Response of radiation-induced fibrosarcoma-1 in mice to cyclophosphamide monitored by in vivo 31P nuclear magnetic resonance spectroscopy

In vivo 31P nuclear magnetic resonance spectroscopy has been used to examine the RIF-1 fibrosarcoma in mice during untreated growth and following chemotherapy with cyclophosphamide. Levels of inorganic phosphate increase relative to phosphocreatine or nucleoside triphosphates during early untreated growth. After the tumor reaches a volume of approximately 1 g, no further decrease in energy level is observed. Following treatment with cyclophosphamide, tumor phosphorus metabolite ratios and pH are significantly altered, compared to untreated age-matched controls. During the growth delay period following chemotherapy there is a significant reduction in the ratio of inorganic phosphate to other phosphate metabolites, compared to age-matched controls. In addition, a more alkaline pH is observed in the tumors of treated animals. When the growth delay period ends, nuclear magnetic resonance spectra return to pretreatment patterns. The magnitude of the differences in 31P nuclear magnetic resonance spectral parameters between treated animals and untreated controls is dose dependent. However, doses of cyclophosphamide above 200 mg/kg do not result in earlier spectroscopic alterations, nor in larger effects by Day 3 after treatment, even though clonogenic cell killing and growth delay are greater at these higher doses.     

Measurements of in vivo 31P nuclear magnetic resonance spectra in neuroectodermal tumors for the evaluation of the effects of chemotherapy

The effects of chemotherapy on living tumor tissue in hamsters and rats were investigated by measuring the 31P nuclear magnetic resonance spectra using topical magnetic resonance. Human neuroblastoma, human glioblastoma, and rat glioma tumor cells were inoculated s.c. in the lumbar region of the animals. After the diameter of the tumors increased to 1.5 cm, in vivo 31P nuclear magnetic resonance spectra were measured selectively in the tumors with a TMR-32 spectrometer. Adenosine triphosphate, inorganic phosphate (Pi), phosphodiester, and phosphomonoester peaks were observed. The phosphocreatine peak was hardly detectable, adenosine triphosphate and phosphomonoester peaks were high, and tissue pH, calculated from the chemical shift of Pi, declined. Regardless of the tumor origin or the histological type, the spectral pattern of each neuroectodermal tumor was found to be essentially the same. After i.v. injection of a large dose of a chemotherapeutic agent, adenosine triphosphate peaks decreased and Pi increased gradually, resulting in a dominant Pi peak pattern after 6 to 12 hours. However, during the same period, there were no observable changes in the spectra of normal organs. These findings indicated that the drugs have a selective and direct action on the energy metabolism of tumor cells. With lower drug doses, no remarkable changes were seen in the spectrum. Measurement of in vivo 31P nuclear magnetic resonance spectra is valuable not only to investigate the energy metabolism in tumor tissue but also to evaluate the effects of chemotherapy.     

In Vivo Determination of ATP in Tumors Using 31P Inversion Spin Transfer

The in vivo exchange kinetics of creatine kinase in the hind leg muscle of rats containing a transplanted mammary adenocarcinoma has been investigated using ³¹P magnetic resonance spectroscopy. Using a solenoid coil, the adenosine triphosphate (ATP) resonances arising from the tumor could be distinguished from ATP resonances arising from the muscle surrounding the tumor by use of inversion spin transfer techniques. This procedure affords a specific method of evaluating ATP metabolism of tumors in vivo.     

Loss of high-energy phosphate following hyperthermia demonstrated by in vivo 31P-nuclear magnetic resonance spectroscopy

We have used in vivo 31P-nuclear magnetic resonance spectroscopy to study the changes in high-energy phosphates following hyperthermia. Immediately after heating, there is a fall in adenosine triphosphate and apparent intracellular pH and an increase in inorganic phosphate. Following sublethal heating (40 degrees for 15 min), these changes were partial, and they resolved over the subsequent 45 hr. With tumors given severe hyperthermia (47 degrees for 15 min), there was complete disappearance of adenosine triphosphate, with no recovery by 24 hr posttreatment. Qualitatively similar effects were seen after heating of normal leg muscle. The degree of fall of the adenosine triphosphate/inorganic phosphate concentration ratio was directly proportional to the heat dose and to thermal cell kill. 31P-Nuclear magnetic resonance spectroscopy may be useful in thermal dosimetry and treatment evaluation following hyperthermia.     

