Abstract
Tumor oxygenation status is an independent prognostic indicator in cancer because it influences tumor progression and treatment outcome. Its quantitative value is determined by a number of tumor vascular parameters such as microvascular density, blood flow, blood volume, blood oxygen saturation, tumor tissue pO₂, and resistance to oxygen diffusion within the tumor. Over the past several years, considerable time and effort have been invested into developing techniques to effectively and reliably measure the oxygenation status of a tumor. The measurement and interpretation of data obtained with currently available methods is complicated by the heterogeneity in tumor oxygenation. Currently available techniques can be broadly classified into direct invasive methods, direct non-invasive methods, and measurement of surrogate endogenous markers of tumor oxygenation. Of these methods, the Eppendorf pO₂ histograph is considered the "gold standard" and even so has several limitations. Given the importance of tumor oxygenation status in therapy and in predicting disease progression, it is imperative that reliable, globally usable, and technically simplistic methods be developed to yield a consistent, comprehensive, and reliable profile of tumor oxygenation. Until newer more reliable techniques are developed, existing independent techniques or appropriate combinations of techniques should be optimized and validated using known endpoints in tumor oxygenation status and/or treatment outcomes.

Keywords: Tumor oxygenation status, prognostic marker, oxygen measurement techniques; validation and optimization.

1. Introduction
Tumor oxygen status is determined by oxygen consumption rate of tumor cells and oxygen supply to tumor tissue. The latter is influenced by a variety of tumor vascular properties which include microvascular density of functional vessels, tumor blood volume, blood flow, blood oxygen saturation, tumor tissue pO₂, and resistance to oxygen diffusion within the tumor. The physiological characteristics of solid tumors differ from those of normal tissue in a number of significant ways. While tumors are known to be more hypoxic and acidic than normal tissue [1], there are several important differences between the two vasculatures as well. Normal tissues have a predictable pattern of blood vessels while tumors are known to have highly abnormal, distended capillaries with leaky endothelial linings and sluggish blood flow [2]. Oxygen supply to the tumor is, therefore, limited by local structural and functional abnormalities in the tumor microcirculation including the transient nature of perfusing and non-perfusing vessels within a tumor, often resulting in perfusion-limited oxygen supply [3]. The size and geometry of the tumor in relation to its functional microvascular architecture frequently leads to diffusion-limited oxygen delivery to tumor tissue that is distal from the blood vessels [3]. Taken together, the above factors contribute to the two main characteristics of tumors with respect to their oxygenation status: first, since supply of oxygen may not always meet the demand, tumors are generally more hypoxic (= 20 mm Hg) [4] than surrounding normal tissue (˜ 24-66 mm Hg) [4] and second, there is temporal and spatial heterogeneity in tissue oxygenation within and between tumors of even the same type [5].

Tumor oxygenation status, important as it is, is an independent prognostic marker because reduced oxygen levels in tumor tissues induces a transcription program that promotes an aggressive tumor phenotype [6-10] that is mostly a consequence of the upregulation of hypoxia inducible factor protein in tumor cells [11, 12]. During the process of hypoxia-driven malignant progression, tumors develop an increased potential for local invasive growth [13], [14], perifocal tumor cell spreading [6, 15], and regional and distant metastases [7, 8, 16-18] . While primary tumors are quite hypoxic, metastatic lesions are similar or even more hypoxic [19]. In recurrent tumors, there is generally a higher proportion of the tumor that is hypoxic when compared with the corresponding primary tumor [19]. Chronic hypoxia is known to increase genomic instability through increased point mutations, chromosomal rearrangements and amplifications [3] and is known to promote the generation and selection of genetic variants that are better adapted to survive the hypoxic environment. Tumor hypoxia is also associated with resistance to radiation therapy [20], chemotherapy [21], photodynamic therapy [22], immunotherapy [23] and even hyperthermia [24]. Since it has been established that the oxygenation status of tumors cannot be predicted by clinical size, stage, grade, histology or site [3], it naturally follows that if we could accurately measure the oxygenation status of individual tumors, we could better predict treatment outcomes and even be able to select individualized treatments such as oxygen enhancement therapies for patients with solid tumors.

