Tumor size dependent changes in a murine fibrosarcoma: use of in vivo 31P NMR for non-invasive evaluation of tumor metabolic status

Tumor tissue contains viable hypoxic regions that are radioresistant and often chemoresistant and may therefore be responsible for some treatment failures. A subject of general interest has been the development of non-invasive means of monitoring tissue oxygen. Pulse Fourier transform 31P NMR spectroscopy can be used to estimate intracellular nucleotide triphosphates (NTP), phosphocreatinine (PCr), inorganic phosphate (Pi) and pH. We have obtained 31P NMR spectra as an indirect estimate of tissue oxygen and metabolic status in a C3H mouse fibrosarcoma FSaII. Sequential spectra were studied during tumor growth in a cohort of animals and peak area ratios for several metabolites were computed digitally by computer. During growth, tumors showed a progressive loss of PCr with increasing Pi, and most tumors greater than 250 mm3 in volume had little or no measurable PCr. The smallest tumors (38 mm3 average volume) had PCr/Pi ratios of 1.03 +/- .24, whereas tumors 250 mm3 or more had an average PCr/Pi ratio of 0.15 +/- .04. Similarly derived NTP/Pi ratios decreased with tumor size, but this change was not significant (p = .17). Radiobiologic hypoxic cell fractions were estimated using the radiation dose required to control tumor in 50% of animals (TCD50) or by the lung colony technique. Tumors less than 100 mm3 had a hypoxic cell fraction of 4% (TCD50) while tumors 250 mm3 had a 40% hypoxic cell fraction (lung colony assay). These hypoxic fraction determinations correlated well with the depletion of PCr and decline in NTP/Pi ratios seen at 250 mm3 tumor volumes. Tumor spectral changes with acute ischemia were studied after ligation of the tumor bearing limb and were similar to changes seen with tumor growth. PCr was lost within 7 minutes, with concurrent increase in Pi and loss of NTP. Complete loss of all high energy phosphates occurred by 40 minutes of occlusion. In vivo tumor 31P NMR spectroscopy can be used to estimate tissue metabolic status and may be useful in non-invasive prediction of hypoxic cell fraction, reoxygenation, and radiation treatment response.     

Size dependent changes in tumor phosphate metabolism after radiation therapy as detected by 31P NMR spectroscopy

In Vivo 31P NMR spectroscopy was used to study changes in phosphate metabolism that occur after irradiation of the C3H fibrosarcoma, FSaII. Previously, we have shown that small FSaII tumors (less than 250 mm3) have a greater phosphocreatinine/inorganic phosphate (PCr/Pi) ratio and a lower hypoxic cell fraction (HCF) than large FSaII tumors (greater than 250 mm3). Six small tumors (113 +/- 26 mm3) were treated with radiation doses chosen to induce local control in greater than 50% of animals, (70-100 Gy, single fraction). Minimal changes in the tumor 31P NMR spectrum were seen over eight days of monitoring. During this interval, tumor regression began a minimum of 36 hours after radiation. This contrasted with large tumors (650-1000 mm3) wherein a significant increase in the Pcr/Pi ratio was seen 44 hr after irradiation. In tumors of this size range, a tumor growth delay of 4 to 7 days is obtained after a single 70 Gy fraction of radiation. Since small FSaII tumors have a minimal HCF (approximately equal to 4%), radiation induced reoxygenation would not be expected to have a large effect on their average cellular metabolism. Large tumors of this histology have a high HCF (greater than or equal to 40%), and may therefore be expected to have a significant average change in tumor cell metabolism with reoxygenation. The 31P NMR observations of small and large tumors after irradiation are compatible with radiation induced reoxygenation in the larger tumors.     

