Assessment of the changes in 9L and C6 glioma pO2 by EPR oximetry as a prognostic indicator of differential response to radiotherapy.

Tumor hypoxia impedes the outcome of radiotherapy. As the extent of hypoxia in solid tumors varies during the course of radiotherapy, methods that can provide repeated assessment of tumor pO2 such as EPR oximetry may enhance the efficacy of radiotherapy by scheduling irradiations when the tumors are oxygenated. The repeated measurements of tumor pO2 may also identify responders, and thereby facilitate the design of better treatment plans for nonresponding tumors. We have investigated the temporal changes in the ectopic 9L and C6 glioma pO2 irradiated with single radiation doses less than 10 Gy by EPR oximetry. The 9L and C6 tumors were hypoxic with pO2 of approximately 5-9 mmHg. The pO2 of C6 tumors increased significantly with irradiation of 4.8-9.3 Gy. However, no change in the 9L tumor pO2 was observed. The irradiation of the oxygenated C6 tumors with a second dose of 4.8 Gy resulted in a significant delay in growth compared to hypoxic and 2 Gy × 5 treatment groups. The C6 tumors with an increase in pO2 of greater than 50% from the baseline of irradiation with 4.8 Gy (responders) had a significant tumor growth delay compared to nonresponders. These results indicate that the ectopic 9L and C6 tumors responded differently to radiotherapy. We propose that the repeated measurement of the oxygen levels in the tumors during radiotherapy can be used to identify responders and to design tumor oxygen guided treatment plans to improve the outcome.

Dynamic changes in oxygenation of intracranial tumor and contralateral brain during tumor growth and carbogen breathing: a multisite EPR oximetry with implantable resonators.

INTRODUCTION: Several techniques currently exist for measuring tissue oxygen; however technical difficulties have limited their usefulness and general application. We report a recently developed electron paramagnetic resonance (EPR) oximetry approach with multiple probe implantable resonators (IRs) that allow repeated measurements of oxygen in tissue at depths of greater than 10mm. METHODS: The EPR signal to noise (S/N) ratio of two probe IRs was compared with that of LiPc deposits. The feasibility of intracranial tissue pO(2) measurements by EPR oximetry using IRs was tested in normal rats and rats bearing intracerebral F98 tumors. The dynamic changes in the tissue pO(2) were assessed during repeated hyperoxia with carbogen breathing. RESULTS: A 6-10 times increase in the S/N ratio was observed with IRs as compared to LiPc deposits. The mean brain pO(2) of normal rats was stable and increased significantly during carbogen inhalation in experiments repeated for 3months. The pO(2) of F98 glioma declined gradually, while the pO(2) of contralateral brain essentially remained the same. Although a significant increase in the glioma pO(2) was observed during carbogen inhalation, this effect declined in experiments repeated over days. CONCLUSION: EPR oximetry with IRs provides a significant increase in S/N ratio. The ability to repeatedly assess orthotopic glioma pO(2) is likely to play a vital role in understanding the dynamics of tissue pO(2) during tumor growth and therapies designed to modulate tumor hypoxia. This information could then be used to optimize chemoradiation by scheduling treatments at times of increased glioma oxygenation.

Synergistic combination of hyperoxygenation and radiotherapy by repeated assessments of tumor pO2 with EPR oximetry.

The effect of hyperoxygenation with carbogen (95% O(2) + 5% CO(2)) inhalation on RIF-1 tumor pO(2 )and its consequence on growth inhibition with fractionated radiotherapy is reported. The temporal changes in the tumor pO(2) were assessed by in vivo Electron Paramagnetic Resonance (EPR) oximetry in mice breathing 30% O(2) or carbogen and the tumors were irradiated with 4 Gy/day for 5 consecutive days; a protocol that emulates the clinical application of carbogen. The RIF-1 tumors were hypoxic with a tissue pO(2) of 5-9 mmHg. Carbogen (CB) breathing significantly increased tumor pO(2), with a maximum increase at 22.9-31.2 min on days 1-5, however, the magnitude of increase in pO(2) declined on day 5. Radiotherapy during carbogen inhalation (CB/RT) resulted in a significant tumor growth inhibition from day 3 to day 6 as compared to 30%O(2)/RT and carbogen (CB/Sham RT) groups. The results provide unambiguous quantitative information on the effect of carbogen inhalation on tumor pO(2) over the course of 5 days. Tumor growth inhibition in the CB/RT group confirms that the tumor oxygenation with carbogen was radiobiologically significant. Repeated tumor pO(2) measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcomes by scheduling doses at times of improved tumor oxygenation.

