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Background: Inspiratory hyperoxia under hyperbaric conditions has been shown to effectively reduce tumor hypoxia and to improve radiosensitivity. However, applying irradiation (RT) under hyperbaric conditions is technically difficult in the clinical setting since RT after decompression may be effective only if tumor pO2 remains elevated for a certain period of time. The aim of the present study was to analyze the time course of tumor oxygenation and perfusion during and after hyperbaric hyperoxia., Materials and Methods: Tumor oxygenation, red blood cell (RBC) flux for perfusion monitoring, and vascular resistance were assessed continuously in experimental rat DS-sarcomas by polarographic catheter electrodes and laser Doppler flowmetry at 1 and 2 atm (bar) of environmental pressure during breathing of pure O2 or carbogen (95 % O2 + 5 % CO2)., Results: During room air breathing, the tumor pO2 followed very rapidly within a few minutes the change of the ambient pressure during compression or decompression. With O2 breathing under hyperbaric conditions, the tumor pO2 increased more than expected based on the rise of the environmental pressure, although the time course was comparably rapid. Breathing carbogen, the tumor pO2 followed with a slight delay of the pressure change, and within 10 min after decompression the baseline values were reached again. RBC flux increased during carbogen breathing but remained almost constant with pure O2, indicating a vasodilation (decrease in vascular resistance) with carbogen but a vasoconstriction (increase in vascular resistance) with O2 during hyperbaric conditions., Conclusion: Since the tumor pO2 directly followed the environmental pressure, teletherapy after hyperbaric conditions does not seem to be promising as the pO2 reaches baseline values again within 5-10 min after decompression.

Electron paramagnetic resonance (EPR) oximetry is a technique which allows accurate and repeatable oxygen measurements. We encapsulated a highly oxygen sensitive particulate EPR spin probe into microparticles to improve its dispersibility and, hence, facilitate the administration. These biocompatible, non-toxic microspheres contained 5-10 % (w/w) spin probe and had an oxygen sensitivity of 0.60 ± 0.01 µT/mmHg. To evaluate the performance of the microparticles as oxygen sensors, they were co-implanted with syngeneic tumor cells in 2 different rat strains. Thus, tissue injury was avoided and the microparticles were distributed all over the tumor tissue. Dynamic changes of the intratumoral oxygen partial pressure during inhalation of 8 %, 21 %, or 100 % oxygen were monitored in vivo by EPR spectroscopy and quantified. Values were verified in vivo by invasive fluorometric measurements using Oxylite probes and ex vivo by pimonidazole adduct accumulation. There were no hints that the tumor physiology or tissue oxygenation had been altered by the microparticles. Hence, these microprobes offer great potential as oxygen sensors in preclinical research, not only for EPR spectroscopy but also for EPR imaging. For instance, the assessment of tissue oxygenation during therapeutic interventions might help understanding pathophysiological processes and lead to an individualized treatment planning or the use of formulations with hypoxia triggered release of active agents., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Inspiratory hyperoxia reduces tumor hypoxia, which is responsible for limited radiosensitivity of tumors. However, very little is known about the heterogeneity of intratumoral oxygenation during this supportive treatment. The study analyzes whether local hypoxia is still present during normobaric and hyperbaric inspiratory hyperoxia and whether the addition of CO2 to the inspiratory gas affects the spatial pO2 distribution., Material and Methods: Tumor oxygenation of experimental DS-sarcomas in rats was assessed by polarographic needle electrodes at 1 and 2 atm (bar) environmental pressure during pure O2 or carbogen (95 % O2 + 5 % CO2) breathing. Up to 320 individual pO2 measurements were performed in a strictly oriented grid resulting in an oxygenation profile in a horizontal tumor layer., Results: In the experimental tumors used the oxygenation showed pronounced heterogeneities with closely adjacent hypoxic and oxygenated regions. This heterogeneity was still visible under normobaric hyperoxia where large confluent hypoxic regions were detectable. At 1 atm, the addition of CO2 improved tumor oxygenation significantly (at least in large tumors). At 2 atm, only very small local regions of hypoxia were detected. However, under this condition hypercapnia had no impact on tumor oxygenation., Conclusions: The data show that even under hyperbaric hyperoxia, hypoxic regions are detectable despite the average pO2 increased by a factor of 100. The results also clearly indicate that the oxygenation pattern improves disproportionally with increasing environmental pressure.

