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.     

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).     

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.     

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.     

Angiogenesis determines blood flow, metabolism, growth rate, and ATPase kinetics of tumors growing in an irradiated bed: 31P and 2H nuclear magnetic resonance studies.

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased and growth rate is slowed, these tumors are assumed to be metabolically deprived with hypoperfusion. In this study, we directly measured the effect of tumor bed irradiation on blood flow, growth rate, rate of nucleoside triphosphate (NTP) turnover, and metabolic state using 31P and 2H nuclear magnetic resonance, and an intradermal assay for angiogenesis. (NTP turnover refers to ATP-synthetase mediated NTP turnover that is visible to 31P nuclear magnetic resonance using the technique of saturation transfer.) A decrease in the number of small blood vessels perfusing tumors in a preirradiated bed was found. Most of the decrease was due to a loss of vessels with diameters less than 0.04 mm. When tumors growing in preirradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed in most tumors. Consistent with these observations, the phosphocreatine/inorganic phosphate and nucleoside triphosphate/inorganic phosphate ratios were significantly lower in the tumors growing in a preirradiated bed compared with tumors in a nonirradiated bed. The blood flow rate was similar to control for tumors less than 100 mm3 (45.8 versus 40.5 ml/100 g/min, P = not significant), but was significantly lower than control for tumors greater than 100 mm3 (40.4 versus 12.2 ml/100 g/min, P less than 0.01). The NTP turnover rates correlated (P less than 0.005, r = 0.66) with the volume doubling rate (1/tumor volume doubling time), but for tumors approximately 100 mm3 in size neither the volume doubling rate nor the NTP turnover rate of tumors growing in an irradiated bed was statistically lower than control [NTP turnover: 14 +/- 3%/s versus 9 +/- 2%/s; volume doubling rate: 0.47 +/- 0.07/day versus 0.33 +/- 0.04/day (mean +/- SE)]. A large intertumor variability of all metabolic parameters was observed.     

Relation between pO2, 31P magnetic resonance spectroscopy parameters and treatment outcome in patients with high-grade soft tissue sarcomas treated with thermoradiotherapy

PURPOSE: In a prior study, the combination of (31)P magnetic resonance spectroscopy (MRS)-based intracellular pH (pHi) and T2 relaxation time was highly predictive of the pathologic complete response (pCR) rate in a small series of patients with soft tissue sarcomas (STSs) treated with thermoradiotherapy. Changes in the magnetic resonance metabolite ratios and pO(2) were related to the pCR rate. Hypoxia also correlated with a greater likelihood for the development of metastases. Because of the limited number of patients in the prior series, we initiated this study to determine whether the prior observations were repeatable and whether (31)P MRS lipid-related resonances were related to a propensity for metastasis. METHODS AND MATERIALS: Patients with high-grade STSs were enrolled in an institutional review board-approved Phase II thermoradiotherapy trial. All tumors received daily external beam radiotherapy (1.8-2.0 Gy, five times weekly) to a total dose of 30-50 Gy. Hyperthermia followed radiotherapy by <1 h and was given two times weekly. Tumors were resected 4-6 weeks after radiotherapy completion. The MRS/MRI parameters included (31)P metabolite ratios, pHi, and T2 relaxation time. The median pO(2) and hypoxic fraction were determined using pO(2) histography. Comparisons between experimental endpoints and the pCR rate and metastasis-free and overall survival were made. RESULTS: Of 35 patients, 21 and 28 had reportable pretreatment MRS/MRI and pO(2) data, respectively. The cutpoints for a previously tested receiver operating curve for a pCR were T2 = 100 and pHi = 7.3. In the current series, few tumors fell below the cutpoints so validation was not possible. The phosphodiester (PDE)/inorganic phosphate (Pi) ratio and hypoxic fraction correlated inversely with the pCR rate in the current series (Spearman correlation coefficient -0.51, p = 0.017; odds ratio of percentage of necrosis > or =95% = 0.01 for a 1% increase in the hypoxic fraction; Wald p = 0.036). The pretreatment phosphomonoester (PME)/Pi ratio also correlated inversely with the pCR rate (odds ratio of percentage of necrosis > or =95% = 0.06 for pretreatment PME/Pi ratio >0.8 vs. < or =0.8, Wald p = 0.023). The pretreatment PME/PDE ratio correlated strongly with metastasis-free survival and overall survival (p = 0.012 and hazard ratio = 5.8, and p = 0.038 and hazard ratio = 6.75, respectively). CONCLUSION: The dual parameter model containing pHi and T2 to predict the pCR in STSs treated with thermoradiotherapy was not verified. However, other parameters were statistically significant, including the PDE/Pi ratio and hypoxic fraction. These relationships may have interfered with our ability to obtain the pCR rate predicted by thermal doses achieved in these patients. The relationship between the PME/PDE ratio and metastasis-free and overall survival was provocative, but requires additional study to verify its predictive capability. Currently, 50% of all STS patients with high-grade tumors develop distant metastasis even when excellent local control is achieved. Parameters that could help select for patients who need adjuvant chemotherapy could have significant clinical benefit.     