Repeated tumor oximetry to identify therapeutic window during metronomic cyclophosphamide treatment of 9L gliomas.

Malignant gliomas are aggressive and angiogenic tumors with high VEGF content. Consequently, approaches such as metronomic chemotherapy, which have an anti-angiogenic effect, are being investigated. However, a lack of an appropriate technique that can facilitate the identification of vascular changes during antiangiogenic treatments has restricted therapeutic optimization. We have investigated the potential of tumor pO2 as a marker to detect vascular changes during metronomic chemotherapy. Electron paramagnetic resonance (EPR) oximetry was used to repeatedly assess tumor pO2 during metronomic cyclophosphamide treatment of subcutaneous 9L tumors. The 9L tumors were hypoxic with a pO2 of 5.6-8 mmHg and a tumor volume of 247-300 mm3 prior to any treatment. Tumor pO2 increased significantly to 19.7 mmHg on day 10 and remained at an elevated level until day 33 during 4 weekly treatments with 140 mg/kg cyclophosphamide. A significant decrease in the tumor volume on days 21-31 occurred in the cyclophosphamide group, while the tumor volume of the control group significantly increased during measurements for two weeks. A significant tumor growth delay was achieved with two weekly treatments of cyclophosphamide plus radiotherapy (4 Gy x 5) as compared to control, cyclophosphamide and radiotherapy alone groups. The results indicate the potential of EPR oximetry to assess tumor pO2 during metronomic chemotherapy. The ability to identify the duration of an increase in tumor pO2, therapeutic window, non-invasively by EPR oximetry could have a significant impact on the optimization of antiangiogenic approaches for the treatment of gliomas. This vital information could also be used to schedule radiotherapy to enhance therapeutic outcome.

Modulation of tumor hypoxia by topical formulations with vasodilators for enhancing therapy.

Tumor hypoxia is a well known therapeutic problem which contributes to radioresistance and aggressive tumor characteristics. Lack of techniques for repeated measurements of tumor oxygenation (pO(2), partial pressure of oxygen) has restricted the optimization of hypoxia modifying methods and their efficacious application with radiotherapy. We have investigated a non-invasive method to enhance tissue pO(2) of peripheral tumors using topical application of formulations with BN (Benzyl Nicotinate), a vasodilator, and have used EPR (Electron Paramagnetic Resonance) oximetry to follow its effect on tumor oxygenation.We incorporated 2.5% BN in both hydrogel and microemulsions and investigated the effects on pO(2) of subcutaneous RIF-1 (Radiation Induced Fibrosarcoma) tumors in C3H mice. The experiments were repeated for five consecutive days. The topical application of BN in hydrogel led to a significant increase from a pre-treatment pO(2) of 9.3 mmHg to 11 - 16 mmHg at 30 - 50 min on day 1. However, the magnitude and the time of significant increase in pO(2) decreased with repeated topical applications. The BN in a microemulsion resulted in a significant increase from a baseline pO(2) of 8.8 mmHg to 13 - 18 mmHg at 10 - 50 min on day 1. Experiments repeated on subsequent days showed a decline in the magnitude of pO(2) increase on repeated applications. No significant change in tumor pO(2) was observed in experiments with formulations without BN (vehicle only).EPR oximetry was successfully used to follow the temporal changes in tumor pO(2) during repeated applications for five consecutive days. This approach can be potentially used to enhance radiotherapeutic outcome by scheduling radiation doses when an increase in tumor pO(2) is observed after topical applications of BN formulations.

Tumor pO2 as a surrogate marker to identify therapeutic window during metronomic chemotherapy of 9L gliomas.

