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The aim of this review is to stimulate readers to undertake appropriate investigations of the mechanism for a possible oxygen effect in FLASH. FLASH is a method of delivery of radiation that empirically, in animal models, appears to decrease the impact of radiation on normal tissues while retaining full effect on tumors. This has the potential for achieving a significantly increased effectiveness of radiation therapy. The mechanism is not known but, especially in view of the prominent role that oxygen has in the effects of radiation, investigations of mechanisms of FLASH have often focused on impacts of FLASH on oxygen levels. We and others have previously shown that simple differential depletion of oxygen directly changing the response to radiation is not a likely mechanism. In this review we consider how time-varying changes in oxygen levels could account for the FLASH effect by changing oxygen-dependent signaling in cells. While the methods of delivering FLASH are still evolving, current approaches for FLASH can differ from conventional irradiation in several ways that can impact the pattern of oxygen consumption: the rate of delivery of the radiation (40 Gy/s vs. 0.1 Gy/s), the time over which each fraction is delivered (e.g., <0.5 s. vs. 300 s), the delivery in pulses, the number of fractions, the size of the fractions, and the total duration of treatment. Taking these differences into account and recognizing that cell signaling is an intrinsic component of the need for cells to maintain steady-state conditions and, therefore, is activated by small changes in the environment, we delineate the potential time dependent changes in oxygen consumption and overview the cell signaling pathways whose differential activation by FLASH could account for the observed biological effects of FLASH. We speculate that the most likely pathways are those involved in repair of damaged DNA., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The delivery of radiation at an ultra-high dose rate (FLASH) is an important new approach to radiotherapy (RT) that appears to be able to improve the therapeutic ratio by diminishing damage to normal tissues. While the mechanisms by which FLASH improves outcomes have not been established, a role involving molecular oxygen (O 2 ) is frequently mentioned. In order to effectively determine if the protective effect of FLASH RT occurs via a differential direct depletion of O 2 (compared to conventional radiation), it is essential to consider the known role of O 2 in modifying the response of cells and tissues to ionising radiation (known as 'the oxygen effect'). Considerations include: (1) The pertinent reaction involves an unstable intermediate of radiation-damaged DNA, which either undergoes chemical repair to restore the DNA or reacts with O 2 , resulting in an unrepairable lesion in the DNA, (2) These reactions occur in the nuclear DNA, which can be used to estimate the distance needed for O 2 to diffuse through the cell to reach the intermediates, (3) The longest lifetime that the reactive site of the DNA is available to react with O 2 is 1-10 μsec, (4) Using these lifetime estimates and known diffusion rates in different cell media, the maximal distance that O 2 could travel in the cytosol to reach the site of the DNA (i.e., the nucleus) in time to react are 60-185 nm. This calculation defines the volume of oxygen that is pertinent for the direct oxygen effect, (5) Therefore, direct measurements of oxygen to determine if FLASH RT operates through differential radiochemical depletion of oxygen will require the ability to measure oxygen selectively in a sphere of <200 nm, with a time resolution of the duration of the delivery of FLASH, (6) It also is possible that alterations of oxygen levels by FLASH could occur more indirectly by affecting oxygen-dependent cell signalling and/or cellular repair., (© 2022. Springer Nature Switzerland AG.)

Reliable measurements using modern techniques and consensus in experimental design have enabled the assessment of novel data sets for normal maternal and foetal respiratory physiology at term. These data sets include (a) principal factors affecting placental gas transfer, e.g., maternal blood flow through the intervillous space (IVS) (500 mL/min) and foeto-placental blood flow (480 mL/min), and (b) O 2 , CO 2 and pH levels in the materno-placental and foeto-placental circulation. According to these data, the foetus is adapted to hypoxaemic hypoxia. Despite flat oxygen partial pressure (pO 2 ) gradients between the blood of the IVS and the umbilical arteries of the foetus, adequate O 2 delivery to the foetus is maintained by the higher O 2 affinity of the foetal blood, high foetal haemoglobin (HbF) concentrations, the Bohr effect, the double-Bohr effect, and high foeto-placental (=umbilical) blood flow. Again, despite flat gradients, adequate CO 2 removal from the foetus is maintained by a high diffusion capacity, high foeto-placental blood flow, the Haldane effect, and the double-Haldane effect. Placental respiratory gas exchange is perfusion-limited, rather than diffusion-limited, i.e., O 2 uptake depends on O 2 delivery., (© 2022. Springer Nature Switzerland AG.)

Measuring molecular oxygen levels in vivo has been the cornerstone of understanding the effects of hypoxia in normal tissues and malignant tumors. Here we discuss the advances in a variety of partial pressure of oxygen ( P O 2 ) measurements and imaging techniques and relevant oxygen thresholds. A focus on electron paramagnetic resonance (EPR) imaging shows the validation of treating hypoxic tumours with a threshold of P O 2  ≤ 10 Torr, and demonstrates utility for in vivo oxygen imaging, as well as its current and future role in cancer studies., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Clinical measurements of O 2 in tissues will inevitably provide data that are at best aggregated and will not reflect the inherent heterogeneity of O 2 in tissues over space and time. Additionally, the nature of all existing techniques to measure O 2 results in complex sampling of the volume that is sensed by the technique. By recognizing these potential limitations of the measures, one can focus on the very important and useful information that can be obtained from these techniques, especially data about factors that can change levels of O 2 and then exploit these changes diagnostically and therapeutically. The clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of O 2 .