Estimation of tumor oxygenation and metabolic rate using 31P MRS: correlation of longitudinal relaxation with tumor growth rate and DNA synthesis

31P MRS longitudinal relaxation times (T1) were determined for C3H murine fibrosarcomas (FSaII), and mammary carcinomas (MCaIV). Tumors were implanted in the foot dorsum, and were 100-300 mm3 in volume. T1s were repeated after the animal was allowed to breathe 100% oxygen for 30 min and then again 36-48 hr following 30 Gy. The spectrum were obtained using an 8.5 T spectrometer with a 8 cm bore and a 1.4 cm single turn antenna coil. The 31P relaxation times for untreated tumors in air breathing animals were: 3.78 sec for phosphomonoesters, 4.37 sec for inorganic phosphate (Pi), 2.73 sec for phosphocreatine, 1.37 sec for gamma ATP, 1.14 sec for alpha ATP, and 1.18 sec for beta ATP. The Pi T1s were 4.37 and 4.70 sec in control and irradiated tumors in air breathing animals. Respiration of oxygen for 30 min reduced the T1s to 3.02 and 2.62 sec in control and irradiated tumors respectively. The Pi T1 of an anoxic tumor, determined on an in situ tumor 60 min after death was 5.93 sec. The oxygen breathing induced decrease in the T1 of Pi is unlikely to have been caused by the paramagnetic properties of oxygen alone, and suggests a component of increased magnetization transfer secondary to the ATPase reaction. Oxygen breathing following 30 Gy, resulted in a decreased growth time (800 mm3 endpoint) and an increased proportion of cells in S-phase. These results support the hypothesis that the decrease in Pi T1 measured with oxygen breathing is a measure of tumor oxygen tension and metabolic rate, and suggests that T1 measurement may indirectly predict tumor growth rate and DNA synthesis.     

Tumors growing in irradiated tissue: oxygenation, metabolic state, and pH

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Animal tumors were early generation isotransplants of a spontaneous fibrosarcoma in a C3Hf/Sed mouse, designated FSa-II. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased, these tumors are assumed to be metabolically deprived with hypoperfusion and acidosis. In this study we directly measured the oxygen partial pressure (pO2) distribution, metabolic state, and pH of tumors growing in an irradiated tumor bed using oxygen sensitive electrodes and 31P-NMR. The results confirmed a three-fold increase in the number of pO2 readings less than or equal to 2.5 mmHg and also showed increased acidosis with a 0.17 unit decrease in pHNMR. When tumors growing in pre-irradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed. Consistent with these observations, the phosphocreatine/inorganic phosphate (PCr/Pi) and nucleoside triphosphate/inorganic phosphate (NTP/Pi) ratios were significantly lower in the tumors in a pre-irradiated bed compared to tumors in a non-irradiated bed (PCr/Pi: 0.51 vs 0.79, p less than 0.05; and NTP/Pi: 0.64 vs 0.93, p less than 0.05). The longitudinal relaxation time (T1) of Pi was numerically shorter in control tumors (consistent with the better tissue oxygenation), but this did not reach statistical significance (2.09 +/- .11 sec vs 2.25 +/- .16 sec).     

Correlations between in vivo tumor weight,oxygen pressure, 31P NMR spectroscopy,hypoxic microenvironment marking by beta-D-iodinated azomycin galactopyranoside (beta-D-IAZGP), and radiation sensitivity

PURPOSE: The purpose of this study is to evaluate the amount of hypoxic fraction in a rodent tumor by means of polarographic oxygen electrode, phosphorus-31 magnetic resonance spectroscopy (31P-MRS), and a newly synthesized hypoxic marker, beta-D-iodinated azomycin galactopyranoside (beta-D-IAZGP). We also investigated the radiosensitivity for tumors of different weights. METHODS AND MATERIALS: Murine mammary carcinoma cells, FM3A, were subcutaneously implanted into the back of 5-week-old male C3H/He mice. beta-D-IAZGP radiolabeled with 123I or with 125I was injected intravenously into tumor-bearing mice, and marker distribution was measured by nuclear medicine procedures. Radiosensitivity of the tumor was measured by the in vivo/in vitro clonogenic assay. Tumor oxygenation status was also measured directly by polarographic oxygen electrodes and indirectly estimated from 31P-MR spectra. RESULTS: Higher accumulation of 123I-beta-D-IAZGP was observed in the tumors than in normal tissues at 24 h after administration. As to biodistribution of 125I-beta-D-IAZGP, the tumor/blood ratio varied widely, but correlated significantly with tumor weight. Mean oxygen pressure (pO2) values and ratios of nucleoside triphosphate beta to inorganic phosphate (beta-ATP/Pi) and of phosphocreatine to inorganic phosphate (PCr/Pi) decreased significantly with the increase in tumor volume. As tumor volume increased, the surviving fraction of cells from tumors irradiated in vivo increased significantly. CONCLUSIONS: The increase in tumor volume was significantly correlated with a reduction in mean pO2, a decrease in the ratios of beta-ATP/Pi or PCr/Pi, an increase in uptake of beta-D-IAZGP, and an increase in radioresistance. Because the uptake of beta-D-IAZGP can be measured noninvasively by nuclear medicine techniques, it could be clinically useful for monitoring hypoxia in human tumors.     