Glioblastomas are aggressive and highly vascularized primary brain tumors with a 5-year survival rate of less than 10%. Approaches targeting tumor vasculature are currently being investigated to achieve therapeutic benefits for this fatal malignancy. However, lack of suitable markers that can be used to monitor therapeutic effects during such treatments has restricted their optimization. We have focused on the development of tumor pO(2) as a surrogate marker to identify the therapeutic window during metronomic chemotherapy.We report the effect of four weekly administrations of cyclophosphamide (140 mg/Kg, i.p), a chemo drug, on tumor pO(2) and growth of subcutaneous 9L tumors in SCID mice. The repeated measurement of tumor pO(2) was carried out using in vivo EPR oximetry. The subcutaneous 9L tumors were hypoxic with a pre-treatment tumor pO(2) of 5.1 ± 1 mmHg and a tumor volume of 236 ± 45 mm3 on day 0. The tumor pO(2) increased significantly to 26.2 ± 2 mmHg on day 10, and remained at an elevated level till day 31 during weekly treatments with cyclophosphamide. The tumor pO(2) then declined to 20 ± 9 mmHg on day 43. The tumor volume of the control group increased significantly with no change in tumor pO(2)over days.Results indicate a transient increase in tumor pO(2) during metronomic chemotherapy of 9L gliomas and could be potentially used as a marker to identify vessel normalization during metronomic chemotherapy. The ability to identify therapeutic window non-invasively using EPR oximetry can have a significant impact on the optimization of clinical protocols. In vivo EPR oximetry is currently being tested for repeated pO(2) measurements in patients with superficial tumors.

Effect of hyperoxygenation on tissue pO2 and its effect on radiotherapeutic efficacy of orthotopic F98 gliomas.

PURPOSE: Lack of methods for repeated assessment of tumor pO(2) limits the ability to test and optimize hypoxia-modifying procedures being developed for clinical applications. We report repeated measurements of orthotopic F98 tumor pO(2) and relate this to the effect of carbogen inhalation on tumor growth when combined with hypofractionated radiotherapy. METHODS AND MATERIALS: Electron paramagnetic resonance (EPR) oximetry was used for repeated measurements of tumor and contralateral brain pO(2) in rats during 30% O(2) and carbogen inhalation for 5 consecutive days. The T(1)-enhanced volumes and diffusion coefficients of the tumors were assessed by magnetic resonance imaging (MRI). The tumors were irradiated with 9.3 Gy x 4 fractions in rats breathing 30% O(2) or carbogen to determine the effect on tumor growth. RESULTS: The pretreatment F98 tumor pO(2) varied between 8 and 16 mmHg, while the contralateral brain had 41 to 45 mmHg pO(2) during repeated measurements. Carbogen breathing led to a significant increase in tumor and contralateral brain pO(2); however, this effect declined over days. Irradiation of the tumors in rats breathing carbogen resulted in a significant decrease in tumor growth and an increase in the diffusion coefficient measured by MRI. CONCLUSIONS: The results provide quantitative measurements of the effect of carbogen inhalation on intracerebral tumor pO(2) and its effect on therapeutic outcome. Such direct repeated pO(2) measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcome by scheduling doses at times of improved tumor oxygenation. EPR oximetry is currently being tested for clinical applications.

Effect of a topical vasodilator on tumor hypoxia and tumor oxygen guided radiotherapy using EPR oximetry.

We sought to reduce tumor hypoxia by topical application of a vasodilator, benzyl nicotinate (BN), and investigated its effect on the growth of tumors irradiated at times when tumor pO(2) increased. EPR oximetry was used to follow the changes in the tissue pO(2) of subcutaneous radiation-induced fibrosarcoma (RIF-1) tumors during topical applications of 1.25-8% BN formulations for 5 consecutive days. The RIF-1 tumors were hypoxic with a tissue pO(2) of 4.6-7.0 mmHg. A significant increase in tumor pO(2) occurred 10-30 min after BN application. The formulation with the minimal BN concentration that produced a significant increase in tumor pO(2) was used for the radiation study. The tumors were irradiated (4 Gy x 5) at the time of the maximum increase in pO(2) observed with the 2.5% BN formulation. The tumors with an increase in pO(2) of greater than 2 mmHg from the baseline after application of BN on day 1 had a significant growth inhibition compared to the tumors with an increase in pO(2) of less than 2 mmHg. The results indicate that the irradiation of tumors at the time of an increase in pO(2) after the topical application of the 2.5% BN formulation led to a significant growth inhibition. EPR oximetry provided dynamic information on the changes in tumor pO(2), which could be used to identify responders and non-responders and schedule therapy during the experiments.

