Response-specific adriamycin sensitivity markers provided by in vivo 31P nuclear magnetic resonance spectroscopy in murine mammary adenocarcinomas.

The in vivo phosphorus-31 nuclear magnetic resonance (NMR) spectra of Adriamycin (ADR)-sensitive murine mammary adenocarcinomas (17/A) and an ADR-resistant subline of this tumor which has been isolated in vivo (17/A/ADR) were compared both before and after i.v. administration of 12 mg/kg ADR. Significant differences between ADR-sensitive and -resistant tumors for the changes observed 1 day after treatment (prior to significant decreases in tumor size) included: the pH increased to greater than 7.3 in response to treatment (or pH remained elevated) in ADR-sensitive tumors only; the inorganic phosphate to nucleoside triphosphates peak height ratio decreased to less than 1 in response to treatment only in ADR-sensitive tumors; glycerophosphocholine to nucleoside triphosphates peak height ratio decreased in response to treatment in ADR-sensitive tumors only; and the phosphocholine to nucleoside triphosphates peak height ratio decreased in response to treatment in ADR-sensitive tumors only. These differences are evidence in support of the hypothesis that in vivo 31P-NMR provides response-specific markers of ADR sensitivity. Because 31P-NMR can be applied to humans, these differences may be of prognostic value in the clinical management of human breast cancer if they are present after treatment with lower, nontoxic doses of ADR.

Deuterium nuclear magnetic resonance imaging of tracer distribution in D2O clearance measurements of tumor blood flow in mice.

Proper implementation of direct injection clearance techniques to measure tumor blood flow (TBF) requires knowledge of the tracer distribution because TBF distribution is often inhomogeneous. Therefore, deuterium nuclear magnetic resonance imaging was used to follow tracer (HOD) distribution after direct injection of 10-40 microliters isotonic saline/D2O into RIF-1 tumors. Within 2 to 4 min after intratumor injection, tracer clearance was imaged by obtaining deuterium images every 1.4 min. The mean volume occupied by HOD in tumors in the first image acquired after injection with 10, 20, or 40 microliters D2O was 56 +/- 37 (SD) mm3, 44 +/- 2.9 mm3, and 174 +/- 83 mm3, respectively (n = 3 for each). In these control tumors, HOD was cleared from that volume without an appreciable increase in tracer distribution. In tumors heated for 45 min at 45 degrees C to greatly reduce TBF, the mean tracer volume in the first image after 10-microliters D2O injection was 41 +/- 10 mm3 and increased to 111 +/- 24 mm3 at 30 min (n = 3). For 10 microliters D2O injected at two distinct sites, the intensity decreased at each site while the sites remained separate (n = 6). The TBF at the two sites, measured independently by fitting the integrated HOD intensity from each site to a monoexponential decay function, was significantly different in only one of the six tumors examined. The use of deuterium nuclear magnetic resonance imaging to measure TBF from two (or more in larger tumors) independent sites provides a practical approach to assess TBF heterogeneity. The direct measurement of the tissue volume labeled with tracer and its dependence on injection volume should aid in determining how best to implement direct injection tracer clearance methods.

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

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

Modulation of murine radiation-induced fibrosarcoma-1 tumor metabolism and blood flow in situ via glucose and mannitol administration monitored by 31P and 2H nuclear magnetic resonance spectroscopy.