It is well understood that the level of molecular oxygen (O 2 ) in tissue is a very important factor impacting both physiology and pathological processes as well as responsiveness to some treatments. Data on O 2 in tissue could be effectively utilized to enhance precision medicine. However, the nature of the data that can be obtained using existing clinically applicable techniques is often misunderstood, and this can confound the effective use of the information. Attempts to make clinical measurements of O 2 in tissues will inevitably provide data that are aggregated over time and space and therefore will not fully represent the inherent heterogeneity of O 2 in tissues. Additionally, the nature of existing techniques to measure O 2 may result in uneven sampling of the volume of interest and therefore may not provide accurate information on the "average" O 2 in the measured volume. By recognizing the potential limitations of the O 2 measurements, one can focus on the important and useful information that can be obtained from these techniques. The most valuable clinical characterizations of oxygen are likely to be derived from a series of measurements that provide data about factors that can change levels of O 2 , which then can be exploited both diagnostically and therapeutically. The clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of O 2 ., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The aim of the paper is to discuss what currently is feasible clinically to measure the level of oxygen and how that measurement can be clinically useful. Because oxygen in tissues is quite heterogeneous and all methods of measurement can only provide an average across heterogeneities at some spatial and temporal resolution, the values that are obtained may have limitations on their clinical utility. However, even if such limitations are significant, if one utilizes repeated measurements and focuses on changes in the measured levels, rather than 'absolute levels', it may be possible to obtain very useful clinical information. While these considerations are especially pertinent in cancer, they also pertain to most other types of pathology.

The success of treatment for malignancies, especially those undergoing radiation therapy or chemotherapy, has long been recognized to depend on the degree of hypoxia in the tumor. In addition to the prognostic value of knowing the tumor's initial level of hypoxia, assessing the tumor oxygenation during standard therapy or oxygen-related treatments (such as breathing oxygen-enriched gas mixtures or taking drugs that can increase oxygen supply to tissues) can provide valuable data to improve the efficacy of treatments. A series of early clinical studies of tumors in humans are ongoing at Dartmouth and Emory using electron paramagnetic resonance (EPR) oximetry to assess tumor oxygenation, initially and over time during either natural disease progression or treatment. This approach has the potential for reaching the long-sought goal of enhancing the effectiveness of cancer therapy. In order to effectively reach this goal, we consider the validity of the practical and statistical assumptions when interpreting the measurements made in vivo for patients undergoing treatment for cancer.

In the early 1920s, Warburg published experimental data on the enhanced conversion of glucose to pyruvate (followed by lactate formation) even in the presence of abundant oxygen (aerobic glycolysis, Warburg effect). He attributed this metabolic trait to a respiratory injury and considered this a universal metabolic alteration in carcinogenesis. This interpretation of the data was questioned since the early 1950s. Realistic causative mechanisms and consequences of the Warburg effect were described only during the past 15 years and are summarized in this article. There is clear evidence that mitochondria are not defective in most cancers. Aerobic glycolysis, a key metabolic feature of the Warburg phenotype, is caused by active metabolic reprogramming required to support sustained cancer cell proliferation and malignant progression. This metabolic switch is directed by altered growth factor signaling, hypoxic or normoxic activation of HIF-1α- transcription, oncogene activation or loss-of-function of suppressor genes, and is implemented in the hostile tumor microenvironment. The 'selfish' reprogramming includes (a) overexpression of glucose transporters and of key glycolytic enzymes, and an accelerated glycolytic flux with subsequent accumulation and diversion of glycolytic intermediates for cancer biomass synthesis, (b) high-speed ATP production that meets the energy demand, and (c) accumulation of lactate which drives tumor progression and largely contributes to tumor acidosis, which in turn synergistically favors tumor progression and resistance to certain antitumor therapies, and compromises antitumor immunity. Altogether, the Warburg effect is the central contributor to the cancer progression machinery.

Positron-emission tomography (PET) with hypoxia specific tracers provides a noninvasive method to assess the tumor oxygenation status. Reaction-diffusion models have advantages in revealing the quantitative relation between in vivo imaging and the tumor microenvironment. However, there is no quantitative comparison of the simulation results with the real PET measurements yet. The lack of experimental support hampers further applications of computational simulation models. This study aims to compare the simulation results with a preclinical [ 18 F]FMISO PET study and to optimize the reaction-diffusion model accordingly. Nude mice with xenografted human squamous cell carcinomas (CAL33) were investigated with a 2 h dynamic [ 18 F]FMISO PET followed by immunofluorescence staining using the hypoxia marker pimonidazole and the endothelium marker CD 31. A large data pool of tumor time-activity curves (TAC) was simulated for each mouse by feeding the arterial input function (AIF) extracted from experiments into the model with different configurations of the tumor microenvironment. A measured TAC was considered to match a simulated TAC when the difference metric was below a certain, noise-dependent threshold. As an extension to the well-established Kelly model, a flow-limited oxygen-dependent (FLOD) model was developed to improve the matching between measurements and simulations. The matching rate between the simulated TACs of the Kelly model and the mouse PET data ranged from 0 to 28.1% (on average 9.8%). By modifying the Kelly model to an FLOD model, the matching rate between the simulation and the PET measurements could be improved to 41.2-84.8% (on average 64.4%). Using a simulation data pool and a matching strategy, we were able to compare the simulated temporal course of dynamic PET with in vivo measurements. By modifying the Kelly model to a FLOD model, the computational simulation was able to approach the dynamic [ 18 F]FMISO measurements in the investigated tumors.

