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Blood oxygen level-dependent (BOLD) contrast-based functional MRI (fMRI) has been reported as a method to assess the evolution of tumor oxygenation after hyperoxic treatments, because of its sensitivity to changes in blood flow and deoxyhemoglobin content. However a number of questions remain: 1) In view of tumor heterogeneity, how good is the correlation between the MR parameters in gradient-echo imaging (signal intensity (SI) or effective transverse relaxation time (T)) and local tumor oxygen partial pressure (pO2)? 2) Is the magnitude of the change in SI or T a quantitative marker for variation in pO2? 3) Is initial T a good marker for initial pO2? To address these questions, murine tumors were imaged during respiratory challenges at 4.7 Tesla, using fiber-optic microprobes to simultaneously acquire tumor pO2 and erythrocyte flux. The BOLD signal response (SI and T) was temporally correlated with changes in pO2. However, the magnitude of the signal bore no absolute relation to pO2 across tumors, i.e., a given change in SI corresponded to a 25 mmHg pO2 change in one tumor, but to a 100 mmHg change in another. The initial T value did not reliably predict tumor oxygenation at the beginning of the experiment. In conclusion, the major advantages of the technique include noninvasiveness, high spatial resolution, and real-time detection of pO2 fluctuations. Information afforded by the BOLD imaging technique is qualitative in nature and may be combined with other techniques capable of providing an absolute measure of pO2. Magn Reson Med 48:980–986, 2002. © 2002 Wiley-Liss, Inc.
Oxygen is a key environmental factor in the development and growth of tumors, and their response to treatment. Because solid tumor hypoxia is associated with resistance to radiotherapy (1, 2), treatments aimed at acutely increasing the amount of oxygen delivered to tumors during radiotherapy have been developed, e.g., inhaling high-oxygen gas. Functional MRI (fMRI) studies based on blood oxygen level-dependent (BOLD) contrast (3) are of potential value in assessing the oxygenation status of tumors, particularly with regard to interventions designed to alter tumor oxygenation. Several groups have monitored the tumor response to high-oxygen-content gases by gradient-echo (GRE) MRI (4–8). Significant increases in intensity of T-weighted GRE images have been observed during hyperoxia using 100% O2 or carbogen (95% O2/5% CO2) breathing. The factors underlying the BOLD contrast have been the subject of intensive research, especially in neuroscience (9–12). In tumors, the BOLD effects observed in 2D GRE images have been attributed to both changes in blood flow and changes in blood oxygenation; the technique is also referred to as flow and oxygenation-dependent (FLOOD) imaging (13).
Since the key factor determining tumor response to radiotherapy is thought to be local pO2, rather than blood flow or hemoglobin saturation, there is a critical need to know how the BOLD signal correlates with tumor pO2. In attempts to understand this relationship, a good correlation was found between the averaged increase in tumor T and the averaged increase in tumor pO2 as measured by oxygen microelectrodes positioned subsequent to removal of the animal from the magnet (14). The simultaneous acquisition of tumor GRE images and pO2 was achieved in another study (15) using an MR-compatible fiber-optic oxygen sensor based on fluorescence quenching of a ruthenium dye. Here (15) carbogen breathing caused an increase in both pO2 and signal intensity (SI) averaged over the whole tumor.
In order to definitively assess the value of the method, however, possible temporal and spatial heterogeneity within tumors must be taken into account. Furthermore, it would be of value to know whether variation in SI or T quantitatively reflects the tissue pO2. The aim of the present study was to simultaneously measure the MR parameters and the tissue pO2 in the same tissue volume. We developed an experimental set-up (comparable to that described by Maxwell et al. (15)) to allow the simultaneous MRI of tumors in which fiber-optic oxygen/flow sensors were implanted. Respiratory gases were used to modulate tumor pO2 (a range of oxygen concentrations and carbogen). In addition to GRE imaging, multi-GRE images were also acquired to calculate the absolute effective transverse relaxation rate (R) and thereby avoid the confounding effects of inflow and oxygenation (16). Because of the frequently heterogeneous distribution of vascular response throughout the tumor (17), data obtained in the immediate vicinity of the fiber-optic probe were compared with those obtained in the whole tumor.