The hyperglycemia-induced in situ metabolism and blood flow changes produced in s.c. implanted murine radiation-induced fibrosarcoma-1 tumors, grown on the flanks of female C3H/HeJ mice, were examined with 31P and 2H nuclear magnetic resonance. Initial experiments verified a hyperglycemic tumor acidification similar to that reported earlier with a different substrain of mice, C3H/AnF (J.L. Evelhoch et al., Proc. Natl. Acad. Sci. USA, 81: 6496-6500, 1984). Changes in the tumor pH, phosphorus metabolites, and blood flow were then compared after administration of saline, glucose, or mannitol (a nonmetabolizable glucose analogue) using a mole-equivalent dose of the sugars (i.e., 0.8 mmol/20g mouse). Neither saline (n = 8) nor mannitol (n = 6) administration had any marked effect upon tumor pH, whereas glucose administration produced a mean maximum tumor pH reduction of 0.74 +/- 0.09 (SE; n = 9) during the 2.5 h post-glucose injection. No significant changes in high energy phosphate concentrations were observed during the same period after saline injection. After glucose injection, the [phosphocreatine] gradually decreased by 64% (P = 0.0001). After the initial 1 h post-glucose injection, the [inorganic phosphate] increased by 58% (P = 0.0001), and the [nucleoside triphosphates] decreased by 29% (P = 0.0001) during the following 1.5 h. After mannitol injection, while there was no change in [inorganic phosphate] over time (P = 0.37), the [phosphocreatine] decreased by 33% (P = 0.0001) and the [nucleoside triphosphates] decreased by 21% (P = 0.0015) within 20 min, then both the [phosphocreatine] and [nucleoside triphosphates] remained at constant levels during the following 2 h. In parallel experiments, the volumetric rate of tumor blood flow and perfusion was measured by 2H nuclear magnetic resonance monitoring of 2H2O washout kinetics (S-G. Kim and J. J. H. Ackerman, Cancer Res., 48: 3449-3453, 1988); tumor blood flow decreased by 80% (P = 0.0001, n = 11), 60% (P = 0.0031, n = 4), and 20% (P = 0.058, n = 10) at 2 h after glucose, mannitol, or saline injections, respectively. These results suggest that anaerobic glycolysis is a requirement for hyperglycemic tumor acidification. However, the decrease in tumor blood flow accompanying hyperglycemic acidification suggests that flow reduction also may be a contributing or a required cofactor for acidification via inhibition of lactic acid egress.(ABSTRACT TRUNCATED AT 400 WORDS)

Flavone acetic acid (NSC 347512)-induced modulation of murine tumor physiology monitored by in vivo nuclear magnetic resonance spectroscopy.

Flavone acetic acid (FAA), a new drug with broad activity against transplanted solid tumors of mice, induces nonrepairable DNA single strand breaks that correlate with therapeutic efficacy. To test the hypothesis that the inability of the cells to repair single strand breaks is associated with a disruption of tumor energy metabolism, in vivo 31P nuclear magnetic resonance (NMR) spectra were acquired from s.c. implanted Glasgow osteogenic sarcomas in C57BL/6 x DBA/2 F1 mice both before and after treatment with FAA i.v. at 100, 150, or 200 mg/kg and from a control (no treatment) group (n = 4 in each group). While FAA produced a dose-dependent decrease in both the nucleoside triphosphates level and pH, only treatment with an efficacious dose of 200 mg/kg resulted in both a reduction in pH and a complete loss of nucleoside triphosphates from the NMR spectrum at 4 h with no recovery until 48 h and little recovery out to 72 h. The ATP concentration determined by high pressure liquid chromatography in a parallel set of experiments was 5.59 +/- 1.16 (SE) mumol/g (wet weight) in control tumors (n = 9) and 0.24 +/- 0.12 mumol/g (wet weight) at 4 h after 200 mg/kg FAA (n = 7). To examine the possibility that the loss of ATP and decreased pH are associated with a reduction in tumor blood flow, we used 2H NMR to monitor the washout of D2O injected directly into the tumor both before and 4 h after treatment with 200 mg/kg FAA. The pretreatment tumor blood flow of 12.4 +/- 1.7 ml/min/100 g was reduced to 1.9 +/- 0.5 ml/min/100 g at 4 h after treatment (n = 3). The FAA-induced reduction of both tumor blood flow and ATP may play an important role in its mechanism of action and should be considered in the combination of FAA with other drugs or therapeutic modalities. In addition, because 31P NMR can be used clinically, it should provide a nonambiguous early indicator of activity for clinical trials of FAA.

Applications of magnetic resonance in model systems: cancer therapeutics.

