Hypoxia in human tumors can significantly influence treatment outcome and the aggressiveness of the tumor . This finding has led to considerable interest in non-invasive means to assess the extent of hypoxia in tumors prior to therapy, for example with MR (BOLD) imaging [14, 15]. Several studies have shown that tumor cell or tumor18F-FDG uptake is associated with hypoxia [16–18]. Glucose transporter (GLUT) receptor proteins and hexokinase activity are elevated in tumors and thought to be responsible for increased18F-FDG tumor uptake compared to normal tissues . Hypoxia has been shown to be a significant factor in the over-expression of these proteins [16, 17, 20]. In the present study we examined whether information concerning hypoxia could be gained using18F-FDG and PET imaging. This was considered possible since the hypoxic nature of most tumors forces tumor glucose metabolism to utilize primarily glycolytic pathways, rather than oxidative metabolism . Very large quantities of glucose are required to produce a given amount of ATP via the glycolytic pathway compared to the oxidative pathway, which supposedly accounts for why many tumors have such high18F-FDG uptake. We hypothesized that by oxygenating the tumor (via breathing carbogen), tumor glucose metabolism would begin to have a significant oxidative component, requiring considerably less glucose per ATP molecule produced. Thus, better oxygenated tumors (i.e. tumors influenced by breathing carbogen) should have less18F-FDG uptake than when in their usual, hypoxic state (i.e. while the animal is breathing air). To examine this possibility,18F-FDG uptake in tumors was examined in tumors when the mouse was breathing air versus carbogen.
The SCC tumor used in the present study is known to be quite hypoxic as determined by previous oxygen electrode measurements in our laboratory . Even though the tumor is extremely hypoxic, there was no evidence of necrosis in the tumors used in the present study (~1 cm diameter). Carbogen breathing has been shown in numerous studies to increase the oxygenation status of tumor-bearing mice [8, 21] and in patients [22–24]. Using the same SCCVII tumor model, tumor size, and experimental conditions with respect to the maintenance to core body temperature as used in the present study, we have previously shown that carbogen breathing increases oxygenation of the SCCVII tumor as measured by Eppendorf oxygen electrodes [8, 8]. In this study the mean tumor pO2 values for animals breathing air was 8.2 mm Hg, which increased to a value of 19.8 for animals breathing carbogen .
As mentioned above, the hypoxic nature of the SCC tumor makes it likely that a large proportion of the tumor cells would utilize glucose by anaerobic glycolysis, and so exhibit enhanced18F-FDG uptake. Indeed, we found glucose utilization as measured by uptake of18F-FDG to be significant in the SCC tumor as evidenced by the tumor images shown in Figure 1. The FDG uptake in the tumor was particularly striking compared to the non-tumor bearing leg where little to no uptake was observed (data not shown). It was anticipated that increasing the oxygenation of the tumor by carbogen breathing would lead to a shift to aerobic metabolism producing much more ATP per molecule of glucose, thereby decreasing the need for glucose, and decreasing18F-FDG tumor uptake. The results of this study only partially supported this hypothesis. Air/carbogen ratios for overall tumor18F-FDG uptake were not significantly different from unity. However, when only the highest18F-FDG uptake region of each tumor was considered (perhaps the most hypoxic regions) the hypothesis was supported. In these presumably metabolically active regions, there was a small (21%) but significant increase in air/carbogen uptake, suggesting reduced18F-FDG uptake under conditions of better oxygenation. The lack of an air/carbogen difference for the whole tumor, and the fairly small air/carbogen difference even for the hottest part of the tumor might suggest that tumors are "programmed" to burn glucose primarily in an anaerobic fashion, regardless of their level of oxygenation. This could be a potential survival mechanism for tissue destined to grow in an anaerobic environment. This potential explanation is of course speculative. Another factor which could play a role is the potential vasodilatory properties of carbon dioxide, although there is conflicting data reported in the literature .
Changes in tumor FDG uptake (hot spots) when breathing carbogen might add another dimension to18F-FDG functional imaging. While small, the 21% change in18F-FDG uptake in small regions may be giving information similar and perhaps complimentary to that obtained by the MRI based oxygen imaging is Blood Oxygen Level Dependent (BOLD) MRI. For this imaging technique, temporal changes in the ratios of deoxy- to oxyhemoglobin can be monitored utilizing the contrast mechanisms provided by deoxyhemoglobin to protons. With this method, it is possible to examine changes in blood flow and tumor oxygenation levels in response to carbogen breathing versus air breathing [14, 15]. The enhancement observed in BOLD MRI images is typically only a few percent. In contrast, the decrease in18F-FDG tumor uptake upon carbogen breathing in small regions of the tumor in the present study was up to 21%, suggesting that in these small regions tumor glucose metabolism is altered as a result of increased perfusion and oxygenation. Should this observation be correct, which will require further validation, the added functional dimension to18F-FDG imaging afforded by carbogen breathing could be useful to clinicians assessing treatment response, particularly for radiation treatment.
Our results did not wholly agree with some previous studies that showed whole tumor18F-FDG uptake increases in more hypoxic conditions [25–28]. Our whole tumor results gave an air/carbogen uptake ratio that did not differ significantly from unity. Only when VOIs were drawn around the hottest part of our tumors were our findings more consistent with these previous studies. The aforementioned studies either examined a different tumor type from our study (C3H mammary carcinomas) , or examined the same tumor type (SCC), but in an in vitro setting . These differences alone could potentially account for the difference in our findings. Additionally, this result could be due to differences in experimental method, or due to the variability in our own results. Previous studies did not attempt to correct for potential differences in input function. The lack of standardization across animal PET studies, both in the means of determining reference input functions, as well as lack of consensus regarding simpler procedures such as controlling animal core temperature, might also explain this difference in result. Future experiments with a greater number of mice, using simultaneous blood sampling, would clarify the results shown in Figure 3.
The ability to examine and quantify regions of greatest change in18F-FDG uptake, which is where a radiation oncologist would presumably concentrate three-dimensional conformal radiation dose painting, has significant implications for cancer diagnosis and treatment. Future studies could further test this phenomenon by shifting to more hypoxic conditions, to see if18F-FDG uptake improves even more.
As an adjunct to our study, we examined the results obtained when mice were allowed to drop core body temperature to 30°C. Past work in our laboratory has demonstrated that animal's core body temperature under isoflurane anesthesia drops ten degrees in an average of 15 min . We found that mice at this lower temperature showed significantly greater18F-FDG accumulation in carbogen than air environments. This could be due to relative vasoconstriction in low temperature conditions, possibly making18F-FDG uptake flow limited in these circumstances. Adding carbogen to this low-temperature state may improve blood flow, because vasculature responds to the high partial pressure of oxygen by vasodilation. Although this reasoning is speculative, our low temperature findings do point definitively to the necessity for strict control of the mice's physiology and the need for standardization across animal PET studies.
One strength of the present study was the ability to measure18F-FDG uptake with air and carbogen breathing in the same tumor. Previous studies have compared uptake across different animals. Differences in tumor heterogeneity across mice potentially confound these results. Our study controlled for this confounding variable. Further studies would benefit from testing multiple tumor types, as well as human tumors.