Prediction of tumor radiosensitivity by hexafluoromisonidazole retention monitored by [1H]/[19F] magnetic resonance spectroscopy

Hexafluoromisonidazole (CCI-103F) is a hypoxic cell label that can be measured by in vivo [1H]/[19F] magnetic resonance spectroscopy (MRS). The retention of CCI-103F in SCCVII tumors was found to be correlated with tumor size, with larger tumors retaining more label. Such a correlation was anticipated, as larger SCCVII tumors have larger hypoxic fractions. A significant but less strong correlation was also found between CCI-103F retention and the fraction of tumor cells surviving after 10 Gy irradiation, with tumors that retained larger amounts of CCI-103F having higher surviving fractions. These results indicated that non-invasive MRS measurement of CCI-103F retention could predict radiosensitivity in SCCVII tumors. The lack of a strong correlation between CCI-103F retention and radiosensitivity, however, indicated that hypoxic fraction was not the only factor influencing radiosensitivity in these tumors.     

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.     

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.     

The response of spontaneous and transplantable murine tumors to vasoactive agents measured by 31P magnetic resonance spectroscopy.

31P magnetic resonance spectroscopy has been used to compare the effects of the vasoactive agents hydralazine and flunarizine on the oxygenation of the transplantable tumors, SCCVII/Ha and 16C, and a range of spontaneous mammary tumors arising in the breeding stock in the Genetics Division at the Radiobiology Unit. The vasodilator hydralazine, previously shown to increase the radiobiological hypoxic fraction of transplantable murine tumors, increased inorganic phosphate to total phosphate (Pi/total) in SCCVII/Ha and 16C tumors. However, only two spontaneous tumors responded to this agent (2/12). The calcium antagonist flunarizine, which sensitizes the SCCVII tumor to X rays, consistent with a reduction in hypoxic fraction, reduced Pi/total in this and the 16C tumor. Further, most spontaneous tumors tested (8/10) responded to this agent, as measured by a reduction in Pi/total. These results point to fundamental differences between transplantable and spontaneously arising tumors in mice in their response to vasoactive agents.     

骨和软组织肿瘤的3.0T磁共振磷谱变化研究

目的 探讨骨和软组织肿瘤磁共振磷谱(31P-MRS)的变化特点.方法 对41例经病理证实的骨和软组织肿瘤患者的18个良性肿瘤病灶、28个恶性肿瘤病灶及其相邻部位正常肌肉组织,应用3.0T MR机进行31P-MRS分析,测量波谱中磷酸单酯(PME)、无机磷(Pi)、磷酸二酯(PDE)、磷酸肌酸(Pcr)、三磷酸腺苷γ-峰(γ-ATP)、α-峰(α-ATP)和β-峰(β-ATP)的峰下面积.分别以β-ATP、三磷酸核苷(NTP)和Pcr为参照,计算各代谢产物的相对比值.根据Pi相对于Pcr化学位移的变化计算细胞内pH值.结果 良、恶性肿瘤组中Pcr/PME、PME/NTP与正常对照组比较,差异均有统计学意义(P<0.05).良、恶性肿瘤组中PME/β-ATP与PME/NTP比较,差异有统计学意义(P<0.05).结论 Pcr/PME和PME/NTP是诊断骨和软组织肿瘤的潜在指标,PME/β-ATP和PME/NTP是鉴别骨和软组织肿瘤良、恶性的潜在指标.     