In this chapter we allude to a series of facts and fallacies often encountered in the description of tumor hypoxia, a relevant trait of the tumor microenvironment and a paramount driver of tumor aggressiveness and treatment resistance. The critical role of diffusion distances, terminological inconsistencies considering O 2 partial pressures vs. O 2 concentrations and with it the use of inept units, the impact of O 2 depletion on proliferation and cell viability, the switch in the Warburg dogma , the distribution of hypoxic subvolumes within a tumor, the involvement of O 2 diffusion shunts in the development of chronic hypoxia, and the role of endogenous biomarkers as surrogates for the assessment of hypoxia are discussed in more detail. Special emphasis is put on the clinical relevance of these misconceptions and misinterpretations and their impact on the assessment of hypoxia as well as hypoxia-targeted treatment planning.

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.

Tumor hypoxia is a hallmark of solid malignant tumor growth, profoundly influences malignant progression and contributes to the development of therapeutic resistance. Pathogenesis of tumor hypoxia is multifactorial, with contributions from both acute and chronic factors. Spatial distribution of hypoxia within tumors is markedly heterogeneous and often changes over time, e.g., during a course of radiotherapy. Substantial changes in the oxygenation status can occur within the distance of a few cell layers, explaining the inability of currently used molecular imaging techniques to adequately assess this crucial trait. Due to the possible importance of tumor hypoxia for clinical decision-making, there is a great demand for molecular tools which may provide the necessary resolution down to the single cell level. Exogenous and endogenous markers of tumor hypoxia have been investigated for this purpose. Their potential use may be greatly enhanced by multiparametric in situ methods in experimental and human tumor tissue.

Molecular imaging of tissue hypoxia generates contrast in hypoxic areas by applying hypoxia-specific tracers in organisms. In cancer tissue, the injected tracer needs to be transported over relatively long distances and accumulates slowly in hypoxic regions. Thus, the signal-to-background ratio of hypoxia imaging is very small and a non-specific accumulation may suppress the real hypoxia-specific signals. In addition, the heterogeneous tumor microenvironment makes the assessment of the tissue oxygenation status more challenging. In this study, the diffusion potential of oxygen and of a hypoxia tracer for 4 different hypoxia subtypes: ischemic acute hypoxia, hypoxemic acute hypoxia, diffusion-limited chronic hypoxia and anemic chronic hypoxia are theoretically assessed. In particular, a reaction-diffusion equation is introduced to quantitatively analyze the interstitial diffusion of the hypoxia tracer [(18)F]FMISO. Imaging analysis strategies are explored based on reaction-diffusion simulations. For hypoxia imaging of low signal-to-background ratio, pharmacokinetic modelling has advantages to extract underlying specific binding signals from non-specific background signals and to improve the assessment of tumor oxygenation. Different pharmacokinetic models are evaluated for the analysis of the hypoxia tracer [(18)F]FMISO and optimal analysis model were identified accordingly. The improvements by model-based methods for the estimation of tumor oxygenation are in agreement with experimental data. The computational modelling offers a tool to explore molecular imaging of hypoxia and pharmacokinetic modelling is encouraged to be employed in the corresponding data analysis.

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.

Heterogeneity is a feature of both normal oxygen supply to tissue and of a supply that is disturbed due to a wide range of different pathologies. Here, the physiological importance of heterogeneity of tissue oxygenation is revisited. The anatomical and functional basis for heterogeneity of blood flow, local and regional regulatory mechanisms in normal tissues and the pathophysiology of the failure of regulation will be examined.Under physiological conditions, regulation of blood flow distributions at global, regional and microregional levels play coordinated roles in ensuring adequate O2 supply to all tissue cells. How this is achieved may be organ-/organ layer-specific, depending on its function and priorities to match local O2 delivery to consumption. Examples where these regulatory mechanisms break down under conditions of ischaemia and shock will also be given.In contrast, pathologic heterogeneity in tissue oxygenation resulting from uncontrolled, chaotic growth as seen in malignant tumours represents a pathophysiological status that is not predictable which, in general, is associated with chronic and acute hypoxia. This can have fatal consequences due to hypoxia- induced (mal-)adaptive processes, malignant tumour progression and treatment resistance.