However, there are many obstacles to obtaining a reliable measurement of tumor oxygenation status. A number of independent direct and indirect, invasive and non-invasive methods have been used for measuring tumor oxygenation. It appears that an appropriate combination of these methods that could provide validation of the measurements will be more useful for prognosis and prediction of the tumor's response to treatment than any one currently available technique. In this review, the presently available methodologies for measuring oxygenation in tumors will be discussed, as well as some of the limitations and challenges to obtaining a comprehensive, consistent, and reliable profile of tumor oxygen status in solid tumors in the clinic. If and when the hurdles are overcome, a reliable measurement of tumor oxygenation status could potentially provide critical information for prognosis and treatment.

2. Direct invasive methods for measuring tumor oxygenation
The polarographic pO₂ electrode has been considered the "gold standard" for measuring tumor oxygen tension [25] although this has been questioned recently [26]. Measurements with the polarographic needle electrode probe are made by rapidly stepping a 300 micron needle electrode through the tissue, thus providing a micro-global assessment of tumor oxygenation. This technology has been utilized for several decades and was the only technique that could directly measure tumor tissue pO₂. However, in spite of the relative ease with which the device can be used, there are a number of important problems that need to be addressed. First, a study on the inter-laboratory variation in oxygen tension measurement using the polarographic histograph showed that while the data could be useful, the median pO₂ values varied widely among the different laboratories using the same tumor model [27]. There were also significant differences in computed hypoxic fractions between laboratories [27]. The needle electrode cannot measure oxygenation in a given microregion over time because it utilizes oxygen in the measurement process. Furthermore, Buerk [28] discusses some of the advantages for using smaller recessed pO₂ microelectrodes (5 to 10 microns in diameter) to make tissue pO₂ measurements. The potential for tissue damage by the polarographic needle is a problem particularly for tumors that are relatively small. Miniaturized electrochemical sensors have two major advantages compared to larger electrodes: faster time responses and finer spatial resolution. Miniaturization can also decrease tissue damage and microcirculatory disturbances by the microsensor, decreasing possible distortion of the physiological measurement [28]. With the much larger dimensions of the polarographic pO₂ electrode, the most likely explanation for the differences in tumor tissue pO₂ distributions is the greater probability for damage to capillaries and small blood vessels compared to damage that might be caused by the smaller tip of the recessed pO₂ microelectrodes. Although multiple sampling sites with the recessed microelectrode may allow the best direct assessment of tumor oxygenation without disruption of the microvasculature, pO₂ measurements using the microelectrode cannot be used clinically in its present form and will need to be further developed before it can be used in patients. This suggests that there is still a persisting requirement for valid and reliable oxygen-sensing probes which combine the advantages of the two types of electrodes. Recently, a fibre-optic based system has been developed with which measurements are made based on pO₂-dependent changes in half-life of an excited luminophore at the tip of the probe [29]. This process does not consume oxygen, but relies solely on the presence of oxygen. The fibre-optic probe can be left at one position for a longer period of time, which makes it possible to measure both temporal pO₂-changes as well as the effect of tissue oxygen modifying agents such as carbogen [29].