Effects of oxygen on the metabolism of murine tumors using in vivo phosphorus-31 NMR

The effect of 100% inspired oxygen on in vivo tumor metabolism was examined using phosphorus-31 (31P) NMR spectroscopy. Isotransplants of two murine tumor histologies, designated MCaIV (C3H mammary adenocarcinoma) and FSaII (C3H fibrosarcoma), were used in syngeneic mice. Tumor volumes ranged from 30 to 1,800 mm3. Both tumor histologies are known to have a high hypoxic cell fraction when tumor volumes exceed 250 mm3. 31P nuclear magnetic resonance (NMR) spectra were obtained at 145.587 MHz, and the signal was detected using a 1.4 cm diameter, single loop coil designed to localize the signal from only the tumor. Spectral parameters for optimal signal-to-noise ratio (SNR) included a 60 degrees pulse and a 2-second recycle delay. Tumors were implanted in the hindfoot dorsum to assure that all detected mobile phosphates were of tumor origin. Phosphocreatine/inorganic phosphate (PCr/Pi) ratios of large tumors (greater than 250 mm3) were reduced compared with small tumors (less than 250 mm3) of the same histology. The increased PCr/Pi response to 100% inspired oxygen was greater for large tumors and for tumors with lower baseline PCr/Pi ratios. When host animals were given 10% oxygen for respiration, there was an increase in Pi and a decrease in both PCr and ATP. The response to 10% oxygen was observed in both large and small tumors of both tumor histologies studied. Resting skeletal muscle exhibited no alteration in the NMR spectrum during either 100 or 10% oxygen breathing. We conclude that the fractional increase in PCr/Pi ratio that occurs after 100% oxygen breathing is a sensitive, noninvasive method of detecting tumor hypoxia.     

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.     

Quantitative changes in tumor metabolism, partial pressure of oxygen, and radiobiological oxygenation status postradiation.

Hypoxia is considered to be a major cause of tumor radioresistance. Reoxygenation of previously hypoxic areas after a priming dose of radiation is associated with an increase in tumor radiosensitivity. In a study of a hypoxic mammary carcinoma, 31P nuclear magnetic resonance spectra showed statistically significant increases in metabolite ratios (phosphocreatine/Pi and nucleotide triphosphate/Pi) after 65 and 32 Gy. The maximum changes in metabolite ratios after 32 Gy occurred at 48 h, although significant changes were detected at 24 h. A corresponding increase in the mean tumor pO2 (polarographic microelectrode measurements) and a decrease in hypoxic cell fraction [changes in paired (clamped versus unclamped) tumor control dose for 50% of tumors] were also shown to occur 48 h after a priming dose of 32 Gy. A significant increase in the mean tumor pO2, phosphocreatine/Pi, and nucleotide triphosphate/Pi, compared to initial values, was noted at 24, 48, and 96 h post 65-Gy radiation. An increase in the downfield component of the phosphomonoester peak relative to the upfield component (phosphoethanolamine), is also noted after doses of 65 and 32 Gy. These are likely to be due to cell kill and/or decreased cell proliferation. In this tumor model, 31P nuclear magnetic resonance spectroscopic changes postradiation are temporally coincident with and may be indicative of tumor reoxygenation as measured by the tumor control dose for 50% of tumors and oxygen-sensitive microelectrodes.     

Relationship of changes in pH and energy status to hypoxic cell fraction and hyperthermia sensitivity

The relative concentrations of nucleotide triphosphates, creatine phosphate, inorganic phosphate, and pH have been evaluated as a function of tumor volume in a murine fibrosarcoma (FSaII) by 31P NMR spectroscopy. As the tumor volume increased from 60-1250 mm3, the ratio of phosphocreatine to inorganic phosphate systemically decreased. This decrease paralleled a decrease in the ratio of nucleotide triphosphate to inorganic phosphate in the same tumor volume range. The tumor pH as measured by 31P NMR decreased slightly with tumor growth. A pH of 7.17 +/- 0.07 (n = 17) was found for tumors between 60 and 150 mm3, whereas for tumors greater than 900 mm3, a pH of 7.05 +/- .03 (n = 6) was noted. Intermediate size tumors (151-900) had a pH of 7.12 +/- 0.09 (n = 18). The change in tumor energy status with tumor volume inversely paralleled the change in tumor radiobiologic hypoxic cell fraction and suggested a causal relationship between tumor nutrient status and energy status. Tumor thermal sensitivity also increased with tumor volume, suggesting a relationship between pH, energy status, and thermal sensitivity, as has been demonstrated under in vitro conditions. Each NMR parameter was found to correlate significantly with tumor volume independent of the other NMR parameters.     