Changes in radiation sensitization induced by Fluosol-DA as measured by 31P nuclear magnetic resonance spectroscopy

Numerous agents have been studied in attempts to sensitize radioresistant hypoxic tumor cells. We have investigated the effect of Fluosol-DA plus carbogen (95% oxygen and 5% CO2) on the sensitivity of a radioresistant mammary carcinoma in C3H/He mice and also on tumor metabolism by 31P nuclear magnetic resonance spectroscopy. Statistically significant increases in phosphocreatine/Pi were noted for small- (150-350 mm3) and medium- (351-650 mm3) sized tumors treated with Fluosol-DA plus carbogen. Small tumors were shown to undergo significant radiosensitization in the presence of Fluosol-DA plus carbogen and medium-sized tumors showed a lesser degree of radiosensitization. Large tumors (greater than 900 mm3) showed no effect. Fluosol-DA or carbogen alone had no effects on animals with any tumor volume, as monitored by significant changes in radiosensitivity or nuclear magnetic resonance parameters. An approximately linear relationship was found between the decrease in the values for radiation dose which yields 50% tumor control and the increase in phosphocreatine/Pi, with a correlation of r = -0.93. 31P nuclear magnetic resonance spectroscopy may be useful for monitoring changes in radiosensitivity induced by agents which alter tumor oxygenation and subsequent metabolic status.     

31P nuclear magnetic resonance spectroscopy studies of tumor energy metabolism and its relationship to intracapillary oxyhemoglobin saturation status and tumor hypoxia

Relationships between tumor bioenergetic status on the one hand and intracapillary oxyhemoglobin (HbO2) saturation status and fraction of radiobiologically hypoxic cells on the other were studied using two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI). Tumor energy metabolism was studied in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy and the resonance area ratio (PCr + NTP beta)/Pi was used as parameter for bioenergetic status. Intracapillary HbO2 saturation status reflects the oxygen supply conditions in tumors and was measured in vitro using a cryospectrophotometric method. The KHT, RIF-1, and MLS lines showed decreasing bioenergetic status, i.e., decreasing PCr and NTP beta resonances and an increasing Pi resonance, with increasing tumor volume, whereas the OWI line showed no changes in these resonances during tumor growth. The volume-dependence of the HbO2 saturation status differed similarly among the tumor lines; HbO2 saturation status decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines and was independent of tumor volume for the OWI line. Moreover, linear correlations were found between bioenergetic status and HbO2 saturation status for individual tumors of the KHT, RIF-1, and MLS lines. These observations together indicated a direct relationship between 31P-NMR spectral parameters and tumor oxygen supply conditions. However, this relationship was not identical for the different tumor lines, suggesting that it was influenced by intrinsic properties of the tumor cells such as rate of respiration and ability to survive under hypoxia. Similarly, there was no correlation between bioenergetic status and fraction of radiobiologically hypoxic cells across the four tumor lines. This indicates that 31P-NMR spectroscopy data have to be supplemented with other data, e.g., rate of oxygen consumption, cell survival time under hypoxic stress, and/or fraction of metabolically active, nonclonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.     