Another approach that has received a great deal of attention in recent years has been the use of fluorescent [30] and radiolabeled bioreductive compounds [31] to mark hypoxic areas within the tumor. The observation that the nitroimidazole compound, misonidazole was preferentially metabolized in oxygen deficient cells and then covalently bound to the macromolecules in those cells by the cell-specific action of cellular nitroreductases such as cytochrome P-450 and cytochrome P-450 reductases led to the utilization of such agents as important hypoxic markers. Since then a number of similar agents such as pimonidazole, EF5, NITP and CCl-103F, have been used in various studies [32] . Antibodies have been raised against these hypoxia markers and the hypoxic cell fraction is determined either by flow cytometry [33] or by image analysis of microscopic sections [34]. Accurate calibration of the marker is pivotal to obtaining reproducible and reliable results. A strength of this technique is that dead cells (necrosis) do not generate a signal because they do not metabolize these compounds and that a correction can be made for necrotic areas of tumors. Although the use of bioreductive hypoxic markers is being evaluated in clinical trials [35-38] such methods have their limitations when used as an independent method of estimating tumor oxygenation. First, the successful use of this technique usually requires an intravenous injection of the marker followed by a biopsy or invasive surgical procedure although more recently, the use of hypoxic markers have been adapted for positron emission tomography (PET) and single photon emission tomography (SPECT) [39]. Second, the methodology limits the study of hypoxia in tumors to one single point in time when in actuality the development of acute tumor hypoxia is, in part, a dynamic process and needs to be studied over time because of the transient nature of perfusing and non-perfusing vessels. The latter property of tumors also raises the question of whether it is reasonable to use a transiently perfusing system to distribute an exogenous hypoxia marker to evaluate tumor oxygenation. This limitation could be overcome by the use of a perfusion marker, but one is not currently available for clinical use. Third, the hypoxic markers are activated between 10 and 20 mm Hg [40, 41] while radiobiologic response falls precipitouslybelow 25 mm Hg and reduced cell death in conventional photodynamic therapy begins at 35 mm Hg [42]. Therefore, the use of hypoxic markers to predict treatment outcome can exclude the measurement of some physiologically relevant tumor tissue oxygen tensions that are outside the range of accurate measurement by these markers. Fourth, the above methodology excludes the possibility of making repeated measurements such as before and after therapy on the same tumor and lastly, since the cellular levels and/or levels of activity of the cytochrome P-450 reductases are important for the activation of the nitroheterocyclics, it is important to establish relationships between the levels of hypoxia, reductase activity and measurement of the bound marker.

3. Direct non-invasive methods for measuring tumor oxygenation
A number of cross-sectional non-invasive imaging techniques have also been recently developed. Contrast enhanced dynamic computerized tomography (CT)-determined tumor perfusion has been suggested as an independent predictor of local control in head-and-neck cancer [43, 44] and also in carcinoma of the cervix [45] treated by radiotherapy. A contrast agent bolus is rapidly injected intravenously while during the first pass a dynamic data acquisition is performed at the level of the largest axial tumor surface. The perfusion in the selected tumor region of interest is calculated by dividing the slope of the tumor-time density curve by the maximal value in arterial density. Primary and nodal tumor volume is calculated from the CT images. A known limitation of this technique, however, is that only one level through the tumor can be examined with the currently available technology and, therefore, the obtained results may not be representative of the entire tumor. Also, this technique does not lend itself well to examining known temporal heterogeneity of tumor perfusion because several hours are needed before the baseline tissue density is restored through renal clearance of the contrast medium [43]. Furthermore, as has been pointed out earlier, tumor perfusion alone cannot provide the most accurate estimate of tumor oxygenation.

Magnetic resonance imaging (MRI) techniques such as blood oxygen level-dependent MRI (BOLD MRI) [46] and dynamic contrast-enhanced MRI (DCE-MRI) [47] have been applied to inaccessible tumors to measure tumor oxygenation. BOLD MRI also referred to as flow and oxygenation dependent (FLOOD) MRI detects relative changes in tumor deoxyhemoglobin concentration and blood flow before and after an intervention. Increased FLOOD image intensity indicates a reduction in blood oxyhemoglobin concentration and an increase in tumor blood flow [48]. Since tumors have a chaotic and poorly regulated blood supply, and a mix of glycolytic and oxidative metabolism, the response to vasoactive agents is not predictable. Changes in blood volume can counteract the effect of blood oxygenation changes and changes in blood pH and glucose levels can alter oxygen unloading. This can lead to changes detected by FLOOD MRI that are smaller or even reverse of those expected [46]. In the last several years, DCE MRI has emerged as a promising method for measuring perfusion including blood flow and blood volume in tumor tissue. Much of the success of DCE MRI to date can be attributed to its ability to provide high resolution images of tumor that depict perfusion and permeability of the smallest vessels, especially the capillary network [47]. The same area of an organ or tissue is imaged before and several times after the administration of contrast medium. In this way it is possible to depict the rate of change in MRI signal intensity due to perfusion of the contrast medium over time with a time-intensity curve. The time-to-peak and the magnitude of enhancement can also be derived. However, the widespread use of DCE MRI is limited by the need for further technical improvements. First, DCE MRI requires flexible acquisition methods to permit a trade-off between temporal and spatial resolution. Quantitative DCE MRI, which is needed to permit widespread use, requires special purpose data acquisition techniques and analysis software not normally included by the instrument manufacturers [49]. For DCE MRI to be useful in a routine clinical setting, acquisition and processing must be straightforward providing a 3D representation of the relevant quantitative information [49]. Lastly, DCE MRI-derived tumor perfusion, blood flow and blood volume data, by itself, cannot provide a comprehensive picture of tumor oxygen status for reasons already discussed earlier.