Hypoxia is a hallmark of tumors leading to (mal-)adaptive processes, development of aggressive phenotypes and treatment resistance. Based on underlying mechanisms and their duration, two main types of hypoxia have been identified, coexisting with complex spatial and temporal heterogeneities. Chronic hypoxia is mainly caused by diffusion limitations due to enlarged diffusion distances and adverse diffusion geometries (e.g., concurrent vs. countercurrent microvessels, Krogh- vs. Hill-type diffusion geometry) and, to a lesser extent, by hypoxemia (e.g., in anemic patients, HbCO formation in heavy smokers), and a compromised perfusion or flow stop (e.g., due to disturbed Starling forces or intratumor solid stress). Acute hypoxia mainly results from transient disruptions in perfusion (e.g., vascular occlusion by cell aggregates), fluctuating red blood cell fluxes or short-term contractions of the interstitial matrix. In each of these hypoxia subtypes oxygen supply is critically reduced, but perfusion-dependent nutrient supply, waste removal, delivery of anticancer or diagnostic agents, and repair competence can be impaired or may not be affected. This detailed differentiation of tumor hypoxia may impact on our understanding of tumor biology and may aid in the development of novel treatment strategies, tumor detection by imaging and tumor targeting, and is thus of great clinical relevance.

Since 1970, the multifactorial pathogenesis of the deficient and heterogeneous oxygenation of transplanted murine tumors and of human cancers (including parameters determining oxygen delivery, e.g., blood flow, diffusion geometry, oxygen transport capacity of the blood) has been investigated in vivo. Hypoxia and/or anoxia was quantitatively assessed and characterized using microtechniques and special preclinical tumor models. Hypoxia subtypes were identified, and critical supply conditions were theoretically analyzed. In the 1980s, first experiments on humans were carried out in cancers of the rectum and of the oral cavity. In the 1990s, the clinical investigations were carried out on cancers of the breast and of the uterine cervix, clearly showing that hypoxia is a hallmark of locally advanced human tumors. In multivariate analysis, hypoxia was found to be an independent, adverse prognostic factor for patient survival due to hypoxia-driven malignant progression and hypoxia-associated resistance to anticancer therapy.

Hypoxia is a characteristic of many solid tumors, can lead to the development of an aggressive phenotype and acquired treatment resistance, and is an independent, adverse prognostic indicator. In this literature review, we show that hypoxia is also a typical feature in prostate cancer (PC), the most commonly diagnosed cancer among men in most western countries. Data on blood flow (a major determinant of oxygenation status in malignancies) and on the oxygenation status (as assessed by O(2)-sensitive electrodes) are presented. Where possible, data on prostate cancers are compared to normal prostate (NP) tissue and benign prostate hyperplasia (BPH). The average blood flow rate in NP is 0.21 vs. 0.28 mL/g/min in BPH. Blood flow in PC is approximately three times higher than in NP (mean flow: 0.64 mL/g/min) and shows pronounced intra- and inter-tumor variability. Despite relatively high flow rates in PC, the overall mean pO(2) in cancers is 6 mmHg compared to 26 mmHg in NP. As was the case with blood flow, tissue oxygenation was extremely heterogeneous with no clear dependency on a series of tumor (Gleason score, clinical size, androgen deprivation) and patient characteristics (serum PSA levels, age).

Contrary to conventional belief, the mitochondria of most cancer cells usually function normally, i.e., their respiratory capacity is not fundamentally impaired as compared to normal cells. Strong evidence against the misconception of mitochondrial dysfunction is provided by in vivo data clearly showing that O(2) availability is the major determinant of the O(2) consumption rate of cancer cells, independent of the means for increasing availability (e.g., by increasing blood flow or by elevating arterial O(2) content, the latter being accomplished either by an increase in the hemoglobin level and/or arterial hyperoxia). Additional support against the Warburg effect in its original concept comes from normal temperature coefficients (Q(10)) for O(2) consumption rates of malignant cells. Thus, the Warburg hypothesis postulating that mitochondrial dysfunction in cancer cells forces them to generate energy with a poor ATP yield through glycolysis appears to be elusive. Instead, due to a "reprogrammed" cancer cell metabolism, glycolysis is used to produce intermediates as building blocks for various biosynthetic pathways of cancer cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Heterogeneously distributed hypoxia is a major characteristic of solid tumors. (Immuno-)fluorescence detection of hypoxia in experimental tumors is frequently assessed in a single central section; however, this may not necessarily be representative of the whole tumor. In order to determine whether analysis of one central section is exemplary of the whole tumor and whether different volumes have an impact on tumor oxygenation, we assessed the fractions of total (TH), chronic (CH), and acute hypoxia (AH) throughout different layers of tumors of varying volumes., Materials and Methods: Xenografted FaDu human squamous cell carcinomas of different volumes were investigated for intra- and intertumor heterogeneities. Tissue blocks located at the apical, central, and basal layer were sliced from individual tumors. Four serial cryosections were analyzed from each tissue block. Vital tumor tissue was explored for the distribution of Hoechst 33342 (perfusion), pimonidazole (hypoxia), and CD31 (endothelium) to assess TH, CH, and AH., Results: Fractions of TH, CH, and AH were consistently similar in the serial sections of individual tissue blocks. However, significant differences were found between the apical, central, and basal blocks that were even opposite depending on the tumor volume. Pooled data from all three tissue blocks revealed significantly higher fractions of hypoxia in the large tumors than in the small tumors., Conclusion: FaDu tumors exhibit a heterogeneous and volume-dependent oxygenation status. Assessing the average fractions of TH, CH, and AH from central blocks corresponds best to the average of the entire tumor. However, information on intratumor heterogeneities is lost, especially when considering tumors of substantially different volumes.

