDs) might enhance tumor radiosensitivity by increasing tumor oxygenation (pO2), via either a decrease in the recruitment of macrophages or from inhibition of mitochondrial respiration. The effect of four NSAIDs (diclofenac, indomethacin, piroxicam, and NS-398) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (t(max), 30 minutes after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo NSAID administration was measured using high-frequency electron paramagnetic resonance. Tumor-infiltrating macrophage localization was done with immunohistochemistry using CD11b antibody. All the NSAIDs tested caused a rapid increase in pO2. At t(max), tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at t(max), global oxygen consumption decreased but the amount of tumor-infiltrating macrophages remained unchanged. Our study strongly indicates that the oxygen effect caused by NSAIDs is primarily mediated by an effect on mitochondrial respiration. When irradiation (18 Gy) was applied at t(max), the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7). These results show the potential utility of an acute administration of NSAIDs for radiosensitizing tumors, and shed new light on the mechanisms of NSAID radiosensitization. These results also provide a new rationale for the treatment schedule when combining NSAIDs and radiotherapy.

Preclinical safety and antitumor efficacy of insulin combined with irradiation.

BACKGROUND AND PURPOSE: We have previously reported that insulin significantly enhances tumor oxygenation (pO(2)) and increases radiation-induced tumor regrowth delay in experimental models. Considering the large radiosensitizing effect, clinical trials might be envisioned. The aim of the present pre-clinical study was to obtain a more complete set of safety and efficacy data which would further justify the commencement of such clinical trials. MATERIAL AND METHODS: Toxicity on normal (early and late-responding) tissues was measured by the intestinal crypt regeneration assay and the late leg contracture assay. Efficacy in terms of enhancement of pO(2) (measured by in vivo EPR oximetry) and increase in radiation-induced tumor regrowth delay was evaluated with a dose-response study on mice bearing FSaII fibrosarcoma. RESULTS: The effect on regrowth delay was directly correlated with the effect on the tumor pO(2), with a maximal effect using 400 mU kg(-1) insulin. Importantly, there was no increase in the radiation toxicity for normal tissues. Finally, we found that the hypoglycaemia induced by insulin can be corrected by simultaneous glucose infusion without modification of efficacy. CONCLUSION: Insulin here demonstrated a therapeutic gain and a lack of toxicity to normal tissues. The results of this study fully justify further larger preclinical assays such as the use of fractionated irradiation and a tumor control dose assay, before determining the utility of insulin as a radiosensitizer for human patients in the clinic.

Changes in the tumor microenvironment during low-dose-rate permanent seed implantation iodine-125 brachytherapy.

PURPOSE: There is a lack of data regarding how the tumor microenvironment (e.g., perfusion and oxygen partial pressure [pO2]) changes in response to low-dose-rate (LDR) brachytherapy. This may be why some clinical issues remain unresolved, such as the appropriate use of adjuvant external beam radiation therapy (EBRT). The purpose of this work was to obtain some basic preclinical data on how the tumor microenvironment evolves in response to LDR brachytherapy. METHODS AND MATERIALS: In an experimental mouse tumor, pO2 (measured by electron paramagnetic resonance) and perfusion (measured by dynamic contrast-enhanced magnetic resonance imaging) were monitored as a function of time (0-6 days) and distance (0-2 mm and 2-4 mm) from an implanted 0.5 mCi iodine-125 brachytherapy seed. RESULTS: For most of the experiments, including controls, tumors remained hypoxic at all times. At distances of 2-4 mm from radioactive seeds ( approximately 1.5 Gy/day), however, there was an early, significant increase in pO2 within 24 h. The pO2 in that region remained elevated through Day 3. Additionally, the perfusion in that region was significantly higher than for controls starting at Day 3. CONCLUSION: It may be advantageous to give adjuvant EBRT shortly (approximately 1 to 2 days) after commencement of clinical LDR brachytherapy, when the pO2 in the spatial regions between seeds should be elevated. If chemotherapy is given adjuvantly, it may best be administered just a little later (approximately 3 or 4 days) after the start of LDR brachytherapy, when perfusion should be elevated.

Early reoxygenation in tumors after irradiation: determining factors and consequences for radiotherapy regimens using daily multiple fractions.