31P NMR spectroscopy and HbO2 cryospectrophotometry in prediction of tumor radioresistance caused by hypoxia

The aim of this study was to search for possible relationships between the fraction of radiobiologically hypoxic cells in tumors and their 31P NMR spectral parameters and intracapillary HbO2 saturations. Four different tumor lines, two murine sarcomas (KHT, RIF-1) and two human ovarian carcinoma xenografts (MLS, OWI), were used. When tumor volume increased from about 200 mm3 to about 2000 mm3, hypoxic fraction increased from 12 to 23% for the KHT line, from 0.9 to 1.7% for the RIF-1 line, and from 9 to 28% for the MLS line. The OWI line showed similar hypoxic fractions at 200 (17%) and 2000 mm3 (15%). Tumor bioenergetic status decreased, that is, the inorganic phosphate (Pi) resonance increased and the phosphocreatine (PCr) and nucleoside triphosphate beta (NTP beta) resonances decreased, with increasing tumor volume for the KHT, RIF-1, and MLS lines, whereas the OWI line did not show any changes in the 31P NMR spectral parameters during tumor growth. Similarly, tumor HbO2 saturation status, that is, the fraction of vessels with HbO2 saturation above 30%, decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines, but remained unchanged during tumor growth for the OWI line. Although the data indicated a relationship between hypoxic fraction and tumor bioenergetic status as well as tumor HbO2 saturation status within a specific line during tumor growth, there was no correlation between hypoxic fraction and tumor bioenergetic status or tumor HbO2 saturation status across the four tumor lines. This may have occurred because cell survival time under hypoxic stress as well as fraction of non-clonogenic, but metabolically active hypoxic cells differed among the tumor lines. This indicates that 31P NMR spectroscopy and HbO2 cryospectrophotometry data have to be supplemented with other data to be useful in prediction of tumor radioresistance caused by hypoxia.     

Energy status in the murine FSaII and MCaIV tumors under aerobic and hypoxic conditions: an in-vivo and in-vitro analysis.

The relationship between energy status and hypoxia was examined in two murine tumors with substantially different hypoxic cell fractions in situ and in cells derived from these tumors in vitro. Parameters of tumor energy status were NTP/Pi and PCr/Pi obtained by 31P-NMR spectroscopy and adenylate energy charge and energy status obtained by high-pressure liquid chromatographic analysis of tumor extracts. Adenylate energy charge and rates of high-energy phosphate degradation were determined on cells obtained from both tumor types (MCaIV and FSaII) under identical nutrient and oxygen conditions, that is, air and nitrogen for various durations (0-6 hr). No consistent or substantial differences were noted in the various parameters of tumor energy status obtained by nuclear magnetic resonance analysis or analysis of tumor extracts, even though the MCaIV contains a substantially larger hypoxic fraction (49% vs 12%). Under in vitro conditions, the two cell lines exhibited different responses to oxygen deprivation, the MCaIV being substantially more refractory to energy changes secondary to hypoxia. Noting with caution that this study is based on only two tumor types, our results suggest that differences in cellular capacity for energy maintenance preclude quantitative inferences regarding tumor oxygen status from energy status between tumor types.     

The effect of hypoxia and hyperoxia on nucleoside triphosphate/inorganic phosphate, pO2 and radiation response in an experimental tumour model.

This study has evaluated the effect of breathing 100% oxygen, carbogen and carbon monoxide (at 660 p.p.m.) on the bioenergetic and oxygenation status and the radiation response of 200-mm3 C3H mammary carcinomas grown in the feet of CDF mice. Bioenergetic status was assessed by 31P magnetic resonance spectroscopy (MRS) using a 7-tesla spectrometer with both short (2 s) and long (6 s) pulse repetition times. Tumour partial pressure of oxygen (PO2) was measured with an Eppendorf polarographic electrode; the oxygenation parameters were the median pO2 and fraction of pO2 values < or = 2.5 mmHg. The radiation response was estimated using a tumour growth delay assay (time to grow three times treatment volume). Carbon monoxide breathing decreased tumour pO2 and compromised the radiation response, but the beta-nucleoside triphosphate (NTP)/Pi ratio was unchanged. Both carbogen and oxygen (100%) increased tumour pO2 and beta-NTP/Pi and enhanced the radiation response, the effects being similar under the two gassing conditions and dependent on the gas breathing time. Thus, in this tumour model, 31P-MRS can detect hyperoxic changes, but because cells can remain metabolically active even at low oxygen tensions the beta-NTP/Pi did not correlate with low tissue oxygenation. An analysis of variance showed that gas breathing time induced a significant systematic effect on beta-NTP/Pi, the MRS pulse repetition time had a significant effect on beta-NTP/Pi change under hypoxic but not under hyperoxic conditions and the type of gas that was inhaled had a significant effect on beta-NTP/Pi.     