Background: Many tumors contain hypoxic regions. Hypoxia, in turn, is known to increase aggressiveness and to be associated with treatment resistance. The two most frequently described and investigated subtypes of tumor hypoxia are acute and chronic. These two subtypes can lead to completely different hypoxia-related responses within the tumor, which could have a direct effect on tumor development and response to treatment. In order to accurately assess the specific biological consequences, it is important to understand which time frames best define acute and chronic hypoxia., Materials and Methods: This article provides an overview of the kinetics of in vitro and in vivo acute and chronic tumor hypoxia. Special attention was paid to differentiate between methods to detect spontaneous in vivo hypoxia and to describe the biological effects of experimental in vitro and in vivo acute and chronic tumor hypoxia., Results and Conclusions: There are large variations in reported spontaneous fluctuations in acute hypoxia that are dependent on the cell lines investigated and the detection method used. In addition to differing hypoxia levels, exposure times used to induce in vitro and in vivo experimental acute and chronic hypoxia range from 30 min to several weeks with no clear boundaries separating the two. Evaluation of the biological consequences of each hypoxia subtype revealed a general trend that acute hypoxia leads to a more aggressive phenotype. Importantly, more information on the occurrence of acute and chronic hypoxia in human tumors is needed to help our understanding of the clinical consequences.

Development of molecular imaging such as positron-emission tomography (PET) offers an opportunity to optimize radiotherapy treatment planning by conforming the dose distribution to physiological details within tumors, so called dose painting. Quantification of the acquired images and an efficient and practical dose prescription remain two key questions in this field. This paper proposes a novel framework to optimize the dose prescription based on dual-pass modeling of dynamic [18F]FMISO PET images. An optimization algorithm for sparse dose painting (SDP) is developed by minimizing a linear combination of two terms corresponding to the efficiency and total variation of the dose distribution with the constraint of a constant mean dose. Dose efficiency is defined using the linear-quadratic model. The radiosensitivity given by the oxygen tension is estimated using a dual-pass kinetic-oxygen mapping strategy. This is achieved by integrating a realistic [18F]FMISO PET imaging simulation model, which can simulate the distribution of oxygen and tracer under the same tumor microenvironment setting. The algorithm was compared with a typical dose painting by number (DPBN) method in one data set of a patient with head and neck cancer.

In malignant urogenital tumors, tissue oxygenation is compromised and very heterogeneous,with steep and fluctuating spatio-temporal oxygen gradients signaling a complex instability in tumor oxygenation (complex "4D-heterogeneity"). Tumor hypoxia is highly dynamic, and rapidly changing pO(2) gradients may be key factors driving hypoxia-dependent adaptive processes leading to malignant progression. The grand median oxygen tension in malignant urogenital tumors is 7-11 mmHg. In contrast, benign leiomyomas of the uterus are severely, but uniformly, hypoxic with only shallow oxygen gradients ("static hypoxia"). In these benign tumors, the median pO(2) is 1 mmHg and signs of hypoxia-driven processes are missing.

Based on studies performed with a selected mouse monoclonal anti-transketolase- like (TKTL)-1 antibody (clone JFC12T10), overexpression of TKTL-1 has been shown to be correlated with poor survival and increased metastatic spread in several human tumor entities. Since the clinical aggressiveness mediated by TKTL-1 has been partially related to resistance to hypoxia,we originally aimed to explore the influence of hypoxia on the expression of TKTL-1. Unexpectedly, results of our experiments indicated that the antibody clone JFC12T10 lacks target specificity. Since the majority of data on the role of TKTL-1 in human cancer is based upon studies performed with this antibody clone, we subsequently re-evaluated the expression of TKTL-1 in six different cancer cell lines (HeLa, MCF-7, A549, HT-1080, M21 and TF-1). Using RT-PCR and consecutive sequence analysis, we show that transketolase (TKT), not TKTL-1, is the dominant isoform of transketolases in the cell lines analyzed. Our data argue against a major role of TKTL-1 for the metabolism of cancer cells.

This study has attempted to address the controversy concerning sustained increases in tumor oxygenation upon localized mild hyperthermia. While some previous studies have reported transient increases, others have reported persistent increases in tumor oxygenation, lasting for upto 2 days after application of mild hyperthermia. In order to determine changes in oxygenation at clinically relevant tumor temperatures, experimental tumors in rats underwent localized hyperthermia at either 40, 41.8 degrees C or 43 degrees C for 1 h using water-filtered infrared-A irradiation. Oxygenation was continuously measured before, during and upto 60 min after hyperthermia in the tumors of anesthetized rats using oxygen-sensitive catheters. The data obtained indicate that localized hyperthermia can lead, on average to an improved tumor oxygenation, although this improvement is generally transient and no longer evident 1 h after heating. Since clinically relevant increases in oxygenation enduring beyond the heating period were rarely seen, it would appear that an improvement in the efficacy of oxygen-dependent cancer therapy is unlikely to be achieved in the post-hyperthermia period.