The aim of this study was to assess the anti-tumor effect of photodynamic therapy (PDT) using a novel bacteriochlorophyll derivative, palladium-bacteriopheophorbide (TOOKAD) on tumor growth, perfusion and oxygenation. Rat DS-sarcomas were treated with either TOOKAD-PDT (2 mg/kg, i.v., immediate illumination) or one of the control treatments (sham-treatment, illumination without photosensitizer, or photosensitizer without illumination). The light source was an infrared-A irradiator fitted with appropriate filters, so that the wavelengths applied (665-800 nm) included the absorption maximum of TOOKAD at 763 nm. Tumor volume was monitored for 90 days after treatment or until a target volume (3.5 ml) was reached. TOOKAD-PDT dramatically inhibited tumor growth with 92% of tumors not reaching the target volume within the observation period. In further experiments, tumor perfusion was assessed using laser Doppler flowmetry. Upon TOOKAD-PDT treatment, a rapid, pronounced decrease in perfusion was seen, down to levels corresponding to only 3% of initial values. Tumor oxygenation monitoring revealed parallel decreases, with levels corresponding to anoxia being reached. The significant anti-tumor effects presented in this report, taken together with the chemical and pharmacokinetic properties of the novel photosensitizer TOOKAD, underline the therapeutic potential of this approach in which flow stasis and induction of anoxia are key elements.

Purpose: In carcinomas of the uterine cervix, the tumor oxygenation status has been shown to be a prognostic indicator that is independent of treatment modality. In vitro studies suggest gene amplification and polyploidization to be among the major consequences of hypoxia (with or without consecutive reoxygenation) and to be associated with treatment resistance and tumor progression. This study analyzed whether hypoxia alters net DNA content in uterine cervix cancer cells to the extent that it is identifiable by DNA image cytometry., Materials and Methods: In 64 patients with primary cervical cancer, tumor oxygenation was assessed polarographically and correlated with cell DNA content (DNA image cytometry) in areas adjacent to the oxygen microsensor tracks in which oxygenation measurements were made., Results: No correlation between DNA content (stemline position, Auer classification, and 2c deviation index) and oxygenation status was observed. However, an association between DNA content and patient age and menopausal status was found., Conclusion: Using DNA cytometry, hypoxia-associated genomic changes in uterine cervix cancer cells could not be detected. The impact of tumor hypoxia on the genome may be masked by the effects of alternative mechanisms of genomic instability that can also influence DNA content.

The aim of this study was to assess pivotal factors determining the oxygenation status of malignant tumors, and, for comparison, of normal tissues. For this purpose, microvascular patterns, blood flow data, O2 consumption rates and O2 extractions were correlated to the median pO2 values of isografted rodent and xenografted human tumors, and of various normal tissues. No correlations were found between median pO2 values and vascular patterns, O2 consumption rate or blood flow rate. When O2 extraction, however, was considered (a comprehensive parameter which encompasses all of the above factors), a strong inverse correlation was found between this parameter and the median pO2 values, both for malignant tumors (r2=0.917) and normal tissues (r2=0.959). At similar O2 extraction values, the median pO2 values in the solid tumors were substantially lower than those in normal tissues, a finding which most probably reflects the chaotic microcirculation found in tumors, the loss of flow regulation which in normal tissues meets the metabolic demands of the cells, and in some cases, tumor-associated anemia.

Since local hyperthermia (HT) affects microenvironmental parameters, the aim of the study was to analyze the impact of 42 degrees C-HT on microcirculatory function, tumor pO2, microregional redox status and ATP distribution in experimental rat tumors. Subcutaneously growing DS-sarcomas were treated with localized HT using infrared-A radiation resulting in a mean tumor temperature of 42 degrees C. The relative red blood cell (RBC) flux in the tumor was assessed using the laser Doppler technique and the mean tumor pO2 measured continuously using O2-sensitive catheter electrodes. In a second series of experiments, the microregional distribution of the mitochondrial redox status and ATP concentration was measured. Although the average RBC flux increased by 63%, tumor pO2 rose only by approx. 6%. No distinct changes were seen in the mitochondrial redox status. The microregional distribution of the redox status as well as of the ATP concentration showed considerable heterogeneity. In conclusion, although 42 degrees C-HT leads to a distinct improvement in tumor perfusion, there is practically no change in the oxygenation status. The latter finding can be explained by an equivalent increase in the oxygen consumption rate of the cells which increases by approx. 58% at 42 degrees C compared to normothermia.