Recently, electron paramagnetic resonance (EPR) spectroscopy and imaging techniques have been used to determine the redox and oxygenation status of tumors [50, 51]. Low molecular weight stable nitroxide free radicals have been used as probes for this purpose. Nitroxides occur in biological systems as a redox pair - the nitroxide free radical form and the diamagnetic hydroxylamine, which is the one-electron reduction product of the nitroxide free radical [52]. It has been shown that nitroxides are reduced to the corresponding hydroxylamine in vivo and that the rate of reduction is significantly increased under hypoxic conditions [53-57]. It is believed that the nitroxide accepts cellular reducing equivalents that would have been otherwise used for the reduction of molecular oxygen. Hence the nature of the nitroxide species in an EPR study would represent the redox and oxygenation status of any given tumor tissue [52]. The characteristics of EPR which appear to be potentially advantageous include the capability of making repeated measurements from the same site, specificity of the information in regard to the site at which the measurement is being made and the level of accuracy that is obtainable. However, although the paramagnetic materials that are likely to be used in human EPR studies such as nitroxides are unlikely to have significant toxicity, they have not yet been fully tested in human patients [58]. Also, with existing instrumentation, the non-invasive sensitivity of the method is restricted to 10 mm from the surface of the body. From a metabolic stand point, it is well known that glutathione plays a central role in the maintenance of enzyme sulphydryl groups in the reduced state [59]. Therefore, alterations of the GSH redox state within a tumor or differences in the amount of glutathione in different tumor types must be factored into the standardization of the EPR method for determining tumor oxygen status. Furthermore, the rate of infusion of the probe into the tumor, metabolic reduction of the probe within tumor tissue and the clearance of the probe from the tumor tissue by blood flow can all be confounding factors in accurately defining the tumor oxygen status using this method [52].

Oxygen dependent quenching of phosphorescence is another direct non-invasive optical method for measuring oxygen pressure in tissues [60-62]. This technique makes use of the ability of oxygen molecules to return from the excited triplet state to the ground state by absorbing energy which would otherwise present as phosphorescence. This quenching process requires collision between oxygen and the excited state molecule. Quenching efficiency is dependent on collisional frequency and the probability of energy transfer in any given collision. The oxygen pressure can be determined by measuring phosphorescence intensity or phosphorescence lifetime [63] although the latter method seems to be the preferred one. A phosphorimeter is used to make such measurements. Results are presented as oxygen histograms and represent dissolved oxygen in the plasma of blood from which blood oxygen saturation in tumor microvasculature is derived. This technique has very good temporal resolution. Because phosphors that are injected intravenously are not cleared readily and exhibit minimal toxicity, repeated measurements can be made. However, the phosphors have not been used in the clinic in human patients as yet and the technique is still being optimized for three-dimensional phosphorescence lifetime global imaging of tissues. One of the weaknesses of this technique is the difficulty in obtaining a good signal-to-noise-ratio. Since the tumor has a relatively leakier blood vascular system than normal tissue, more tests are required to ensure that tumor specific vascular properties will not interfere with the obtaining of reliable tumor oxygenation profiles.

