An ex vivo model for the study of tumor metabolism by nuclear magnetic resonance: characterization of the phosphorus-31 spectrum of the isolated perfused Morris hepatoma 7777

We have developed an isolated perfused tumor model to study the metabolism of solid tumors by nuclear magnetic resonance spectroscopy. Morris hepatomas (7777) were implanted in the inguinal region of Buffalo rats, such that they developed an isolated blood supply. These tumors were perfused with a RBC perfusate, removed from the animal, and studied by 31P nuclear magnetic resonance spectroscopy. ATP levels, as determined from the spectra, were stable for as long as the tumors were maintained in the magnet (7 h) only if the perfusate contained inosine, adenosine, and insulin. The adenosine and inosine were also required for recovery from ischemia. Under these conditions, ischemia did not result in a change in tumor pH. The gamma nucleoside triphosphate resonance was significantly larger than the beta nucleoside triphosphate resonance in spectra of some of the perfused tumors, suggesting that ADP above about 300 nmol/g wet weight was not complexed in these tumors. The adenylate levels determined from extracts, O2 consumption, histology, and 31P nuclear magnetic resonance spectra of extracts of perfused tumors and tumors in situ were all similar, indicating the perfused tumor is a reasonable model of the tumor in vivo.     

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.     

31P-nuclear magnetic resonance studies of the effect of recombinant human interleukin 1 alpha on the bioenergetics of RIF-1 tumors

The effect of a single injection of human recombinant interleukin 1 alpha (IL-1 alpha) on s.c. RIF-1 tumors in mice was studied by in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were obtained before and up to 24 h after IL-1 alpha. At 2, 4, 6, and 8 h after IL-1 alpha injection, RIF-1 tumors exhibited a reduction in bioenergetic status compared to untreated controls. The Pi to beta-nucleoside triphosphate and the phosphomonoester to beta-nucleoside triphosphate ratios increased, while the phosphocreatine to Pi and phosphodiester to phosphomonoester ratios decreased. Tumor blood flow, estimated by 86RbCl uptake, decreased within 30 min after IL-1 alpha treatment. Minimum perfusion was detected at 4 h, with recovery between 6 and 12 h after IL-1 alpha treatment. Histological sections of the RIF-1 tumors revealed intravascular congestion by 2 h, extravascular hemorrhage by 4 h, and necrosis by 12 h after treatment with IL-1 alpha. The time course of bioenergetic changes in RIF-1 tumors determined by 31P-NMR spectroscopy was found to parallel the reduction and subsequent recovery of tumor blood flow.     

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.     

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.     

Assessment of tumor energy and oxygenation status by bioluminescence, nuclear magnetic resonance spectroscopy, and cryospectrophotometry

The energy and oxygenation status of tumors from two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) were assessed using three independent techniques. Tumor energy metabolism was investigated in vivo by 31P nuclear magnetic resonance spectroscopy. After nuclear magnetic resonance measurements, tumors were frozen in liquid nitrogen to determine the tissue ATP concentration by imaging bioluminescence and to register the intracapillary oxyhemoglobin (HbO2) saturation using the cryospectrophotometric method. There was a positive correlation between the nucleoside triphosphate beta/total resonance ratio or a negative correlation between the Pi/total resonance ratio and the model ATP concentration obtained by bioluminescence, respectively. This was true for small tumors with no extended necrosis irrespective of tumor type. Moreover, a positive correlation was obtained between the HbO2 saturations and the ATP concentration measured with bioluminescence. The results demonstrate the potential of combined studies using noninvasive, integrating methods and high-resolution imaging techniques for characterizing the metabolic milieu in tumors.     

31P magnetic resonance spectroscopic profiles of neoplastic human breast tissues

Phosphorus-containing metabolites of human breast tissues from malignant, benign, and noninvolved breast parenchymal specimens were examined by using techniques of perchloric acid extraction and 31P magnetic resonance spectroscopy. Twenty-four separate resonances arising from the established phosphorylated metabolites of high-energy- and low-energy-phosphate intermediary metabolism were identified and quantitated. Subsequent to magnetic resonance spectroscopic analysis, the data from the three tissue groups were compared and contrasted on a statistical basis by using Scheffé simple and complex contrast procedures. Theories of tumor metabolism and biochemical interactions were invoked, including the tissue high-energy-/low-energy-phosphate modulus, the phosphomonoester/Pi ratio, and 10 other metabolic indices. The data demonstrated the ability of 31P magnetic resonance spectroscopy to differentiate among the three tissue groups. Both benign and malignant tumors demonstrated comparable Warburg effects. Phosphomonoester metabolism was shown to be altered in neoplastic tissues relative to the noninvolved tissues. Phosphocreatine was elevated in benign tumors. This elevation in phosphocreatine plus a parallel elevation in an uncharacterized phosphate resonating at a chemical shift of 3.66 delta permits the important differentiation between malignancy and benignancy in human breast disease. The tissue energy modulus indicated that benign tissue is relatively more aerobic than noninvolved tissue and significantly more aerobic than malignant tissue.     