Hypoxia, a characteristic feature of locally advanced solid tumors, has emerged as a key factor of the tumor pathophysiome, since it can promote tumor progression and resistance to therapy. Independent of established prognostic parameters, such as clinical tumor stage, histology, histological grade and nodal status, hypoxia has been identified as an adverse prognostic factor for patient outcome. Studies of pretreatment tumor hypoxia involving direct assessment (polarographic oxygen tension measurements) have suggested a poor prognosis for patients with hypoxic tumors. These investigations indicate a worse disease-free survival for patients with hypoxic cancers of the uterine cervix or soft tissue sarcomas. In head & neck cancers, the studies suggest that pretherapeutic hypoxia is prognostic for survival and local control.

Hypoxia is now established as a key factor influencing the pathophysiology of malignant growth. Among other effects, hypoxia modulates the expression of a multitude of genes through the induction of hypoxia-inducible transcription factors. This differential gene expression favors angiogenesis, cell survival, an invasive/metastatic phenotype, and resistance to anticancer therapies. Because benign tumors do not exhibit these traits, one might expect these entities to be neither hypoxic nor to induce the genetic hypoxia response program. To test this hypothesis, an investigation of the oxygenation status of 17 leiomyomas and 1 leiomyosarcoma of the uterus using polarographic needle electrodes (Eppendorf pO(2) sensor) and the expression of hypoxia-related markers in biopsy specimens of the same tumors was carried out. Marker expression in eight additional archival leiomyosarcomas was also assessed. Leiomyoma tissue was generally found to be severely hypoxic, with median oxygen (O(2)) partial pressure values ranging from 1 to 5 mm Hg. In contrast, none of the hypoxia-related markers hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, glucose transporter-1, or carbonic anhydrase IX were expressed in any leiomyoma. Larger intercapillary distances were correlated with a poorer oxygenation status. Conversely, the expression of hypoxia-related markers was abundant in the leiomyosarcomas and they also exhibited a high-turnover phenotype (significantly increased proliferation and apoptosis). Uterine leiomyoma might therefore represent a state of oxygen-limited proliferation. Malignancy in the same organ system is associated with growth and metabolism beyond tissue-inherent limitations leading to the induction of hypoxia-related markers, thereby contributing to a self-perpetuating aggressive phenotype.

Tumor hypoxia, mostly resulting from poor perfusion and anemia, is one of the key factors in inducing the development of cell clones with an aggressive and treatment-resistant phenotype that leads to rapid progression and poor prognosis. Studies in patients with solid tumors suggest that there is a range of hemoglobin (Hb) concentrations that is optimum for tumor oxygenation. When used to achieve an Hb level within this range, erythropoiesis-stimulating agents (ESAs) can be expected to increase tumor oxygenation, and this may favorably influence sensitivity to treatment as well as quality of life. There is no robust evidence that ESAs, when used as indicated, have a negative effect on survival in patients with solid tumors. When used outside the indications recommended, the rise in Hb level that results may reduce tumor blood flow and tissue oxygenation because of a raised viscosity within the abnormal tumor microvasculature. In the current situation, it remains important to use ESAs within the approved indications and according to treatment guidelines such as those developed by the European Organization for Research and Treatment of Cancer.

Data from 125 studies describing the pretreatment oxygenation status as measured in the clinical setting using the computerized Eppendorf pO2 histography system have been compiled in this article. Tumor oxygenation is heterogeneous and severely compromised as compared to normal tissue. Hypoxia results from inadequate perfusion and diffusion within tumors and from a reduced O2 transport capacity in anemic patients. The development of tumor hypoxia is independent of a series of relevant tumor characteristics (e.g., clinical size, stage, histology, and grade) and various patient demographics. Overall median pO2 in cancers of the uterine cervix, head and neck, and breast is 10 mm Hg with the overall hypoxic fraction (pO2

Purpose: The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable., Results: Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques., Conclusions: This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

Background: Tumor hypoxia is considered to be relevant for several aspects of tumor pathophysiology, for acquired treatment resistance, and tumor progression (e. g., in cancers of the uterine cervix). Therefore, there is a demand for simple and universally applicable methods allowing the estimation of oxygenation status in patient material obtained during pretherapeutic diagnostic procedures (biopsies) or surgical treatment. Protein members of the transcriptional response to hypoxia expressed in tumor tissue, e. g., hypoxia-inducible factor-1alpha (HIF-1alpha), glucose transporter-1 (GLUT-1) and carbonic anhydrase IX (CA IX) are currently being discussed as "endogenous hypoxia markers"., Material and Methods: The (hypothetic) suitability of different markers for the assessment of the oxygenation status is reviewed on the basis of current knowledge., Results: Data from studies investigating the suitability of different markers are conflicting. Although a robust induction of HIF-1alpha, GLUT-1 and CA IX by hypoxia has been demonstrated in vitro, this reaction is modulated both by confounding factors of the tumor microenvironment and intrinsic traits of malignant cells in vivo., Conclusion: On the basis of the available data, the suitability of "endogenous hypoxia markers" for the estimation of the oxygenation status of advanced cervix cancers seems questionable.