Correlations between in vivo 31P NMR spectroscopy measurements, tumor size, hypoxic fraction and cell survival after radiotherapy

Phosphorus metabolite levels were measured non-invasively using 31P magnetic resonance spectroscopy (MRS) in SCCVII/SF tumors, subcutaneously transplanted into the legs of unanesthetized C3Hf/Sed mice. Shortly after MRS measurements, tumors were irradiated with a single dose of 20 Gy, and cell survival and radiobiologic hypoxic fraction were determined with an in vitro cloning assay. Significant correlations were found between tumor size and surviving fraction, hypoxic fraction, pH, and phosphorus metabolite ratios. With increase of tumor size, surviving fraction and hypoxic fraction both increased, the ratios of inorganic phosphate and phosphomonoesters to nucleoside triphosphates (Pi/NTP and PME/NTP, respectively) and inorganic phosphate to phosphocreatine (Pi/PCr) increased and pH decreased. However, considerable heterogeneity of MRS spectral parameters, even in tumors of similar size, precluded accurate prediction of hypoxic fraction and cell survival after radiotherapy.     

Influence of hormono- and/or chemotherapy on the MXT mouse mammary tumor as monitored by 31P MRS

We describe the early in vivo modifications that occurred in the MXT mouse hormone sensitive mammary tumor following various treatments which were monitored by ³¹P NMR spectroscopy. The MXT mouse mammary tumor was subjected to clinically relevant low-dose chemotherapy, i.e. seven cycles of 20 mg/kg cyclophosphamide (CPA) with or without an attempt at estrogenic cell recruitment prior to the CPA treatment. NMR measurements were begun at the end of the CPA treatment in order to evaluate the remaining ‘long-term’ chemotherapy-induced modifications within the MXT tumors. Statistical analyses performed on the ³¹P NMR parameters revealed that treatment had induced significant effects on bATP/PCr, Pi/PCr and PME/PDE only, with PCr being the most discriminating index. Its presence within MXT tumors was verified by means of an analysis of perchloric extracts. The results indicate a relative decrease of PME/PDE and a better conservation of PCr within the CPA-treated group as compared to the control one. This feature appeared even prior to any macroscopic modifications, as was the case within the group which contained tumors smaller than 120 mm², and where no significant differences appeared between the mean sizes of the MXT cancers. In contrast, within the G2 group, which contained tumors equal to or larger than 120 mm², CPA significantly slowed down tumor growth, while the administration of estradiol (E2) prior to CPA treatment antagonized the positive CPA-induced therapeutic effect. In conclusion, the non-invasive follow-up of the chemotherapeutic treatment of a clinically relevant mammary tumor model by ³¹P NMR spectroscopy backed up by statistical analyses revealed metabolic changes that appeared well before any modifications in histopathology or growth.     

Growth studies of subcutaneous rat tumours: comparison of 31P-NMR spectroscopy, acid extracts and histology

31P-NMP, surface coil spectra of three subcutaneously implanted rat tumours (Morris hepatoma 7777, GH3 prolactinoma, Walker carcinosarcoma) and an N-methyl-N-nitrosourea induced rat mammary adenocarcinoma at different stages of growth were obtained and compared with histological sections taken immediately after NMR acquisitions. Metabolite ratios (phosphocreatine (PCr)/beta nucleoside triphosphate (beta NTP), PCr/Pi, beta NTP/Pi) calculated from the NMR spectra were compared with ratios obtained from acid extracts of tumours of similar size. Measurements of creatine and ADP were also made. Three of the tumours showed positive correlations between increasing tumour size and decreasing metabolite ratios measured both by NMR and in extracts, whereas the Walker carcinosarcoma showed no correlation between size and any parameters measured. Phosphorus metabolite ratios, measured in extracts of skin overlying the tumours, indicated a fall in high energy phosphate when there was histological evidence of skin invasion by the tumour. Surface coil 31P-NMR spectra of subcutaneously grown or induced tumours in the rat represent a slowly changing steady state as the tumour increases in size. We conclude that increasing numbers of hypoxic tumour cells, rather than large areas of necrotic tissue, contribute largely to the NMR spectrum.     