Relative blood flow and blood oxygen saturation can also be measured non-invasively in preclinical and human tissues by diffuse light spectroscopy utilizing a combination of two near-infrared diffuse optical techniques [64]. Briefly, diffuse correlation spectroscopy (or flowmetry) has been used to determine relative blood flow; this method essentially measures the optical phase-shifts caused by moving blood cells. Near-infrared (NIR) absorption spectroscopy has been used, concurrently, to measure tissue absorption at two different wavelengths. These measurements of diffuse light reflection enable the determination of the hemoglobin concentration and blood oxygen saturation of the same tumor tissue probed simultaneously by the flow method. The instrument employs non-contacting sources of near-infrared light derived from laser diodes; source photons are delivered centrally into the tumors. Detection fibers are arranged around the central source fiber. The source-detector separation distance is fixed. The diffuse light transmitted through the tumors is captured by this radial distribution of light detectors. Blood flow is computed from the temporal decay of the diffuse light intensity temporal autocorrelation function. Typically this autocorrelation function decays exponentially. Its decay rate, G (sec-1), depends on a parameter a, proportional to the tissue blood volume fraction, and on the motion of blood cells. Depending on the physical model used, the decay rate is proportional to one of the following parameters which are related to the average motion of the red blood cells: a Brownian diffusion coefficient, DB (cm2/sec), or a mean-square flow velocity, V2 (cm/sec)2 [64]. It has been generally found that the autocorrelation function fits are better when using the model containing the Brownian diffusion coefficient, DB. Relative changes in DB correspond to relative changes in blood flow. The blood oxygen saturation is computed from the diffusely reflected light signals at 830 nm (oxyHb peak) and at 750 nm (deoxyHb peak). However, this calculation assumes that the dominant absorbers at these wavelengths are oxyHb and deoxyHb, and that the smaller, water and lipid contributions to the signal, remain constant. Hence it is an approximation and not an absolute measurement of blood flow and blood oxygen saturation. The technique is also limited by light penetration and by the heterogeneous and not clearly understood optical properties of tissues in general.

Tumor blood flow and blood volume have also been determined by yet another non-invasive technique, sonography and Doppler ultrasound [65, 66]. Using this method, color Doppler and power Doppler images of tumors are obtained using ultrasound imaging systems. The flow indices, vascular density (VD), mean color level (MCL), and color-weighted fractional area (CWFA), are calculated for each color and power Doppler image. The physical meaning of the measurement VD represents the relative area of perfusion. The meaning of mean color level (MCL) varies depending on whether it is derived from color or power Doppler images. The MCL measurements from color Doppler images are a measure of mean velocity of local blood flow. The MCL measurements derived from power Doppler images represented the number of red blood cells moving above a threshold velocity. If one assumes local hematocrit in these small blood vessels to be equal to systemic hematocrit, power Doppler MCL can be related to blood volume (or, more appropriately related to the log of blood volume because the signals are often log compressed) moving above a threshold velocity. The relationship between MCL and physiological parameters (mean flow velocity and blood volume) should be viewed as semi-quantitative because of several variables: the angle between a blood vessel and the direction of ultrasound, the choice of scale maximum, filters and color write priority threshold, and the interpolation, averaging, and other image processing algorithms used internally within a scanner to display images for optimal viewing. However, if the imaging parameters are kept constant throughout the study the influence of these variables can be significantly reduced. Under these circumstances the data provides a meaningful comparison of the flow characteristics through two different tissue types or before and after treatment with various agents. Color-weighted fractional area (CWFA) combines the MCL and VD information. In the case of color Doppler, CWFA (flow mean velocity X area of perfusion) corresponds to the blood flow through the tumor. Similarly the color-weighted fractional area for power Doppler images (product of MCL and VD) is a measure of the "moving" blood volume within the tissue. Doppler ultrasound is good for making repeated measures before, after and also during some types of treatments. Some of the limitations of this technique are that usually only the larger blood vessels can be visualized and studied. Also, while this technique can be used for measuring blood flow and blood volume, it does not give much information on blood oxygen saturation which is an important factor in making any conclusions on tumor oxygenation status.

4. Surrogate endogenous markers of tumor oxygenation
Many independent studies have focussed their attention on tumor vascularization and have measured such parameters as intercapillary distance [67, 68], microvascular density [69, 70] and the distance from tumor cells to the nearest vessel [71]. While some of these studies have yielded positive correlations between tumor vascularization and treatment outcome, others have failed to establish such a relationship. Such an outcome is to be expected given the fact that measurements of tumor vascularization alone give very limited information about the actual oxygenation status of tumors which is also dependent on such parameters as the blood oxygenation status, blood flow and blood volume and also on whether the blood vessels are patent. Thus, it is quite plausible that an apparently well-vascularized tumor could be effectively poorly oxygenated.