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.     

Evaluation of the effects of photodynamic therapy with phosphorus 31 magnetic resonance spectroscopy.

Magnetic resonance spectroscopy in situ was used to study changes in phosphorus 31 metabolism after photodynamic therapy (PDT) of transplanted HeLa cell tumours. Tumours were irradiated 2 h after administration of ATX-S10 (8-formyloximethylidene-7-hydroxy-3-ethenyl-2,7,12,18, tetramethyl-porphyrin-13,17-bispropionil aspartate), a new photosensitizer and chlorin derivative. Nuclear magnetic resonance spectra were measured prior to illumination and 1, 3, 7, 14, 21 and 28 days after PDT on each mouse. A drastic decrease in adenosine triphosphate (ATP) and a concomitant increase in inorganic phosphate (Pi) were evident on the first day after PDT in all cases. The beta-ATP/total phosphate (P) ratio was 0.64 +/- 0.29% (average +/- s.d.) in complete response, 0.67 +/- 0.30% in recurrence and 2.45 +/- 0.93% in partial response. Comparison of this ratio to the histological findings revealed that the beta-ATP/total P ratio reflects the HeLa cell tumours which survived PDT. In other words, partial response on the one hand was distinguished from complete response and recurrence on the other with this ratio 1 day after PDT (P < 0.05). In addition, the ratio of phosphomonoester (PME) to Pi rose beyond 1.0 when macroscopic recurrence occurred, while it stayed under 1.0 in complete response. This finding suggests that the recurrence of HeLa cell tumours can be detected by the PME/Pi ratio.     

Noninvasive Magnetic Resonance Spectroscopic Pharmacodynamic Markers of a Novel Histone Deacetylase Inhibitor, LAQ824, in Human Colon Carcinoma Cells and Xenografts

The aim of this work was to use phosphorus magnetic resonance spectroscopy (³¹P MRS) to investigate the pharmacodynamic effects of LAQ824, a histone deacetylase (HDAC) inhibitor. Human HT29 colon carcinoma cells were examined by ³¹P MRS after treatment with LAQ824 and another HDAC inhibitor, suberoylanilide hydroxamic acid. HT29 xenografts and tumor extracts were also examined using ³¹P MRS, pre- and post-LAQ824 treatment. Histone H3 acetylation was determined using Western blot analysis, and tumor microvessel density by immunohistochemical staining of CD31. Phosphocholine showed a significant increase in HT29 cells after treatment with LAQ824 and suberoylanilide hydroxamic acid. In vivo, the ratio of phosphomonoester/total phosphorus (TotP) signal was significantly increased in LAQ824-treated HT29 xenografts, and this ratio was inversely correlated with changes in tumor volume. Statistically significant decreases in intracellular pH, β-nucleoside triphosphate (β-NTP)/TotP, and β-NTP/inorganic phosphate (Pi) and an increase in Pi/TotP were also seen in LAQ824-treated tumors. Tumor extracts showed many significant metabolic changes after LAQ824 treatment, in parallel with increased histone acetylation and decreased microvessel density. Treatment with LAQ824 resulted in altered phospholipid metabolism and compromised tumor bioenergetics. The phosphocholine and phosphomonoester increases may have the potential to act as pharmacodynamic markers for noninvasively monitoring tumor response after treatment with LAQ824 or other HDAC inhibitors.     

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.     

Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids

We have measured the 31P nuclear magnetic resonance spectra of EMT6/Ro multicellular tumor spheroids over a wide range of sizes under constant nutrient conditions which matched those used for culturing the spheroids. The amount of nucleotide triphosphate per cell decreased with spheroid growth, roughly in proportion to the decrease in cell volume. There was no correlation between the intracellular pH, the nucleotide triphosphate:Pi ratio, or the phosphocreatine:Pi ratio and either the spheroid cellularity, the mean cell volume, the S-phase fraction, the clonogenic capacity, or the amount of central necrosis. The phosphoryethanolamine:phosphorylcholine ratio also increased with increasing spheroid size. There was a negative correlation between the phosphoryethanolamine:phosphorylcholine ratio and the S-phase cell fraction or the mean cell volume; this ratio was positively correlated with the extent of central necrosis. The membrane degradation components glycerophosphorylcholine and glycerophosphorylethanolamine showed no significant changes with increasing spheroid size. These results imply that spheroid necrotic areas induced by chronic nutrient deficiencies are "invisible" to 31P nuclear magnetic resonance and that the development of cellular quiescence in spheroids is not caused by a decrease in the steady-state level of high-energy phosphates or a reduced intracellular pH. Together, these data support a model in which cells maintain normal steady-state levels of high energy phosphates until they are very close to necrotic cell death. This implies that the deterioration of 31P nuclear magnetic resonance spectra of tumors with increasing size is not caused by chronic nutrient deficiencies resulting from cells outgrowing the capillary supply, but rather is more related to transient nutrient deprivation phenomena.     