Background and Purpose: Tumor hypoxia has been linked to tumor progression, the development of treatment resistance, and thus poor prognosis. Since anemia is a major factor causing tumor hypoxia, the association between blood hemoglobin concentration (cHb) and tumor oxygenation status has been examined., Patients and Methods: Published data on the relationship between pretreatment cHb values and tumor oxygenation (in terms of median pO(2) values, hypoxic fractions) have been summarized. Pretreatment O(2) tension measurements were performed in histologically proven experimental tumors, human breast cancers, squamous cell carcinomas of the head and neck, and cancers of the uterine cervix and of the vulva. In order to allow for a comparison between solid tumors and normal tissues, pO(2) measurements were also performed in healthy tissue in anemic and nonanemic patients. cHb was determined at the time of the pO(2) measurements., Results: Based on current information from experimental and clinical studies there is increasing evidence that anemia is associated with a detrimental tumor oxygenation status. Increasing cHb values are correlated with significantly higher pO(2) values and lower hypoxic fractions. Maximum tumor oxygenation in squamous cell carcinomas is observed at normal (gender-specific) cHb values (approximately 14 g/dl in women and approximately 15 g/dl in men). Above this "optimal" Hb range, the oxygenation status tends to worsen again. In anemic patients, tumor oxygenation is compromised due to a decreased O(2) transport capacity of the blood. At the upper edge of the Hb scale, a substantial increase in the blood's viscous resistance to flow in "chaotic" tumor microvessels is thought to be mainly responsible for the observed restriction of O(2) supply., Conclusion: Review of relevant clinical data suggests that a maximum oxygenation status in solid tumors is to be expected in the range 12 g/dl < cHb < 14 g/dl for women and 13 g/dl < cHb < 15 g/dl for men. Considering the "optimal" cHb range with regard to tumor oxygenation, the concept of "the higher, the better" is therefore no longer valid. This finding has potentially far-reaching implications in the clinical setting (e. g., inappropriate erythropoietin treatment of nonanemic tumor patients).

Introduction: Cancers of the vulva are relatively rare and, therefore, little is known about the pathophysiological role of tumor oxygenation in this entity., Methods: Data are presented on the oxygenation status of primary (n = 15) and recurrent (n = 19) cancers of the vulva, as measured by the Eppendorf pO2 histography system., Results: Contrary to other tumor entities, no significant differences in the oxygenation status between primary (median pO2 = 13 mmHg; hypoxic fraction < or = 5 mmHg = 37%) and recurrent (median pO2 = 11 mmHg; hypoxic fraction < or = 5 mmHg = 45%) tumors were found. Oxygenation was significantly lower in cancers of the vulva than in the subcutis. Anemic patients had significantly poorer tumor oxygenation compared with patients whose cHb values were within the normal range (p = 0.02)., Conclusions: The oxygenation of vulvar cancers is similar to other tumor entities, but does not show more severe hypoxia in recurrent cases. Anemia is associated with a poorer oxygenation status in vulvar cancers, whereas in the normal tissue no impact of cHb values on the median pO2 was observed.

Background: Anemic tissue hypoxia can decrease the effects of chemotherapy in bladder cancer. Hypoxia leads to overexpression of hypoxia-inducible factor (HIF) and increased synthesis of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Tumor cell survival, invasion and angiogenesis thereby compromise treatment., Materials and Methods: Monolayer cultures of RT112, RT4, T24 and TCC SUP cells were incubated with or without gemcitabine and different gas mixtures (hypoxia, normoxia or hyperoxia). Cell proliferation (microculture tetrazolium assay), VEGF (enzyme-linked immunosorbent assay) and HIF-1alpha (Western blot analysis) were determined., Results: Hypoxia led to increased proliferation of transitional cell carcinoma (TCC) cells and elevated levels of HIF-1alpha and VEGF. Hyperoxia inhibited cell growth and lowered the concentration of VEGF. Treatment with gemcitabine was less effective under hypoxia., Conclusion: Hypoxia enhances TCC growth and may intensify angiogenesis mediated by VEGF. Hypoxia compromises treatment with gemcitabine. Correction of anemia might provide advantages in chemotherapeutic strategies for TCC.

Purpose: Glucose transporter-1 (GLUT-1), a target gene of hypoxia-inducible factor-1, has been considered a candidate endogenous marker of tumor hypoxia. Expression of GLUT-1 may also serve as an indicator for the induction of the transcriptional response to hypoxia, which has been linked to enhanced proliferation, resistance to therapy, and metastatic propagation of cancer cells. Overexpression of GLUT-1 has been shown to correlate with poor prognosis in several tumor entities, among them cancers of the uterine cervix. The validity of these hypotheses is investigated., Experimental Design: The expression of GLUT-1 was assessed in 80 biopsies of Eppendorf oxygenation measurement tracks from locally advanced cervical cancers in 47 patients using immunohistochemistry., Results: No correlation was found between the expression of GLUT-1 and oxygenation variables (median pO2, HF 2.5 and HF 5). Expression of GLUT-1 was found greater in larger tumors (P = 0.0001) and to exhibit a linear increase with Federation Internationale de Gynecologie et d' Obstetrique stage (P = 0.002). Overall survival (P = 0.004) and recurrence-free survival (P = 0.007) were significantly shorter for patients with expression of GLUT-1. In the subgroup of patients treated with surgery, this effect on prognosis was not independent when pT stage or pN stage were included in a multivariate Cox proportional hazards model., Conclusions: The suitability of GLUT-1 as an endogenous marker of tumor hypoxia seems questionable. The association with prognosis may partially depend on confounding factors.