In vivo 31P nuclear magnetic resonance spectroscopy of subcutaneous 9L gliosarcoma: effects of tumor growth and treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea on tumor bioenergetics and histology

In vivo 31P nuclear magnetic resonance spectroscopy was used to examine the bioenergetics of the rat 9L gliosarcoma during untreated growth and in response to chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea. Tumor growth was associated with a decline in the phosphocreatine and nucleoside triphosphate resonances, consistent with an increase in tumor hypoxia during untreated growth. Following chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (10 mg/kg), tumor levels of phosphocreatine and nucleoside triphosphate rebounded while the level of inorganic phosphate in the tumor declined. Histological comparison of treated and untreated tumor sections 4 days posttreatment showed that the treated tumor had a lower proportion of necrotic cells, a higher proportion of viable cells, and a 5-fold higher level of interstitial space than the control tumor.     

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.     

Nuclear magnetic resonance spectroscopy and sensitizer-adduct measurements of photodynamic therapy-induced ischemia in solid tumors.

Dunning R3327-AT prostate carcinomas growing in Fischer X Copenhagen rats were treated with interstitial photodynamic therapy (PDT--15 mg/kg Photofrin II 4 hours before illumination with 630-nm light via four parallelly implanted optical fibers) at different light intensities. Forty to 60 minutes after treatment, 31P-nuclear magnetic resonance spectra of tumors in anesthetized animals were obtained at 2.35 Tesla using surface coil localization. Areas under resonance peaks were normalized to the area under the peak of a phosphorus standard positioned at a fixed distance on the opposite side of the surface coil. Tumor concentrations of phosphomonoesters and phosphodiesters showed no change after tumor light doses up to 3000 J. Phosphocreatine, alpha-adenosine triphosphate (ATP), beta-ATP, and gamma-ATP signals decreased and inorganic phosphate signals increased with increasing light doses. The intratumor pH did not change significantly at these short times after PDT. In other R3327-AT and R3327-H tumor-bearing animals, [3H]misonidazole was administered 30 minutes prior to PDT treatments of both tumors. Twenty-four hours later, the tumors were resected in toto, and levels of retained [3H]misonidazole were determined in lased tumor specimens by liquid scintillation procedures. The amount of [3H]misonidazole activity in tumor tissue (covalently bound after hypoxic reduction) increased with light doses up to 3000 J. Sensitizer-adduct formation was found to correlate with the ratio of the concentration of inorganic phosphate to that of beta-ATP, both of which are presumed measures of tumor oxygenation status. These measurements have high-lighted the heterogenous nature of the oxygenation status of these experimental tumors. The precision of each assay for estimating tumor oxygenation is discussed.     

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.     

Integrated magnetic resonance imaging and phosphorus spectroscopy of soft tissue tumors

Eighteen patients with soft tissue masses underwent integrated magnetic resonance imaging (MRI) and phosphorus spectroscopy (31P-MRS) to evaluate benign and malignant tumor morphology and metabolism. Spectra from soft tissue tumors had a significantly higher proportion of phosphate in the low-energy portion of the 31P spectrum (P less than 0.001) with a concomitant decrease in phosphocreatine (P less than 0.01) compared with 31P spectra from normal muscle. Malignant tumors had a mean pH of 7.35 +/- 0.13 which was greater than that of muscle tissue with a mean pH of 7.08 +/- 0.07 (P less than 0.001). All tumors had greater relative levels of phosphomonoesters, inorganic phosphate, and phosphodiesters compared with those in muscle tissue but considerable variability among tumors was noted due to tumor size, extent of tumor necrosis, and muscle contamination. Integrated MRI/MRS studies are necessary to provide exact localization of the tumor and a more correct interpretation of the 31P-MRS data.     