The lack of information regarding the metabolism and pathophysiology of individual tumors limits, in part, both the development of new anti-cancer therapies and the optimal implementation of currently available treatments. Magnetic resonance [MR, including magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and electron paramagnetic resonance (EPR)] provides a powerful tool to assess many aspects of tumor metabolism and pathophysiology. Moreover, since this information can be obtained nondestructively, pre-clinical results from cellular or animal models are often easily translated into the clinic. This review presents selected examples of how MR has been used to identify metabolic changes associated with apoptosis, detect therapeutic response prior to a change in tumor volume, optimize the combination of metabolic inhibitors with chemotherapy and/or radiation, characterize and exploit the influence of tumor pH on the effectiveness of chemotherapy, characterize tumor reoxygenation and the effects of modifiers of tumor oxygenation in individual tumors, image transgene expression and assess the efficacy of gene therapy. These examples provide an overview of several of the areas in which cellular and animal model studies using MR have contributed to our understanding of the effects of treatment on tumor metabolism and pathophysiology and the importance of tumor metabolism and pathophysiology as determinants of therapeutic response.

Direct magnetic resonance determination of aortic distensibility in essential hypertension: relation to age, abdominal visceral fat, and in situ intracellular free magnesium.

To investigate the contribution of vascular compliance to essential hypertension (EH), we developed magnetic resonance imaging (MRI) techniques to directly measure aortic distensibility (AD) in the ascending and descending thoracic and abdominal aorta of fasting normal (n= 10) and EH (n=20) subjects. These results were compared with concurrent MR-based measurements of left ventricular mass index (LVMI) and abdominal subcutaneous and visceral fat and with 31P-MR spectroscopic measurement of in situ intracellular free magnesium levels (Mgi) in brain and skeletal muscle. Aortic distensibility in EH was consistently and significantly reduced at all measured sites (2.5+/-0.4, 2.2+/-0.4, 2.3+/-0.4 versus 7.0+/-1.6, 5.1+/-0.3, 7.3+/-0.8 mm Hg(-1) x 10(-3), P<.05), as was Mgi in the brain (284+/-22 versus 383+/-34 micromol/L, P<.05) and skeletal muscle (397+/-10 versus 527+/-36 micromol/L, P<.05). For all subjects, systolic blood pressure (r=-.662, P<.0001) and LVMI (r=-.484, P<.01) were inversely related to AD. AD and brain Mgi were inversely related to age (AD, r=-.792, P<.0001; brain Mgi: r=-.673, P<.05). AD was inversely related to fasting blood glucose (r=-.413, P<.05) and to abdominal visceral fat (r=-.416, P<.05) but not to body mass index (BMI: r=-.328, P=NS) or subcutaneous fat (r=-.157, P=NS). AD was also significantly and positively related to in situ Mgi, both in the brain and skeletal muscle (brain: r=.712, P<.01; skeletal muscle: r=.632, P<.01). We conclude that (1) MR techniques can be used to coordinately and noninvasively assess cardiac, vascular, metabolic, and ionic aspects of hypertensive disease in humans; (2) increased systolic blood pressure and LVMI in EH may at least in part result from decreased AD; (3) decreased Mgi contributes to arterial stiffness in hypertension and may help to explain the characteristic age-related decreases in AD; and (4) decreased AD may be one mechanism by which abdominal visceral fat contributes to cardiovascular risk.

pH and therapy of human cancers.

Studies in model systems have demonstrated that tumour pH can be a determinant of treatment response. The potential that tumour pH differs from that of normal tissues may provide a basis for selective killing of tumour cells. Although the data are limited, pH measurements in humans indicate a difference between tumour and normal tissues. In general, electrode pH (generally considered to reflect primarily extracellular pH, pHe) is lower in tumour than normal tissue. However, pH measured by magnetic resonance spectroscopy (MRS) or positron emission tomography (PET; both are generally considered to reflect primarily intracellular pH, pHi) is equal to or slightly higher in tumours than normal tissues. Hence, not only may pHe and pHi differ between normal and malignant tissues, but the pH gradient (which determines the distribution of chemotherapeutic agents that are weak acids or bases) is also reduced or reversed in tumours. To date, the majority of treatment-related studies conducted have focused on hyperthermia (combined with radiotherapy) due to the recognized importance of acidic pH as a thermal sensitizer. However, the results have been somewhat surprising: patients with a better response to hyperthermia radiotherapy have higher pH (as measured by electrode or 31P MRS) prior to treatment.