PURPOSE: To characterize changes in the tumor microenvironment early after irradiation and determine the factors responsible for early reoxygenation. METHODS AND MATERIALS: Fibrosarcoma type II (FSaII) and hepatocarcinoma transplantable liver tumor tumor oxygenation were determined using electron paramagnetic resonance oximetry and a fiberoptic device. Perfusion was assessed by laser Doppler, dynamic contrast-enhanced MRI, and dye penetration. Oxygen consumption was determined by electron paramagnetic resonance. The interstitial fluid pressure was evaluated by the wick-in-needle technique. RESULTS: An increase in oxygen partial pressure was observed 3-4 h after irradiation. This increase resulted from a decrease in global oxygen consumption and an increase in oxygen delivery. The increase in oxygen delivery was due to radiation-induced acute inflammation (that was partially inhibited by the antiinflammatory agent diclofenac) and to a decrease in interstitial fluid pressure. The endothelial nitric oxide synthase pathway, identified as a contributing factor at 24 h after irradiation, did not play a role in the early stage after irradiation. We also observed that splitting a treatment of 18 Gy into two fractions separated by 4 h (time of maximal reoxygenation) had a greater effect on tumor regrowth delay than when applied as a single dose. CONCLUSION: Although the cell cycle redistribution effect is important for treatment protocols using multiple daily radiation fractions, the results of this work emphasize that the oxygen effect must be also considered to optimize the treatment strategy.

Potentiation of radiotherapy by a localized antiangiogenic gene therapy.

BACKGROUND AND PURPOSE: We hypothesized that electrotransfer of a plasmid encoding an antiangiogenic factor, the recombinant disintegrin domain of ADAM-15, (pRDD) could modify the tumor microenvironment and radiosensitize tumor. MATERIALS AND METHODS: pRDD was injected in the TLT tumor or FSaII fibrosarcomas before electroporation. pO2 in tumors and oxygen consumption in vitro were measured by electronic paramagnetic resonance (EPR) oximetry. Tumor perfusion was assessed by laser doppler imaging and patent blue assay. RESULTS: pRDD electrotransfer caused a significant delay in TLT growth and an anti-angiogenic effect. It significantly increased tumor pO2 in TLT and FSaII for at least 4 days. pRDD electrotransfer and radiotherapy were more effective than either treatment alone. Modifications of tumor microenvironment were evaluated: tumor perfusion and interstitial fluid pressure were not modified. Oxygen consumption by the cells was decreased resulting both from a decrease in oxygen consumption rate and from a decrease in cell viability. CONCLUSION: The combination of localized antiangiogenic gene therapy and radiotherapy applied in the time of maximal oxygenation could be a promising alternative for cancer treatment.

Impact of cyclic hypoxia on HIF-1alpha regulation in endothelial cells--new insights for anti-tumor treatments.

Heterogeneities in tumor blood flow are associated with cyclic changes in pO2 or cyclic hypoxia. A major difference from O2 diffusion-limited or chronic hypoxia is that the tumor vasculature itself may be directly influenced by the fluctuating hypoxic environment, and the reoxygenation phases complicate the usual hypoxia-induced phenotypic pattern. Here, we determined the cyclic hypoxia-driven pathways that modulate hypoxia inducible factor (HIF)-1alpha abundance in endothelial cells to identify possible therapeutic targets. We found that exposure of endothelial cells to cycles of hypoxia/reoxygenation led to accumulation of HIF-1alpha during the hypoxic periods and the phosphorylation of protein kinase B (Akt), extracellular regulated kinase (ERK) and endothelial nitric oxide synthase (eNOS) during the reoxygenation phases. We identified stimulation of mitochondrial respiration and activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway during intervening reoxygenation periods as major triggers of the stabilization of HIF-1alpha. We also found that the NOS inhibitor nitro-l-arginine methyl ester further stimulated the cyclic hypoxia-driven HIF-1alpha accumulation and the associated gain in endothelial cell survival, thereby mirroring the effects of a PI3K/Akt inhibitor. However, combination of both drugs resulted in a net reduction in HIF-1alpha and a dramatic in decrease in endothelial cell survival. In conclusion, this study identified cyclic hypoxia, as reported in many tumor types, as a unique biological challenge for endothelial cells that promotes their survival in a HIF-1alpha-dependent manner through phenotypic alterations occurring during the reoxygenation periods. These observations also indicate the potential of combining Akt-targeting drugs with anti-angiogenic drugs, in particular those interfering with the NO pathway.

Targeting of tumor endothelium by RGD-grafted PLGA-nanoparticles loaded with paclitaxel.

Paclitaxel (PTX)-loaded PEGylated PLGA-based nanoparticles (NP) have been previously described as more effective in vitro and in vivo than taxol. The aim of this study was to test the hypothesis that our PEGylated PLGA-based nanoparticles grafted with the RGD peptide or RGD-peptidomimetic (RGDp) would target the tumor endothelium and would further enhance the anti-tumor efficacy of PTX. The ligands were grafted on the PEG chain of PCL-b-PEG included in the nanoparticles. We observed in vitro that RGD-grafted nanoparticles were more associated to human umbilical vein endothelial cells (HUVEC) by binding to alpha(v)beta(3) integrin than non-targeted nanoparticles. Doxorubicin was also used to confirm the findings observed for PTX. In vivo, we demonstrated the targeting of RGD and RGDp-grafted nanoparticles to tumor vessels as well as the effective retardation of TLT tumor growth and prolonged survival times of mice treated by PTX-loaded RGD-nanoparticles when compared to non-targeted nanoparticles. Hence, the targeting of anti-cancer drug to tumor endothelium by RGD-labeled NP is a promising approach.