Isolated limb perfusion allows the direct application of therapeutic agents to a tumour-bearing extremity. The present study investigated whether the dihydropyridine-type Ca(2+)-channel blocker nifedipine could improve blood flow and oxygenation status of experimental tumours during isolated limb perfusion. Perfusion was performed by cannulation of the femoral artery and vein in rats bearing DS-sarcoma on the hind foot dorsum. Perfusion rate was adjusted to maintain a perfusion pressure of 100-140 mmHg throughout the experiment. Following equilibration, nifedipine was continuously infused for 30 min (8.3 microg min(-1) kg(-1) BW). During constant-pressure isolated limb perfusion, nifedipine can significantly increase perfusion rate (+100%) and RBC flux (+60%) through experimental leg tumours. "Steal phenomena" in favour of the surrounding normal tissue and oedema formation were not observed. Despite the increased oxygen availability (+63%) seen upon application of this calcium channel blocker, nifedipine does not result in a substantial reduction of tumour hypoxia, most probably due to an increase in O(2) uptake with rising O(2) supply to the tumour-bearing hind limb. Nifedipine application during isolated limb perfusion can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled isolated limb perfusion., (Copyright 2002 Cancer Research UK)

The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT-PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia., (Copyright 2002 Cancer Research UK)

Background and Purpose: Increasing arterial oxygen partial pressure (pO2) by breathing hyperoxic gases is an effective means of improving tumor oxygenation, although the efficacy of adding CO2 to the inspiratory gas has been discussed controversially. This study aimed at analyzing the impact of different inspiratory CO2 fractions on the time course of oxygenation and perfusion changes in experimental tumors during and after inspiratory hyperoxia., Material and Methods: Perfusion and oxygenation of rat DS-sarcomas were studied during spontaneous breathing of pure oxygen or hyperoxic gas mixtures containing different CO2 fractions (1, 2.5 or 5%). Red blood cell (RBC) flux was assessed as a measure of tumor perfusion using the laser Doppler technique and temporal changes in mean tumor pO2 were measured polarographically., Results: Mean tumor pO2 increased 3.6-fold with pure oxygen, approx. 3.3-fold when 1 or 2.5% CO2 was added and 2.7-fold during carbogen breathing. RBC flux also increased by 25-30% with all gases. With pure oxygen and with 1% CO2 (+99% O2), perfusion changes paralleled those of the mean arterial blood pressure whereas with higher CO2 fractions, a decrease in resistance to flow was observed. The differences found with the various gas mixtures were more pronounced after the end of hyperoxia. With pure oxygen, perfusion immediately returned to pretreatment values whereas with higher CO2 fractions perfusion remained elevated for at least 30 min., Conclusions: Higher inspiratory CO2 fractions (2.5 or 5%) lead to a prolonged improvement of tumor perfusion after the end of inspiratory hyperoxia when compared with pure oxygen breathing. Since no principal differences in oxygenation and perfusion were seen between the gases containing 2.5 and 5% CO2, the former may be preferable for inspiratory hyperoxia.

The oxygen deficiency seen in solid tumors is predominantly caused by an insufficient O2 supply as a result of inadequate tumor perfusion. The aim of this study was to analyze whether a number of vasodilator drugs might be suitable to increase tumor perfusion and consequently improve the oxygenation status of experimental tumors. Rats with s.c. DS-sarcomas were treated with either Na+-nitroprusside (7-25 microg x min(-1) x kg(-1) BW) or nifedipine (10 microg x min(-1) x kg(-1) BW). Red blood cell (RBC) flux was assessed continuously using laser-Doppler flowmetry and mean tumor pO2 was measured polarographically using O2-sensitive catheter electrodes. Systemic application of the vasodilator drugs resulted in a dose-dependent decrease in MABP. In parallel, tumor perfusion was reduced linearly with falling MABP resulting in a decrease in RBC flux by approximately 40%. Resistance to flow did not change during the infusion indicating that these drugs have no impact on tumor vessel diameter. With decreasing tumor perfusion, tumor pO2 was reduced parallel to the MABP drop. This effect was more distinct with Na+-nitroprusside than with nifedipine due to the more pronounced fall in MABP. The vasodilator drugs studied are not suitable for dilation of tumor vessels either because the tumor vasculature lacks contractile wall elements or because the vessels are already maximally dilated.