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Can the method be used to quantitatively assess the evolution of pO2? What is the relationship between SI and pO2? In vivo, the observed increase in SI was more pronounced when passing from 5 to 15 mmHg than when passing from 40 mmHg to 50 mmHg. A plateau was reached at high pO2 (typically above 30 mmHg). At least four parameters may be at the origin of this complex relationship between the BOLD response and tumor pO2: 1) the curvilinear dependence of blood R on blood deoxyHb content, 2) the hemoglobin-O2 dissociation curve, 3) the “inflow” effect, and 4) the blood volume fraction.
To shed some light on this issue, it is interesting to look at the BOLD effect that was assayed in vitro as part of this study. At 4.7 Tesla, we found a quadratic dependence of blood R on the deoxyHb fraction (Fig. 1). A similar relationship was previously reported between both blood R2 (22, 23) and R (24), and oxygen saturation. In normal tissues (pO2 above 15 mmHg), the quadratic term is not relevant and the relationship is often assumed to be linear. However, very low HbO2 saturations may be typical in tumors compared to normal tissue (25, 26). The quadratic term is more relevant in studies of tumor oxygenation, which indicates that the technique should be more sensitive in deoxygenated regions. Moreover, in this study, the BOLD contrast was correlated directly to tumor pO2 and not to blood oxygen saturation. The decreased sensitivity that was observed above 30 mmHg tumor pO2, could probably be explained by the fact that at this tumor pO2, most of the microvessel hemoglobin already exists in oxygenated form. A further increase in blood pO2 would not further increase hemoglobin saturation (no further change in SI or R would be observed), while it would continue to improve tumor oxygenation. Overall, this means that small changes in pO2 can be better detected in tissue with low pO2, e.g., tumors or renal medulla (27).
Another source of nonlinearity in the relationship between SI and pO2 is the influence of the so-called “inflow effect.” We tried to evaluate the influence of this parameter using blood flow data (OxyFlo™) and the SI at TE = 0 (Io) (16). No significant changes were observed in our model in response to the respiratory challenges. However, this does not exclude an inflow effect, as subtle changes in blood flow may go undetected by these methods. Is T a better marker for pO2 because this parameter is independent of the inflow effect? T values and tumor pO2 were positively time-correlated. However, no correlation was found between the T values and the initial tumor pO2 level, indicating that T is a bad predictor of tumor oxygenation. It should be emphasized that the T value for a given voxel is not only determined by microscopic field heterogeneity due to the presence of deoxyHb, but also by other factors such as the macroscopic field homogeneity and the T2 relaxation process. Without any baseline pO2 estimation, and because of the curvilinear relationship, the variation of T could not be quantitatively related to the response in pO2. Finally, a comparison of responses between different locations within the same tumor, or using different tumors, should be made with caution: in addition to the question of magnetic field homogeneity, deoxyHb content can be influenced by the local blood volume fraction, which may vary within tumors (and necessarily between tumors) and thus may influence the BOLD sensitivity (28–30).
In conclusion, GRE sequences and T measurements offer the capability to monitor oxygenation changes in tumors. The major advantages of the technique include its noninvasive nature, high spatial resolution, and real-time detection of changes in oxygenation. This method should be considered qualitative because it does not provide an absolute measure of pO2, and because calibration of the BOLD effect to tissue oxygenation is not straightforward. Individually, SI and T changes are positively correlated with tumor pO2, but the magnitude of change could not easily be compared between voxels of the same tumor or necessarily between tumors. In order to evaluate the basal level of pO2 in the tumor, BOLD imaging should be combined with additional techniques that can provide absolute quantification of pO2. Among MR techniques these include 19F NMRI (20), electron paramagnetic resonance (EPR) spectroscopy (31, 32), EPR imaging (33), and Overhauser imaging (34).