The role of vessel maturation and vessel functionality in spontaneous fluctuations of T2*-weighted GRE signal within tumors.

Acute hypoxia (transient cycles of hypoxia-reoxygenation) is known to occur in solid tumors and is generally believed to be caused by tumor blood flow instabilities. It was recently demonstrated that T2*-weighted (T2*w) gradient echo (GRE) MRI is a powerful non-invasive method for investigating periodic changes in tumor pO2 and blood flow associated with acute hypoxia. Here, the possible correlation between tumor vessel immaturity, vessel functionality and T2*w GRE signal fluctuations was investigated. Intramuscularly implanted FSa II fibrosarcoma-bearing mice were imaged at 4.7 T. Maps of spontaneous fluctuations of MR signal intensity in tumor tissue during air breathing were obtained using a T2*w GRE sequence. This same sequence was also employed during air-5% CO2 breathing (hypercapnia) and carbogen breathing (hypercapnic hyperoxia) to obtain parametric maps representing vessel maturation and vessel function, respectively. Vascular density, vessel maturation and vessel perfusion were also assessed histologically by using CD31 labeling, alpha-smooth muscle actin immunoreactivity and Hoechst 33242 labeling, respectively. About 50% of the tumor fluctuations occurred in functional tumor regions (responsive to carbogen) and 80% occurred in tumor regions with immature vessels (lack of response to hypercapnia). The proportion of hypercapnia-responsive voxels were found to be twice as great in fluctuating than in non-fluctuating tumor areas (P: 0.22 vs 0.13). Similarly, the proportion of functional voxels was somewhat greater in fluctuating tumor areas (P: 0.54 vs 0.43). The mean values of MR signal changes during hypercapnia (VD) and during carbogen breathing (VF) (significant voxels only) were also larger in fluctuating than in non-fluctuating tumor areas (P < 0.05). This study demonstrated that adequate vessel functionality and advanced vessel maturation could explain at least in part the occurrence of spontaneous T2*w GRE signal fluctuations. Functionality and maturation are not required for signal fluctuations, however, because a large fraction of fluctuations could still occur in non-perfused and/or immature vessels.

Complex relationship between changes in oxygenation status and changes in R*2: the case of insulin and NS-398, two inhibitors of oxygen consumption.

Insulin and NS-398 have been reported to inhibit oxygen consumption in experimental tumor models, thereby increasing oxygenation and radiosensitization. The aim of this work was to use MRI to study changes in murine FSaII tumor hemodynamics after administration of those oxygen consumption inhibitors. A multiple-echo gradient-echo (GRE) MRI sequence (4.7 T) was used to map changes in three factors: the GRE signal (at TE=20 ms), the parameter S0 (theoretical signal at TE=0 ms), and the relaxation rate R*2. Perfusion maps were obtained by dynamic contrast-enhanced (DCE) MRI. Insulin caused a significant decrease in the tumor blood oxygen level-dependent (BOLD) signal over time. factor This was likely the result of decreased blood flow, since both S0 and the percentage of perfused tumor decreased as well. Tumor R*2 did not change significantly in response to the treatments, which is surprising considering that other non-MRI techniques (electron paramagnetic resonance (EPR) oximetry and fiber-optic probes) have shown that tumor oxygenation increases after treatment. This suggests that metabolic changes associated with vasoactive challenges may have an unpredictable influence on blood saturation and R*2. In conclusion, this study further emphasizes the fact that changes in BOLD signal and R*2 in tumors do not depend uniquely on changes in oxygenation status.

Mechanism of reoxygenation after antiangiogenic therapy using SU5416 and its importance for guiding combined antitumor therapy.

Emerging preclinical studies support the concept of a transient "normalization" of tumor vasculature during the early stage of antiangiogenic treatment, with possible beneficial effects on associated radiotherapy or chemotherapy. One key issue in this area of research is to determine whether this feature is common to all antiangiogenic drugs and whether the phenomenon occurs in all types of tumors. In the present study, we characterized the evolution of the tumor oxygenation (in transplantable liver tumor and FSAII tumor models) after administration of SU5416, an antagonist of the vascular endothelial growth factor receptor. SU5416 induced an early increase in tumor oxygenation [measured by electronic paramagnetic resonance (EPR)], which did not correlate with remodeling of the tumor vasculature (assessed by CD31 labeling using immunohistochemistry) or with tumor perfusion (measured by dynamic contrast enhanced-magnetic resonance imaging). Inhibition of mitochondrial respiration (measured by EPR) was responsible for this early reoxygenation. Consistent with these unique findings in the tumor microenvironment, we found that SU5416 potentiated tumor response to radiotherapy but not to chemotherapy. In addition to the fact that the characterization of the tumor oxygenation is essential to enable correct application of combined therapies, our results show that the long-term inhibition of oxygen consumption is a potential novel target in this class of compounds.