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.     

Characterization of the 31P nuclear magnetic resonance spectrum from human melanoma tumors implanted in nude mice

31P nuclear magnetic resonance spectra of human melanoma (BRO) cells implanted in nude mice were obtained both in vitro and in vivo. The tumors were allowed to grow in the right axillary region of six adult Swiss nude mice to a transverse diameter of 1.5-2 cm, at which point the in vivo 31P nuclear magnetic resonance spectra were obtained. The animals were subsequently sacrificed and the tumor perchloric acid extract was studied in vitro. Relative peak areas are comparable in the two experiments with the exception of inorganic phosphate, which is more abundant in vivo than in vitro by a factor of 4. This difference may be attributed to a greater contribution of the necrotic portion of the tumor to the in vivo spectra. Resonance peaks in the spectrum of the extract were identified on the basis of their coincidence with standards added at pH 7 and 10. Non-energy phosphorylated metabolites present in the tumor at high levels include phosphoethanolamine, phosphocholine, glycerol phosphocholine, and uridine-5'-diphospho-N-acetyl glucosamine. Sugar phosphates and 2,3-diphosphoglycerate from blood made minor contributions to the spectrum. The tumor also contained substantial amounts of pyrimidine triphosphates accounting for 34% of the total nucleoside triphosphate pool.     

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.     

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.     

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.     

High microvascular blood volume is associated with high glucose uptake and tumor angiogenesis in human gliomas.

The purpose of this investigation was to elucidate the association between microvascular blood volume and glucose uptake and to link these measures with tumor angiogenesis. We demonstrate a regionally specific correlation between tumor relative microvascular blood volume (CBV), determined in vivo with functional magnetic resonance imaging techniques, and tumor glucose uptake determined with fluorodeoxyglucose positron emission tomography. Regions of maximum glucose uptake were well matched with maximum CBV across all patients (n = 21; r = 0.572; P = 0.023). High-grade gliomas showed significantly elevated CBV and glucose uptake compared with low-grade gliomas, (P = 0.009 and 0.008, respectively). Correlations between CBV and glucose uptake were then determined on a voxel-by-voxel basis within each patient's glioma. Correlation indices varied widely, but in 16 of 21 cases of human glioma, CBV and glucose uptake were correlated (r > 0.150). These measures were well correlated in all cases when comparing healthy brain tissue in these same patients. Tumor vascularity, as determined immunohistochemically and morphometrically on clinical samples, revealed statistically significant relationships with functional imaging characteristics in vivo. Regional heterogeneities in glucose uptake were well matched with functional magnetic resonance imaging CBV maps. Our findings support the concept that there is an association of microvascular density and tumor energy metabolism in most human gliomas. In addition, the findings are likely to have important clinical applications in the initial evaluation, treatment, and longitudinal monitoring of patients with malignant gliomas.     

Partial inhibition of the growth of transplanted dunning rat prostate tumors with the long-acting somatostatin analogue sandostatin (SMS 201-995)

The growth-inhibiting effects of the long-acting somatostatin analogue Sandostatin on the transplanted Dunning R3327-H androgen-sensitive rat prostate tumor were investigated. Recipient animals were male Copenhagen x Fischer F1 rats (N = 36). When mean tumor volume reached 700 mm3 (20 weeks following transplantation), the rats were divided into four groups: control; Sandostatin (100 micrograms/kg s.c. twice a day); castrate; castrate/Sandostatin. Tumor size was assessed by magnetic resonance imaging 21, 42, 63, 105, and 138 days subsequently. Administration of Sandostatin was interrupted between days 43 and 62. As assessed by transplant volume, Sandostatin caused a moderate (up to 50%) but highly significant (P less than 0.001) suppression of tumor growth in the intact rats; the effect was reversed when drug administration was stopped. In the castrates, in which tumor growth was markedly less than in intact rats, no significant effect of Sandostatin was seen. Analysis of the tumor growth rate demonstrated that Sandostatin led to a 19% reduction (P less than 0.05) in growth rate in intact rats and a 9% decrease (not significant) in castrates. These findings extend previous reports of partial suppression of various types of tumors in vivo with Sandostatin and other somatostatin analogues. Their relevance with regard to the possible use of Sandostatin in the treatment of prostatic carcinoma in humans is discussed.     