Implantable resonators--a technique for repeated measurement of oxygen at multiple deep sites with in vivo EPR.

EPR oximetry using implantable resonators allows measurements at much deeper sites than are possible with surface resonators (> 80 vs. 10 mm) and achieves greater sensitivity at any depth. We report here the development of an improved technique that enables us to obtain the information from multiple sites and at a variety of depths. The measurements from the various sites are resolved using a simple magnetic field gradient. In the rat brain multi-probe implanted resonators measured pO(2) at several sites simultaneously for over 6 months under normoxic, hypoxic, and hyperoxic conditions. This technique also facilitates measurements in moving parts of the animal such as the heart, because the orientation of the paramagnetic material relative to the sensing loop is not altered by the motion. The measured response is fast, enabling measurements in real time of physiological and pathological changes such as experimental cardiac ischemia in the mouse heart. The technique also is quite useful for following changes in tumor pO(2), including applications with simultaneous measurements in tumors and adjacent normal tissues.

Radiotherapy in conjunction with 7-hydroxystaurosporine: a multimodal approach with tumor pO2 as a potential marker of therapeutic response.

Checkpoint inhibitors potentially could be used to enhance cell killing by DNA-targeted therapeutic modalities such as radiotherapy. UCN-01 (7-hydroxystaurosporine) inhibits S and G2 checkpoint arrest in the cells of various malignant cell lines and has been investigated in combination with chemotherapy. However, little is known about its potential use in combination with radiotherapy. We report the effect of 20 Gy radiation given in conjunction with UCN-01 on the pO2 and growth of subcutaneous RIF-1 tumors. Multisite EPR oximetry was used for repeated, non-invasive tumor pO2 measurements. The effect of UCN-01 and/or 20 Gy on tumor pO2 and tumor volume was investigated to determine therapeutic outcomes. Untreated RIF-1 tumors were hypoxic with a tissue pO2 of 5-7 mmHg. Treatment with 20 Gy or UCN-01 significantly reduced tumor growth, and a modest increase in tumor pO2 was observed in tumors treated with 20 Gy. However, irradiation with 20 Gy 12 h after UCN-01 treatment resulted in a significant inhibition of tumor growth and a significant increase in tumor pO2 to 16-28 mmHg from day 1 onward compared to the control, UCN-01 or 20-Gy groups. Treatment with UCN-01 12 h after 20 Gy also led to a similar growth inhibition of the tumors and a similar increase in tumor pO2. The changes in tumor pO2 observed after the treatment correlated inversely with the tumor volume in the groups receiving UCN-01 with 20 Gy. This multimodal approach could be used to enhance the outcome of radiotherapy. Furthermore, tumor pO2 could be a potential marker of therapeutic response.

Tissue pO2 of orthotopic 9L and C6 gliomas and tumor-specific response to radiotherapy and hyperoxygenation.

PURPOSE: Tumor hypoxia is a well-known therapeutic problem; however, a lack of methods for repeated measurements of glioma partial pressure of oxygen (pO(2)) limits the ability to optimize the therapeutic approaches. We report the effects of 9.3 Gy of radiation and carbogen inhalation on orthotopic 9L and C6 gliomas and on the contralateral brain pO(2) in rats using a new and potentially widely useful method, multisite in vivo electron paramagnetic resonance oximetry. METHODS AND MATERIALS: Intracerebral 9L and C6 tumors were established in the left hemisphere of syngeneic rats, and electron paramagnetic resonance oximetry was successfully used for repeated tissue pO(2) measurements after 9.3 Gy of radiation and during carbogen breathing for 5 consecutive days. RESULTS: Intracerebral 9L gliomas had a pO(2) of 30-32 mm Hg and C6 gliomas were relatively hypoxic, with a pO(2) of 12-14 mm Hg (p < 0.05). The tissue pO(2) of the contralateral brain was 40-45 mm Hg in rats with either 9L or C6 gliomas. Irradiation resulted in a significant increase in pO(2) of the 9L gliomas only. A significant increase in the pO(2) of the 9L and C6 gliomas was observed in rats breathing carbogen, but this effect decreased during 5 days of repeated experiments in the 9L gliomas. CONCLUSION: These results highlight the tumor-specific effect of radiation (9.3.Gy) on tissue pO(2) and the different responses to carbogen inhalation. The ability of electron paramagnetic resonance oximetry to provide direct repeated measurements of tissue pO(2) could have a vital role in understanding the dynamics of hypoxia during therapy that could then be optimized by scheduling doses at times of improved tumor oxygenation.