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.     

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.     

The use of magnetic resonance imaging and spectroscopy in the assessment of patients with head and neck and other superficial human malignancies

The proper demarcation of diseased tissue is important for radiation therapy planning and treatment. The volume to be irradiated is usually identified on radiographs or on x-ray computed tomography (CT) sections. Magnetic resonance (MR)-derived images of the proton T2 relaxation times in small pixel elements, typically 0.5 mm2 or less, provide significantly sharper differentiation between normal and diseased tissue. The T2 values in tissue depend on the tissue composition, histologic condition, and physiologic environment within the tumor. Furthermore, for many tumors the histogram of T2 values has a clear biphasic distribution suggesting that T2 maps may be useful for the identification of necrotic or hypoxic regions within tumors. The distribution of T2 values within the tumor bed shows the general pattern that the T2 values are elevated with a range greater than that seen in normal muscle. Elevated T2 values are not by themselves diagnostic of malignancy; however, they demonstrate the heterogeneity of the microenvironment present within a tumor. The spatial distribution of T2 values is being explored as a method for computer assistance in the delineation of the target volume for treatment planning. In addition, MR P-31 spectroscopic examinations were performed on 30 patients with squamous cell carcinomas of the head and neck. Although hampered by muscle contamination in some P-31 spectra obtained with surface coil profile localization techniques, significant trends can still be appreciated in our data. These trends include the following: (1) the P-31 spectra from malignant tissue have well-resolved spectral lines in the upfield region that correspond to Pi, phosphomonoester (PME), and phosphodiester (PDE) not usually seen in normal muscle; (2) the PDE/B-ATP and PME/B-ATP ratios are greater than unity in all cases; and (3) most of the tumors have higher PME peaks than PDE peaks. The P-31 spectra from patients treated with ionizing radiation changed during and after therapy. Some of the changes could be associated with alteration of the tumor metabolic activity or synthesis and breakdown of lipoproteins. These studies suggest that magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) studies may be useful for both radiotherapy treatment planning and the noninvasive monitoring of patients both before and during treatment.     

Phosphorus-31 metabolism of post-menopausal breast cancer studied in vivo by magnetic resonance spectroscopy.

We have studied the metabolism of 31P-containing metabolites of post-menopausal breast cancers in vivo using magnetic resonance spectroscopy (MRS) and a 5.5 cm surface coil. Spectra were acquired from 23 diameter. The spectra of the 19 previously untreated tumours had significantly higher phosphomonoester (PME) 31P relative peak areas than the normal breasts of eight post-menopausal women (11.7% and 7.7% respectively, P = 0.002). Although an increased PME relative peak area was characteristic of malignancy, PME relative peak area is similarly raised in lactating breast and, therefore, not a specific feature of cancer. An apparently lower nucleotide triphosphate (NTP) relative peak area in tumours than healthy postmenopausal breast was secondary to the differences in PME relative peak area; contamination by signal from chest wall muscle probably accounts for the ostensibly higher phosphocreatine (PCr) relative peak area of the tumours. Spectroscopy was repeated following chemotherapy in six women. An increase in PCr relative peak area was seen in all five patients who responded, but again this may represent increased contamination secondary to changes in tumour size. A fall in PME relative peak area was noted in four responders, but also one non-responder, so this finding may not be sufficiently specific to be of use clinically. Further studies are need to elucidate fully the role of MRS in breast cancer.     

Use of high-resolution 31P-labeled topical magnetic resonance spectroscopy to monitor in vivo tumor metabolism in rats

A probe using a single-tuned solenoid coil has been constructed to study in vivo metabolism of rats in a wide-bore Bruker nuclear magnetic resonance spectrometer. Transplantable rat mammary adenocarcinomas (estrogen receptor negative) were implanted into the hind leg muscle of 8-week-old rats. The other leg without tumor was used as a control. Tumor metabolism could be distinguished from that of surrounding muscle by the appearance of inorganic phosphate and sugar phosphate resonances, reflecting tissue necrosis, and increased glycolysis. Tumor growth was accompanied by an increase in the size of these peaks, and the chemical shifts of the inorganic phosphate peak indicated that the intracellular pH became more acidic. Administration of methotrexate (i.v.) reversed these patterns and decreased tumor volume. Changes in the phosphocreatine peaks indicated changes in tumor volume rather than in tumor metabolism. These studies show that topical magnetic resonance not only can monitor the growth of tumors in vivo but can be also used to evaluate the efficacy of chemotherapeutic drugs.     