Use of high resolution in vivo volume selected 1H-magnetic resonance spectroscopy to investigate leukemia in humans

In vivo high resolution volume-selected 1H magnetic resonance spectroscopy of human tibia has been undertaken using spatial coordinates obtained from magnetic resonance images. Adult tibial marrow has a 1H spectrum rich in fatty acid resonances and is readily distinguished from the 1H spectra of surrounding leg muscle. In all four leukemic patients examined, infiltration of fat cells of tibial marrow by proliferating cells rich in mobile H2O protons was evident by magnetic resonance imaging. Selective examination of volumes of tibial marrow (1 cm3) by 1H magnetic resonance spectroscopy confirmed marked differences in the 1H spectra of marrow from these patients. Increases in the H2O peak of the 1H spectra were correlated with infiltration of blast cells and lack of control of the neoplastic disease. These studies are the first to report the use of volume selected magnetic resonance spectroscopy to selectively monitor leukemia in humans.     

Volume Reconstruction Techniques Improve the Correlation Between Histological and in vivo Tumor Volume Measurements in Mouse Models of Human Gliomas

Assessment of therapy efficacy using animal models of tumorigenic cancer requires the ability to accurately measure changes in tumor volume over the duration of disease course. In order to be meaningful, in vivo tumor volume measurements by non-invasive techniques must correlate with tumor volume measurements from endpoint histological analysis. Tumor volume is frequently assessed by endpoint histological analyses approximating the tumor volume with geometric primitives such as spheroids and ellipsoids. In this study we investigated alternative techniques for quantifying histological volume measurements of tumors in a xenograft orthotopic mouse model of human glioblastoma multiforme, and compared these to in vivo tumor volume measurements based on magnetic resonance imaging (MRI) data. Two techniques leveraging three-dimensional (3D) image analysis methods were investigated. The first technique involves the reconstruction of a smoothed polygonal model representing the tumor volume from histological section images and is intended for accuracy and qualitative assessment of tumor burden by visualization, while a second technique which approximates the tumor volume as a series of slabs is presented as an abbreviated process intended to produce quantitatively similar volume measurements with a minimum of effort required on behalf of the investigator. New software (QuickVol) designed for use in the first technique, is also discussed. In cases where tumor growth is asymmetric and invasive, we found that 3D analysis techniques using histological section images produced volume measurements more consistent with in vivo volume measurements based on MRI data, than approximation of tumor volume using geometric primitives. Visualizations of the volumes represented by each of these techniques qualitatively support this finding, and suggest that future research using mouse models of glioblastoma multiforme (genetically engineered or xenograft) will benefit from the use of these or similar alternative tumor volume measurement techniques.     

Early metabolic response to tumor necrosis factor in mouse sarcoma: a phosphorus-31 nuclear magnetic resonance study

To investigate the effects of recombinant human tumor necrosis factor alpha (rHuTNF-alpha) on high-energy phosphate metabolism of cancer cells, 31P nuclear magnetic resonance (NMR) studies were performed on a murine methylcholanthrene-induced sarcoma. Injection of 15 micrograms of rHuTNF-alpha caused progressive depletion of ATP and phosphocreatine within 90 min, together with an increase in inorganic phosphate. Metabolic changes were correlated with the early histological appearance of thrombosis and hemorrhage. A spatially localized NMR technique demonstrated that these changes were specific for the tumor. Acute ischemia of the tumor produced similar metabolic changes; thus the metabolic effects of rHuTNF-alpha could be due to either a primary action on tumor biochemistry or a secondary action produced by ischemia. These findings indicate that rHuTNF-alpha has a very rapid onset of action, which can be detected by 31P NMR. Furthermore, the results suggest that 31P NMR spectroscopy will be extremely useful for detecting early biochemical changes produced by rHuTNF-alpha or other treatments in animal and human cancers.     

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.