Pulse waveform analysis of arterial compliance: relation to other techniques, age, and metabolic variables.

To assess the physiologic and clinical relevance of newer noninvasive measures of vascular compliance, computerized arterial pulse waveform analysis (CAPWA) of the radial pulse was used to calculate two components of compliance, C1 (capacitive) and C2 (oscillatory or reflective), in 87 normotensive (N1BP, n = 20), untreated hypertensive (HiBP, n = 21), and treated hypertensive (HiBP-Rx, n = 46) subjects. These values were compared with two other indices of compliance, the ratio of stroke volume to pulse pressure (SV/PP) and magnetic resonance imaging (MRI)-based aortic distensibility; and were also correlated with demographic and biochemical values. The HiBP subjects displayed lower C1 (1.34 +/- 0.09 v. 1.70 +/- 0.11 mL/mm Hg, significance [sig] = .05) and C2 (0.031 +/- 0.003 v 0.073 +/- 0.02 mL/mm Hg, sig = .005) than N1BP subjects. This was not true for C1 (1.64 +/- 0.08 mL/mm Hg) and C2 (0.052 +/- 0.005 mL/mm Hg) values in HiBP-Rx subjects. The C1 (r = 0.917, P < .0001) and C2 (r = 0.677, P < .0001) were both closely related to SV/PP, whereas C1 (r = 0.748, P = .002), but not C2, was significantly related to MRI-determined aortic distensibility. Among other factors measured, age exerted a strong negative influence on both C1 (r = -0.696, P < .0001) and C2 (r = -0.611, P < .0001) compliance components. Positive correlations were observed between C1 (r = 0.863, P = .006), aortic distensibility (r = 0.597, P = .19) and 24-h urinary sodium excretion, and between C1- and MR spectroscopy-determined in situ skeletal muscle intracellular free magnesium (r = 0.827, P = .006), whereas C2 was inversely related to MRI-determined abdominal visceral fat area (r = -0.512, P = .042) and fasting blood glucose (r = -0.846, P = .001). Altogether, the close correspondence between CAPWA, other compliance techniques, and known cardiovascular risk factors suggests the clinical relevance of CAPWA in the assessment of altered vascular function in hypertension.

Correlations between 31P NMR spectroscopy and 15O perfusion measurements in the RIF-1 murine tumor in vivo.

The tumor physiological environment is one of the least understood and most important factors in determining the response of solid tumors to cancer therapy. To examine several important characteristics of the tumor physiological environment we have used in situ photon activation-15O decay measurements (perfusion characteristics) and 31P surface coil-NMR spectroscopy (metabolic characteristics) to observe in vivo subcutaneous RIF-1 tumors grown in female C3H/Anf mice. The following correlations between the 15O perfusion characteristics and the 31P NMR metabolic characteristics in individual tumors were observed: a negative correlation between pH, as measured by NMR (pHNMR), and the inorganic phosphate to nucleosides triphosphate peak height ratio (Pi:NTP); for the well-perfused fraction of the tumor there is a positive correlation with both pHNMR and the phosphocreatine to nucleosides triphosphate peak height ratio (PCr:NTP), and a negative correlation with Pi:NTP. These correlations are interpreted as evidence for a direct relationship between the distribution of cellular physiological environments and the tumor metabolic state. Because these physiological characteristics affect tumor response to various therapeutic modalities and both measurements can be made on humans, it is suggested that these techniques may be of prognostic value in the clinical management of human cancer.

Magnetic resonance imaging to measure therapeutic response using an orthotopic model of human pancreatic cancer.