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.     

Enhancement of antitumor effect of hyperthermia with glucose administration in murine mammary carcinoma

Antitumor effects of hyperthermia are enhanced by lowering the pH in the tumor tissue with administration of glucose. This decreased pH in the tumor tissue with glucose administration was determined using mouse experimental tumors. 31P-MRS microelectrodes were used for the measurement of pH. By using these two measurement methods, time course change in the tumor tissue was determined in the controls and the groups treated with 6 g/kg of intraperitoneal glucose. The determination of pH with 31P-MRS was calculated from the chemical shift of the peak of creatine phosphate (Pcr) and that of inorganic phosphate (Pi). Following glucose administration, the tumor tissue showed a decrease of 0.3 pH units with the microelectrode method, but did not show any significant decrease in pH with the MRS determination. This finding suggested that 31P-MRS showed intracellular pH (pHi) due to the localization of Pi and that the microelectrode indicated interstitial or extracellular pH (pHe). The ATP/Pi ratio obtained in tumor tissue 24 h after heat treatment (with, without glucose) was correlated with tumor inhibition.     

Magnetic resonance spectroscopy studies on experimental murine and human tumors: comparison of changes in phosphorus metabolism with induced changes in vascular volume.

The responses of two experimental murine tumors and two human tumor xenografts to the vasodilator hydralazine were compared using two magnetic resonance spectroscopy endpoints. Changes in tumor metabolism were determined using 31P MRS where inorganic phosphate levels relative to total phosphate (Pi/total) were measured, and alteration in tumor blood volume was examined using 19F MRS with perfluorooctylbromide (PFOB) as tracer. The integrated 19F signal from PFOB is dose dependent and stable for at least 2 hr after injection. The murine tumors SCCVII/Ha and KHT both showed changes in tumor metabolism after hydralazine, as an increase in Pi/total. However, hydralazine reduced vascular volume in the KHT tumor, demonstrated by reduced 19F signal from PFOB, but no such reduction was seen in the SCCVII/Ha tumor. In contrast, hydralazine had no effect on phosphorus metabolism in the HT29 and HX118 human tumor xenografts, but reduced vascular volume in both tumors. These results demonstrate that the effects of vasoactive agents such as hydralazine on tumor phosphorus metabolism are only partially consistent with changes in vascular volume, measured by the 19F MRS technique.     

The assessment of treatment response in non-Hodgkin's lymphoma by image guided 31P magnetic resonance spectroscopy

Serial image guided 31P magnetic resonance spectroscopy (MRS) studies were performed in eight patients with non-Hodgkin's lymphoma to determine the changes in phosphorus metabolites that occur in vivo in response to chemotherapy. Pre-treatment spectral characteristics were different in high and low grade lymphoma. A larger inorganic phosphate (Pi) peak was seen in high grade NHL relative to phosphomonoesters (PME) or beta adenosine triphosphate (beta ATP), producing significant differences in the PME/Pi and Pi/beta ATP metabolite ratios, and probably reflecting a larger hypoxic cell fraction within the high grade lymphomas. Consistent metabolite changes were seen with treatment, and before reductions in tumour bulk had occurred. Alterations in tumour energetics with changes in Pi and beta ATP, and increases in phospholipid turnover reflected as an increase in the phosphodiester (PDE) resonance were detected. Changes were seen between days 10 and 27 in low grade lymphoma treated with oral alkylating therapy and between days 1 and 5 in lymphoma treated with intensive combination chemotherapy. Increases in the PDE/beta ATP metabolite ratio may be an early indicator of response to chemotherapy in human tumours. These studies illustrate the feasibility and clinical potential of image guided 31P MRS as a means of assessing response to therapy.     

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.