Perfusion and oxygenation of experimental tumours were studied during angiotensin II (AT II) administration whereby the rate of the continuous AT II infusion was chosen to increase the mean arterial blood pressure (MABP) by 50-70 mmHg. In subcutaneous DS-sarcomas the red blood cell (RBC) flux was assessed using the laser Doppler technique and the mean tumour oxygen partial pressure (pO2) was measured polarographically using O2-sensitive catheter and needle electrodes. Changes in RBC flux with increasing MABP depended mainly on tumour size. In small tumours, RBC flux decreased with rising MABP whereas in larger tumours RBC flux increased parallel to the MABP. As a result of these volume-dependent effects on tumour blood flow, the impact of AT II on tumour pO2 was also mainly tumour volume-related. In small tumours oxygenation decreased with increasing MABP during AT II infusion, whereas in large tumours a positive relationship between blood pressure and O2 status was found. This disparate behaviour might be the result of the co-existence of two functionally distinct populations of tumour vessels. In small tumours, perfusion decreases presumably due to vasoconstriction of pre-existing host vessels feeding the tumour. In larger malignancies, newly formed tumour vessels predominate and seem not to have this vasoresponsive capability (lack of smooth muscle cells and/or AT receptors), resulting in an improvement of perfusion which is not tumour-related per se, but is due to the increased perfusion pressure.

Objectives: Patients with cystic fibrosis (CF) represent a special risk for commercial airlines. Even on ground conditions the oxygen partial pressure (paO subset2) of these patients is partially clearly reduced. The reduced air pressure on board of an airplane can cause a drop of the paO subset2 to a critical point (below 50 mmHg) during a flight. Therefore, medical assistance or at least oxygen-supply over a longer time period could be necessary. Aim of this study was to investigate reaction and clinical outcome of patients with CF during a hypobaric-chamber-flight at altitudes of 2. 000 and 3.000 m to appraise their risk for a flight-trip., Methods: 10 CF-patients (age 19-35 years, mean age 30 y) were investigated in a hypobaric chamber prior to an already booked flight-trip to the Baleares (Spain). Lung-function, oxygen saturation (SO subset2) and paO subset2 by pressure adjusted blood gas analysis were measured on ground level, at 2.000 m and 3.000 m pressure-altitude., Results: Forced expiratory vital capacity (FVC) over the entire group was 2. 9 l (range 1.4 to 4.0 l), forced expiratory 1-second volume (FEV subset1) 2.08 l/sec (range: 1.22 to 3.61 l/sec). Values dropped slightly at 3.000 m chamber altitude (VC=2.7 l, FEV subset1=1.95 l/sec). SO subset2 decreased from 95 % on ground to 89% at 2.000 m and 86 % at 3.000 m chamber altitude. paO2 decreased from 79.5 mmHg at ground level to 60 mmHg at 2000m and 45.5 mmHg at 3.000 m. Only one patient with a paO subset2 of 52 mmHg didn t fall below the critical flight limit of 50 mmHg. No patient felt below a paO subset2 of 40 mmHg. No patient experienced dyspnea during the chamber flight. Two patients without subjective symptoms before the chamber flight developed mild ear blocks during descent presumably due to swollen polyps. Complaints improved quickly by applying decongestant nose-spray. -, Conclusion: The results of the chamber flights indicate that chronically adapted adult lung disease patients without accompanying heart disease and a paO subset2 of > 40 mmHg during flight can anticipate a safe flight trip. These results could be confirmed by the consecutive flight trip to Spain.

Differentiation of a tumor plays an important role in terms of biological aggressiveness. The question arises as to whether this is reflected in differences in the metabolic and energetic status of solid tumors. The aim of this study was to analyze the influence of clonal tumor cell differentiation on the microenvironment of rat rhabdomyosarcomas. Two distinct lines of a rhabdomyosarcoma (BA-HAN-1) with different histomorphological properties were used (line F1, co-existence of mononuclear stellate cells and multinuclear myotube-like giant tumor cells; G8, polygonal, mononuclear tumor cells). Solid tumors were grown s.c. on the hind food dorsum of Lewis rats. Tumor oxygenation was measured using O2-sensitive needle electrodes. For determining tumor blood flow, the 133Xe clearance method was used. Global glucose and lactate concentrations were measured enzymatically, global ATP, ADP, and AMP were analyzed by HPLC. The regional distribution of metabolic and energetic parameters within the tumors was analyzed using quantitative bioluminescence and image analysis. Tumor growth rate was significantly different between the two lines. The volume doubling time was 2.5 days for the F1 and 3.0 days for G8 tumors. No differences in blood flow were seen between the two lines investigated, oxygenation was slightly poorer, glucose and ATP levels slightly higher, and lactate concentration somewhat lower in the F1 line as compared to the G8 line. From these differences - although marginal - it is concluded that the G8 line presumably relies on glycolysis whereas the F1 line seems to prefer oxidative glucose turnover. Despite these different metabolic profiles between the two tumor lines, the histopathology of the rhabdomyosarcomas seems to be only of limited significance for the tissue oxygenation status as already postulated for various tumors in the clinical setting.