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.     

Metabolic signatures associated with a NAD synthesis inhibitor-induced tumor apoptosis identified by 1H-decoupled-31P magnetic resonance spectroscopy.

PURPOSE: Attempts to selectively initiate tumor cell death through inducible apoptotic pathways are increasingly being exploited as a potential anticancer strategy. Inhibition of NAD+ synthesis by a novel agent FK866 has been recently reported to induce apoptosis in human leukemia, hepatocarcinoma cells in vitro, and various types of tumor xenografts in vivo. In the present study, we used 1H-decoupled phosphorus (31P) magnetic resonance spectroscopy (MRS) to examine the metabolic changes associated with FK866 induced tumor cell death in a mouse mammary carcinoma. EXPERIMENTAL DESIGN: Induction of apoptosis in FK866-treated tumors was confirmed by histology and cytofluorometric analysis. FK866-induced changes in mammary carcinoma tumor metabolism in vivo were investigated using 1H-decoupled 31P MRS. To discern further the changes in metabolic profiles of tumors observed in vivo, high-resolution in vitro 1H-decoupled 31P MRS studies were carried out with perchloric acid extracts of mammary carcinoma tumors excised after similar treatments. In addition, the effects of FK866 on mammary carcinoma tumor growth and radiation sensitivity were studied. RESULTS: Treatment with FK866 induced a tumor growth delay and enhanced radiation sensitivity in mammary carcinoma tumors that was associated with significant increases in the 31P MR signal in the phosphomonoester region and a decrease in NAD+ levels, pH, and bioenergetic status. The 31P MRS of perchloric acid extracts of treated tumors identified the large unresolved signal in the phosphomonoester region as the resultant of resonances originating from intermediates of tumor glycolysis and guanylate synthesis in addition to alterations in pyridine nucleotide pools and phospholipid metabolism. CONCLUSION: The present results suggest that FK866 interferes with multiple biochemical pathways that contribute to the increased cell death (apoptosis) and subsequent radiation sensitivity observed in the mammary carcinoma that could be serially monitored by 31P MRS.     

Effects of chemotherapy by 1,3-bis(2-chloroethyl)-1-nitrosourea on single-quantum- and triple-quantum-filtered 23Na and 31P nuclear magnetic resonance of the subcutaneously implanted 9L glioma

The effects of chemotherapy [25 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea administered with a single i.p. injection] on cellular energetics by 31P nuclear magnetic resonance (NMR) spectroscopy, total tissue sodium by single-quantum (SQ) 23Na NMR spectroscopy, and intracellular sodium by triple-quantum-filtered (TQF) 23Na NMR spectroscopy were studied in the s.c. 9L glioma. Animals were studied by NMR 2 days before therapy and 1 and 5 days after therapy. Destructive chemical analysis was also performed 5 days after therapy to validate the origin of changes in SQ and TQF 23Na signals. One day after treatment, there was no significant difference between control and treated tumors in terms of tumor size or 23Na and 31P spectral data. Five days after therapy, treated tumors had 28 +/- 16% (P < 0.1) lower SQ 23Na signal intensity, 46 +/- 20% (P < 0.05) lower TQF 23Na signal intensity, 125 +/- 51% (P < 0.05) higher ATP:Pi ratio, 186 +/- 69% (P < 0.05) higher phosphocreatine:Pi ratio, and 0.17 +/- 0.06 pH units (P < 0.05) higher intracellular pH compared with control tumors. No significant differences in TQF 23Na relaxation times were seen between control and treated tumors at any time point. Destructive chemical analysis showed that the relative extracellular space of control and treated tumors was identical, but the treated tumors had 21 +/- 8% (P < 0.05) lower total tissue Na+ concentration and 60 +/- 24% (P < 0.05) lower intracellular Na+ concentration compared with the controls. The higher phosphocreatine:Pi and ATP:Pi ratios after 1,3-bis(2-chloroethyl)-1-nitrosourea treatment indicate improved bioenergetic status in the surviving tumor cells. The decrease in SQ and multiple-quantum-filtered 23Na signal intensity was largely attributable to a decrease in Na(i)+ because the treatment did not change the relative extracellular space. The improved energy metabolism could decrease the intracellular concentration of Na+ by increasing the activity of Na+-K+-ATPase and decreasing the activity of Na+/H+. Although both 23Na and 31P spectra were consistent with improved cellular metabolism in treated tumors, the 23Na methods may be better suited for monitoring response to therapy because of higher signal:noise ratio and ease of imaging the single 23Na resonance.