Effects of hydralazine-induced vasodilation on the energy metabolism of murine tumors studied by in vivo 31P-nuclear magnetic resonance spectroscopy

The effects of hydralazine on tumor energy metabolism and on some cardiovascular parameters were measured. Tumor energy metabolism was studied in C3Hf/Sed mice with isotransplants of a spontaneous murine fibrosarcoma (FSaII, congruent to 100 mm3 in volume) and 31P-NMR. Cardiovascular parameters were measured in anesthetized C3Hf/Sed mice via intracarotid catheter. Hydralazine doses of 0.25 mg/kg given ip caused an increase of the phosphocreatine to inorganic phosphate ratio (PCr: Pi) in 5 of 6 animals. These doses had minimal effects on mean arterial blood pressure, though there may have been an increased cardiac output due to a decreased afterload. Hydralazine doses greater than or equal to 2.0 mg/kg given ip were associated with a decrease in PCr, nucleotide triphosphate, and pH, and an increase in Pi (P less than .01 for control vs. 10 mg hydralazine/kg). This substantial decrease in high-energy phosphates was associated with a pronounced decrement in mean arterial blood pressure. These findings provide a rational basis for the study in experimental systems of hydralazine-induced enhancement of cell killing by hyperthermia and by agents toxic to hypoxic cells. Further, these results can be taken as a sign that hydralazine should be used with care in patients undergoing radiation treatment.     

Phosphorus-31 magnetic resonance spectroscopy and blood perfusion of the RIF-1 tumor following X-irradiation

Phosphorus-31 magnetic resonance spectra were obtained from the RIF-1 tumor in C3H mice before and up to 2 days after various doses of X rays. Parallel studies were performed to measure relative changes in tumor blood perfusion using [14C]iodo-antipyrine and changes in % tumor necrosis using Chalkley's method. Tumor ratios of phosphocreatine to inorganic phosphate (PCr/Pi) and nucleotide triphosphates to inorganic phosphate (NTP/Pi) as well as pH as measured by 31P-MRS increased significantly at most time points after irradiation with doses of 5, 10, and 20 Gy. Tumor blood perfusion was found to significantly improve after a dose of 20 Gy but not after a dose of 2 Gy. Percent tumor necrosis increased to about 3 times its control level at 1 day after a dose of 20 Gy and then declined to about twice its control value at 2 days. The magnitude of the changes in the 31P-MRS parameters makes it unlikely that any of them are entirely due to radiation-induced changes in the radiobiologically hypoxic fraction of these tumors. Changes in the necrotic fraction did not appear to influence the tumor spectra. However, the observed improvement in tumor blood perfusion may have resulted in an increase in oxidative phosphorylation of the whole tumor population as well as a clearance of inorganic phosphate and acid metabolites, so that 31P-MRS changes may indirectly reflect changes in tumor blood perfusion.     

Tumor-dependent kinetics of partial pressure of oxygen fluctuations during air and oxygen breathing