Endogenous molecular markers of tumor oxygenation can potentially be very powerful indicators of tumor oxygenation status. However, although a number of potential candidates have been suggested and studied, none have presented themselves as reliable and consistent universal markers across different tumor types. Hypoxia inducible factor (HIF-1) is known to induce the expression of several proteins linked to the maintenance of oxygen homeostasis and tumor progression. It is hypothesized that since its alpha subunit (HIF-1 alpha) is stabilized under hypoxic conditions, it might represent a reliable intrinsic marker for tumor hypoxia and prognosis [72]. This has been shown to be the case in cervical cancer patients receiving curative radiation therapy [73], in non-small cell lung cancer patients [74]. However, other studies have shown that HIF-1 alpha is not a reliable marker of tumor oxygenation [75-78]. Recently, the expression of the facilitative glucose transporter, GLUT-1 has also been shown to be an endogenous marker of tumor oxygenation and, therefore, of prognosis in oral squamous cell carcinomas [79] as well in rectal carcinoma [80] but there appears to be some justified questions about its reliability [75]. A much cited tumor oxygenation endogenous marker is carbonic anhydrase 9. This is a transmembrane enzyme with an extracellular active site and is involved in the reversible metabolism of carbon dioxide to carbonic acid and has been shown to be a significant prognostic factor in non-small cell lung cancer [74, 81]. Its up-regulation occurs in highly hypoxic and necrotic regions of the tumor. Carbonic anhydrase 9 has also been marked as an adverse prognostic indicator in cervical cancer [82], and in some head and neck cancer studies as well as in invasive breast cancer studies [83]. Other head and neck cancer studies show no such prognostic importance of the molecule [75, 84]. Various other molecules such as lactate dehydrogenase-5 [85] , vascular endothelial growth factor [86], blood hemoglobin [87], osteonectin [88], and differentiated embryo chondrocyte [89], as well as measurements of DNA damage [90, 91] have all been studied as potential endogenous surrogate markers of tumor oxygenation and, therefore, of prognosis. It does not appear that any one of these markers can be universally used across many tumor types because they are either tumor cell-type specific, or are not 100% reliable even among human patients carrying the same type of cancer. Therefore, more studies have to be conducted and/or alternative molecules have to be explored as potential endogenous markers.

5. Future Directions
While there is no denying the importance of tumor oxygenation status as an independent prognostic marker that is important not only for predicting treatment outcomes, for predicting the rate of progression of disease and its metastatic potential and also for selecting patients for oxygen enhancing treatments such as carbogen breathing, hyperbaric oxygen therapy, and photodynamic therapy, the independent techniques to measure tumor oxygenation status have yet to develop to a level such that they could be readily used by cancer trials across the globe to yield consistent, reproducible and reliable information on tumor oxygenation. It appears that it would be more advantageous to use non-invasive direct methods or endogenous molecular markers to measure tumor oxygenation among currently available techniques as independent methods of measurement. While the non-invasive direct methods that have been enumerated above have yet to be fully optimized, validated, and technically simplified for universal use in the clinic, the use of endogenous markers of tumor oxygenation status, although an appealing alternative, requires further exploration. Under these limiting circumstances, it would be worthwhile to investigate the possibility of using a selective combination of existing methods that would validate each other to provide adequate reliable information on the tumor tissue pO₂ profile that can be used for prognosis and therapy in the clinic.

In an attempt to validate some of the currently available techniques for measuring tumor oxygenation, a study from our laboratory used a VEGF-transfected hypervascular human melanoma xenograft and its non-transfected counterpart as endpoints to measure the predictable tumor oxygenation status of these two tumor types. The studies in this reproducible preclinical model obtained results that validated each other by using several independent techniques to measure the various parameters that constitute a tumor's oxygenation status [92]. The consistency of these measurements using entirely different techniques gives hope that the information from these assessments of blood flow and oxygenation provide reliable data. While the combination of techniques used in the study cannot be directly used in the clinic presently, it is the first study of its kind that validates data obtained by using several different methodologies to obtain a reliable tumor oxygenation profile with the help of predictable endpoints in the form of the hypervascular VEGF-transfected tumor and its non-transfected counterpart in a preclinical setting. Such a validation with known preclinical and/or treatment outcome endpoints offers greater reliability than the use of combinations of independent techniques that have not been fully validated individually to establish a correlation between two tumor vascular parameters.

Acknowledgments
The authors would like to thank Antoinette Ghartey for technical help with preparation of the manuscript

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