Heterogeneously distributed hypoxic areas are a characteristic property of locally advanced breast cancers. Hypoxia results from an imbalance between the supply and consumption of oxygen (O2). Major pathogenetic mechanisms for the emergence of hypoxia are (i) structural and functional abnormalities in the tumor microvasculature, (ii) an adverse diffusion geometry, and (iii) tumor-related and therapy-induced anemia leading to a reduced O2 transport capacity of the blood. There is pronounced intertumor variability in the extent of hypoxia, which is independent of clinical size, stage, histology and grade. Hypoxia is intensified in anemic patients, especially in tumor (areas) with low perfusion rates. Tumor hypoxia is a therapeutic problem since it makes solid tumors resistant to sparsely ionizing radiation, some forms of chemotherapy, and photodynamic therapy. However, besides more direct mechanisms involved in the development of therapeutic resistance, there are, in addition, indirect machineries that can cause barriers to therapies. These include hypoxia-mediated alterations in gene expression, proteomic and genomic changes, and clonal selection. These in turn can drive subsequent events that are known to further increase resistance to therapy in addition to critically affecting long-term prognosis.

Hypoxia inducible factor-1alpha (HIF-1alpha) has been proposed as a candidate endogenous marker of tumor hypoxia and as a molecular mediator of hypoxia-driven malignant progression and acquired treatment resistance. In this study, HIF-1alpha expression in 68 biopsies of oxygenation measurement tracks from squamous cell carcinomas of the uterine cervix of 38 patients was assessed. Expression of HIF-1alpha was commonly found to increase as a function of distance from microvessels, at the center of tumor cell aggregations, and in the vicinity of necrotic areas. However, there was no correlation of HIF-1alpha expression with median oxygen tension (oxygen partial pressure; pO2) and hypoxic fractions (hypoxic fraction < 2.5 mm Hg, hypoxic fraction < 5 mm Hg). The results indicate that HIF-1alpha should not be used as an endogenous marker of tumor hypoxia in locally advanced squamous cell carcinomas of the uterine cervix. Additionally, no significant prognostic impact of HIF-1alpha expression was found in this group of patients.

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.

Tumor hypoxia has been linked to acquired treatment resistance, tumor progression, and poor prognosis. Because anemia is a major causative factor for the development of hypoxia, the association between blood hemoglobin concentration (cHb) and breast cancer oxygenation was examined in this study. In addition, a novel parameter characterizing the relationship between oxygenation status and rising cHb is introduced: the oxygenation gain factor (OGF). In breast cancer patients, median cHb over the range 8.5-14.7 g/dl correlated positively with the median pO(2) (3-15 mm Hg), yielding an average OGF of 2 mm Hg.dl/g. In contrast, in normal tissues (normal breast, subcutis, and skeletal muscle) the median pO(2) values were substantially higher (52 mm Hg, 51 mm Hg, and 37 mm Hg, respectively) and remained constant irrespective of the hemoglobin level over the range from 10 to 16 g/dl (OGF = 0 in grade I anemia and nonanemic patients). Moderately lower median pO(2) values in subcutis and skeletal muscle were only observed in grade II anemia (8 g/dl < cHb < or =10 g/dl), although this would appear to be of no biological relevance. Conversely, in breast cancers, even mild anemia (grade I anemia) is a major causative factor for the development of hypoxia or anoxia.

Background and Purpose: To date, no information is available concerning the impact of spinal anesthesia on the oxygenation status of carcinomas of the uterine cervix. The aim of this study was therefore to determine the influence of spinal anesthesia on the oxygenation status of cervical carcinomas., Patients and Methods: In ten patients with cervical carcinoma who received spinal anesthesia for a first application of brachytherapy, intratumoral pO2 measurements (pO2 histography system, Eppendorf-Netheler-Hinz, Hamburg, Germany) were performed. Systemic parameters were documented prior to and during spinal anesthesia. Patients breathed room air spontaneously. For further evaluation, all intratumoral pO2 values were pooled, and overall median pO2 values and fractions of hypoxic pO2 values < or = 5 mmHg were calculated. Overall median pO2 values in the subcutis were also calculated., Results: There were no significant changes of systemic parameters, median subcutaneous pO2 values, median intratumoral pO2 values, and the fractions of hypoxic pO2 values < or = 5 mmHg in the tumor upon administration of spinal anesthesia. The variability of measured pO2 values increased during spinal anesthesia, although substantial changes in the oxygenation status were only seen in individual cases (n = 2)., Conclusion: This study shows for the first time that the oxygenation status of cervical carcinomas, in general, is not influenced by spinal anesthesia prior to application of brachytherapy. To conclude, the data presented suggest that reliable pO2 measurements can be performed under spinal anesthesia. At the same time, since no substantial changes in tumor oxygenation were observed, spinal anesthesia should not affect the O2-related efficacy of high-dose-rate brachytherapy.

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.