The effects of respiratory hyperoxia (RH) and xanthine oxidase (XO) during localized hyperthermia (HT) were investigated by determining markers of oxidative damage to lipids and proteins and tumor growth. Anesthetized rats with s.c. DS-sarcomas underwent one of the following treatments: (a) localized saline-bath HT (60 min, 44 degrees C); (b) HT + RH (100% O2); and (c) HT + RH + XO (15 units/kg i.v.). Sham-treated animals served as controls. Tumors were investigated for: (a) thiobarbituric acid-reactive substance formation and protein-bound 4-hydroxynonenal, as indicators of lipid peroxidation; (b) reactive oxygen-mediated protein modifications; (c) apoptosis; and (d) tumor volume growth. Upon treatment, increases in thiobarbituric acid-reactive substances, protein-bound 4-hydroxynonenal, protein-associated carbonyl functions, and number of cells undergoing apoptosis were found in tumor tissue, together with an inhibition of tumor growth. When treatment groups were compared, effects in the group HT + RH + XO were generally most pronounced. These findings indicate that the antitumor effect of HT is at least partially mediated through the selective induction of lipid peroxidation and oxidative injury in tumor cells, leading to apoptosis. This effect was enhanced by adding RH or RH + XO, presumably due to enhanced tissue damage following an increased formation of reactive oxygen species, with higher levels of lipid peroxidation and protein oxidation.

Aim: Several studies using different methods of pO2 measurement have shown that in many tumor entities tissue oxygenation is quite heterogeneous and thus cannot be sufficiently described by a single value. This study presents those distribution parameters which are mandatory to describe the complete oxygenation status of a solid tumor., Methods: In determining the oxygenation status different aspects of relevancy have to be taken into account. First, several parameters are necessary to characterize the location, the spread, and the shape of the pO2 distribution from a statistical point of view (statistical parameters). In addition, parameters for the description of the biological relevance of the tumor oxygenation have to be considered (biological parameters). These parameters reflect the role of oxygenation for the metabolic microenvironment and the biological behavior of the tumor as well as the significance under therapeutic and diagnostic aspects. Finally, some parameters are necessary to assess the quality of pO2 measurement itself (quality parameters)., Results: By combining all pO2 readings from one tumor a statistical distribution is obtained, which is in many tumors asymmetrically shifted towards low pO2 values. In order to describe the location, spread, and shape of this distribution the following parameters should be stated: median, mean, 10% and 90% percentile, minimum, maximum and 10 to 90% interpercentile range. For data presentation these values can be combined in a box-whiskers plot. In order to reflect the importance of tumor oxygenation under diagnostic, therapeutic or cell-biological aspects the fraction of pO2 readings < or = 2.5 mm Hg, < or = 5 mm Hg, and < or = 10 mm Hg should be stated. Where negative pO2 readings occur in a study, the fraction of pO2 readings < 0 mm Hg as well as the most negative pO2 value should be presented in order to assess the quality of the pO2 measurement., Conclusions: For a comprehensive description of the oxygenation status of solid tumors the following distribution parameters should be stated: median, mean, 10% and 90% percentile, minimum, maximum, 10 to 90% interpercentile range, fraction of pO2 readings < 0 mm Hg, < or = 2.5 mm Hg, < or = 5 mm Hg, and < or = 10 mm Hg as well as the most negative pO2 reading.

Tumor oxygenation has been measured polarographically in s.c. implanted DS-sarcomas on the dorsum of the hind foot of male Sprague-Dawley rats. pO2 was determined in all 3 spatial dimensions and 3-dimensional pO2 distributions as well as the mean extent of confluent areas with pO2 < 5 mmHg were calculated. Finally, the effect of elevating arterial pO2 (by carbogen breathing) as well as of increasing tumor blood flow (by angiotensin infusion) on the spatial pO2 distribution was analyzed. Depending on the tumor volume, the spatial pO2 distribution is more or less anisotropic. In smaller tumors, areas with physiological pO2 values are found adjacent to large hypoxic areas whereas larger tumors are almost completely hypoxic/anoxic. With carbogen breathing, the mean tissue pO2 is elevated although hypoxia is not eradicated in larger tumors. In small tumors, angiotensin leads to a vasoconstriction of tumor vessels followed by a worsening of tumor oxygenation whereas in large tumors the increased systemic perfusion pressure resulted in an improvement of oxygenation. Thus, carbogen predominantly affects pO2 diffusion by increasing the arterial pO2 whereas angiotensin influences tumor perfusion and leads to an increased oxygen supply to the tumor tissue.