Repeated tumor pO(2) measurements by multi-site EPR oximetry as a prognostic marker for enhanced therapeutic efficacy of fractionated radiotherapy.

PURPOSE: To investigate the temporal effects of single or fractionated radiotherapy on subcutaneous RIF-1 tumor pO(2) and to determine the therapeutic outcomes when the timing of fractionations is guided by tumor pO(2). METHODS: The time-course of the tumor pO(2) changes was followed by multi-site electron paramagnetic resonance (EPR) oximetry. The tumors were treated with single 10, 20, and 10 Gy x 2 doses, and the tumor pO(2) was measured repeatedly for six consecutive days. In the 10 Gy x 2 group, the second dose of 10 Gy was delivered at a time when the tumors were either relatively oxygenated or hypoxic. The changes in tumor volumes were followed for nine days to determine the therapeutic outcomes. RESULTS: A significant increase in tumor pO(2) was observed at 24h post 10 Gy, while 20 Gy resulted in a significant increase in tumor pO(2) at 72-120 h post irradiation. The tumors irradiated with a second dose of 10 Gy at 24h, when the tumors were oxygenated, had a significant increase in tumor doubling times (DTs), as compared to tumors treated at 48 h when they were hypoxic (p<0.01). CONCLUSION: Results indicate that the time of tumor oxygenation depends on the irradiation doses, and radiotherapeutic efficacy could be optimized if irradiations are scheduled at times of increased tumor oxygenation. In vivo multi-site EPR oximetry could be potentially used to monitor tumor pO(2) repeatedly during fractionated schemes to optimize radiotherapeutic outcome. This technique could also be used to identify responsive and non-responsive tumors, which will facilitate the design of other therapeutic approaches for non-responsive tumors at early time points during the course of therapy.

Arsenic as an endocrine disruptor: arsenic disrupts retinoic acid receptor-and thyroid hormone receptor-mediated gene regulation and thyroid hormone-mediated amphibian tail metamorphosis.

BACKGROUND: Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors. OBJECTIVES: The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid regulation. METHODS AND RESULTS: Human embryonic NT2 or rat pituitary GH3 cells were treated with 0.01-5 microM sodium arsenite for 24 hr, with or without RA or TH, respectively, to examine effects of As on receptor-mediated gene transcription. At low, noncytotoxic doses, As significantly altered RAR-dependent gene transcription of a transfected RAR response element-luciferase construct and the native RA-inducible cytochrome P450 CYP26A gene in NT2 cells. Likewise, low-dose As significantly altered expression of a transfected TR response element-luciferase construct and the endogenous TR-regulated type I deiodinase (DIO1) gene in a similar manner in GH3 cells. An amphibian ex vivo tail metamorphosis assay was used to examine whether endocrine disruption by low-dose As could have specific pathophysiologic consequences, because tail metamorphosis is tightly controlled by TH through TR. TH-dependent tail shrinkage was inhibited in a dose-dependent manner by 0.1- 4.0 microM As. CONCLUSIONS: As had similar effects on RAR- and TR-mediated gene regulation as those previously observed for the steroid receptors, suggesting a common mechanism or action. Arsenic also profoundly affected a TR-dependent developmental process in a model animal system at very low concentrations. Because RAR and TH are critical for both normal human development and adult function and their dysregulation is associated with many disease processes, disruption of these hormone receptor-dependent processes by As is also potentially relevant to human developmental problems and disease risk.