The primary purpose of this study was to examine the kinetics of partial pressure of oxygen (pO2) fluctuations in fibrosarcoma (FSA) and 9L tumors under air and O2 breathing conditions. The overall hypothesis was that key factors relating to oxygen tension fluctuations would vary between the two tumor types and as a function of the oxygen content of the breathing gas. To assist in the interpretation of the temporal data, spatial pO2 distributions were measured in 10 FSA and 8 9L tumors transplanted into the subcutis of the hind leg of Nembutal-anesthetized (50 mg/kg) Fischer 344 rats. Recessed-tip oxygen microelectrodes were inserted into the tumor, and linear pO2 measurements were recorded in 50-microm steps along a 3-mm path, and blood pressure was simultaneously measured via femoral arterial access. Additionally, pO2 was measured at a single location for 90 to 120 minutes in FSA (n=11) or 9L tumors (n=12). Rats were switched from air to 100% O2 breathing after 45 minutes. Temporal pO2 records were evaluated for their potential radiobiological significance by assessing the number of times they crossed a 10-mm-Hg threshold. In addition, the data were subjected to Fourier analysis for air and O2 breathing. FSA and 9L tumors had spatial median pO2 measurements of 4 and 1 mm Hg, respectively. 9L had more low pO2 measurements < or =2.5 mm Hg than did FSA, whereas between 2.5 and 10 mm Hg this pattern was reversed. Pimonidazole staining patterns in FSA and 9L tumors supported these results. Temporal pO2 instability was observed in all experiments during air and O2 breathing. Threshold analyses indicated that the 10 mm Hg threshold was crossed 2 to 5 times per hour, independent of tumor type. However, the magnitude of 9L pO2 fluctuations was approximately eight times greater than FSA fluctuations, as assessed with Fourier transform analysis (Wilcoxon, P < 0.005). O2 breathing significantly increased median pO2 in FSA from 3 to 8 mm Hg (P < 0.005) and caused a significant increase in frequency and magnitude of pO2 fluctuations. One hundred percent O2 breathing had no effect on 9L tumor pO2, and it decreased the magnitude of pO2 fluctuations with borderline significance. These results show that these two tumors differ significantly with respect to spatial and temporal oxygenation conditions under air and O2 breathing. Fluctuations of pO2 of the type reported herein are predicted to significantly affect radiotherapy response and could be a source for genetic instability, increased angiogenesis, and metastases.     

Effects of hydralazine on in vivo tumor energy metabolism, hematopoietic radiation sensitivity, and cardiovascular parameters

Energy metabolism of murine FSaII foot tumors was studied by in vivo 31P-MRS in C3Hf/Sed mice. Spectroscopy was performed following exposure to escalating doses of hydralazine (HYD) ip. At 0.25 mg/kg, HYD caused a 20% increase in PCr/Pi and had no significant effect on mean arterial blood pressure. HYD doses greater than or equal to 2 mg/kg lead to hypotension which was associated with a decrease in PCr, NTP, pH, and an increase in Pi (p less than 0.01 for control vs 10 mg/kg HYD). When mice were given ip injections of HYD (0.25, 1, 2 and 10 mg/kg) 10 min prior to whole body irradiation, spleen stem cell survival after 6 Gy was increased (2.19 colonies in control animals vs 6.74 colonies per spleen in animals treated with greater than or equal to 2 mg/kg HYD), as was the LD50/30 dose (6.49 Gy [control] vs 9.00 Gy [10 mg/kg HYD]). The data provide evidence that PCr/Pi is a useful indicator of perfusion efficiency (and indirectly of hypoxic cell fraction) in FSaII tumors. These observations suggest that HYD may be a useful adjuvant for hyperthermic treatment of tumors and for potentiation of agents specifically toxic to hypoxic or nutrient-deprived cancer cells. HYD should be used with care in patients receiving radiation treatments or other therapies for which hypoxia can unfavorably affect treatment outcome.     

Nonglycolytic acidification of murine radiation-induced fibrosarcoma 1 tumor via 3-O-methyl-D-glucose monitored by 1H, 2H, 13C, and 31P nuclear magnetic resonance spectroscopy.

The effects of 3-O-methyl-D-glucose (3-OMG) on subcutaneously implanted murine radiation-induced fibrosarcoma 1 tumor were examined with 2H, 13C, and 31P nuclear magnetic resonance (NMR) in situ. Using 31P NMR, changes in tumor high-energy phosphate metabolism were monitored for 2.5 h after i.p. administration of 3-OMG (8.1 g/kg body weight); tumor pH decreased by a mean maximum of 0.52 +/- 0.05 (SE) (n = 10), [PCr] decreased by 54%, [NTP] decreased by 35%, and [Pi] increased by 36%. Tumor blood flow, as measured by 2H NMR monitoring of D2O washout kinetics, decreased by 40% at 1 h and by 47% at 2 h after 3-OMG injection (n = 4). This substantial tumor acidification (pH decrease much greater than 0.1), expected to require a glycolytic substrate (Hwang et al., Cancer Res., 51: 3108-3118, 1991), is surprising in light of the previously documented metabolically inert nature of 3-OMG. In situ 13C NMR spectroscopy, following [6-13C]3-OMG i.p. injection, examined the possibility of the glycolytic metabolism of 3-OMG. However, only the C-6 resonance of 3-OMG was detected (n = 6); no resonances from [6-13C]3-OMG-6-phosphate or [3-13C]lactate were observed. These results confirmed that 3-OMG was not metabolized in radiation-induced fibrosarcoma 1 tumor. At the completion of the in situ 13C NMR experiments, tumors were freeze clamped, and perchloric acid extraction was performed. High-resolution 1H NMR measurement of lactate concentrations showed no statistically significant difference in control tumor extracts (from mice not receiving i.p. injection; n = 5) and in tumor extracts from mice administered i.p. [6-13C]3-OMG (n = 5), indicating that there was no significant increase in lactate level in the tumor extracts from mice administered i.p. 3-OMG due to increased plasma glucose concentration. The results of these 1H and 13C NMR studies indicated that the radiation-induced fibrosarcoma 1 tumor acidification caused by i.p. administration of 3-OMG was not due to a direct (3-OMG----lactate) or an indirect (systemic glucose----lactate) increase in tumor lactic acid levels.     

The potential for prazosin and calcitonin gene-related peptide (CGRP) in causing hypoxia in tumours

Using 31P NMR spectroscopy, changes in tumour metabolic status were studied in a transplanted rat fibrosarcoma following the administration of vasodilators. Mean Arterial Blood Pressure (MABP) was monitored simultaneously. Two vasodilators were studied, prazosin and CGRP, which altered the NMR parameters Pi/sigma P, beta NTP,Pi, PCr/Pi and PME/Pi in a dose dependent manner. There was a good correlation between the various NMR parameters; for analysis, Pi/sigma P was used for convenience. With increasing doses of vasodilator, Pi/sigma P increased and the MABP decreased. Reduction in pHNMR showed a correlation with decreasing MABP following the administration of prazosin but not after CGRP. Both prazosin and CGRP produced changes in 31P NMR spectra consistent with a reduction in tumour blood flow. The results for prazosin and CGRP were comparable and showed a 15-20% increase in Pi/sigma P for a 20% reduction in MABP. These results were compared with those from hydralazine. With hydralazine an acceptable reduction in blood pressure (up to approximately 25%) has little effect and may even alter NMR parameters consistent with an increase in blood flow, a reduction of approximately 40% is required for a significant decrease in flow. Both prazosin and CGRP are shown to be far more effective than hydralazine in causing tumour hypoxia at a clinically acceptable reduction in blood pressure. CGRP may be the more suitable for clinical use because of its short half life, its capability to achieve controlled hypotension and the relatively few side effects associated with its use.     

Evaluation of tumor energy metabolism and microvascular blood flow after glucose or mannitol administration using 31P nuclear magnetic resonance spectroscopy and laser Doppler flowmetry

The effects of intraperitoneally administered glucose or mannitol (5 mg/g body weight, 25% solutions) on tumor energy metabolism and tumor red blood cell flux were studied using 31P-nuclear magnetic resonance spectroscopy and laser Doppler flowmetry. Isotransplants of a spontaneous murine fibrosarcoma growing in the hind foot dorsum were used. 31P-nuclear magnetic resonance and laser Doppler flowmetry studies in glucose treated animals were performed on small (congruent to 100 mm3) and large (congruent to 300 mm3) tumors. In mannitol treated animals, tumors with an average volume of congruent to 200 mm3 were used. Using this tumor model, intraperitoneally administration of the hypertonic sugar solutions caused similar declines in tumor microcirculation (mannitol, 60 +/- 8% flow reduction; glucose, 72 +/- 4% flow reduction; t = 60 min). These changes were not glucose-specific and can primarily be explained by a water shift into the abdominal cavity and an associated hypovolemic hemoconcentration. A stable (small tumors) or transiently increased (large tumors) tumor energy metabolism which occurred after glucose administration was probably caused by a transiently increased glucose availability. The decline in energy metabolism after mannitol, a non-metabolized sugar alcohol, and the earlier decline in tumor pH seen in the glucose treated animals, supports this conclusion. The differences in the high energy phosphate response to glucose seen in small compared with large tumors, suggests that the baseline metabolic state of larger tumors includes a glucose deficiency in addition to tumor hypoxia.