Pancreatic cancer is one of the most incurable and lethal human cancers in the United States. To facilitate development of novel therapeutic agents, we previously established an orthotopic pancreatic tumor model that closely mimics the natural biological behavior of human pancreatic cancer. In this study, magnetic resonance imaging (MRI) techniques were developed to detect tumor formation noninvasively and monitor serially tumor growth kinetics in this orthotopic model used for experimental drug testing. By using an optimized T2-weighted imaging method, we were able to distinguish human pancreas cancer from normal mouse pancreas. Orthotopic tumor formation was detected as early as day 1 after tumor cell implantation with a tumor volume as small as 12 mm3. Mice with evidence of tumor were separated into four treatment groups: control, auristatin-PE, gemcitabine, and their combination. After treatment, the mice were imaged at least three times before termination of the experiment. Comparison between MRI tumor volume measurements and tumor weights made at biopsy resulted in a correlation coefficient of 0.98. The tumor growth curves constructed from serial magnetic resonance imaging (MRI) measurements clearly showed tumor growth inhibition in treated mice compared with the control group. As expected, the group treated with the combination had the highest response rate compared with either auristatin-PE or gemcitabine alone, and the data were statistically highly significant (p < 0.004). From these results, we conclude that noninvasive MRI can be used to monitor serially therapeutic response in this orthotopic human pancreatic tumor model and can be used in the future to evaluate novel antitumor agents before human studies.

Proton magnetic resonance spectroscopy in patients with glial tumors: a multicenter study.

The authors represent a cooperative group of 15 institutions that examined the feasibility of using metabolic features observed in vivo with 1H-magnetic resonance (MR) spectroscopy to characterize brain tumors of the glial type. The institutions provided blinded, centralized MR spectroscopy data processing long with independent central review of MR spectroscopy voxel placement, composition and contamination by brain, histopathological typing using current World Health Organization criteria, and clinical data. Proton 1H-MR spectroscopy was performed using a spin-echo technique to obtain spectra from 8-cc voxels in the tumor and when feasible in the contralateral brain. Eighty-six cases were assessable, 41 of which had contralateral brain spectra. Glial tumors had significantly elevated intensities of choline signals, decreased intensities of creatine signals, and decreased intensities of N-acetylaspartate compared to brain. Choline signal intensities were highest in astrocytomas and anaplastic astrocytomas, and creatine signal intensities were lowest in glioblastomas. However, whether expressed relative to brain or as intratumoral ratios, these metabolic characteristics exhibited large variations within each subtype of glial tumor. The resulting overlaps precluded diagnostic accuracy in the distinction of low-and high-grade tumors. Although the extent of contamination of the 1H-MR spectroscopy voxel by brain had a marked effect on metabolite concentrations and ratios, selection of cases with minimal contamination did not reduce these overlaps. Thus, each type and grade of tumor is a metabolically hetero-geneous group. Lactate occurred infrequently and in all grades. Mobile lipids, on the other hand, occurred in 41% of high-grade tumors with higher mean amounts found in glioblastomas. This result, coupled with the recent demonstration that intratumoral mobile lipids correlate with microscopic tumor cell necrosis, leads to the hypothesis that mobile lipids observed in vivo in 1H-MR spectroscopy may correlate independently with prognosis of individual patients.

In vivo 31P MR spectral patterns and reproducibility in cancer patients studied in a multi-institutional trial.

The standardization and reproducibility of techniques required to acquire anatomically localized 31P MR spectra non-invasively while studying tumors in cancer patients in a multi-institutional group at 1.5 T are reported. This initial group of patients was studied from 1995 to 2000 to test the feasibility of acquiring in vivo localized 31P MRS in clinical MR spectrometers. The cancers tested were non-Hodgkin's lymphomas, sarcomas of soft tissue and bone, breast carcinomas and head and neck carcinomas. The best accrual and spectral quality were achieved with the non-Hodgkin's lymphomas. The initial analysis of the spectral values of the sum of phosphoethanolamine plus phosphocholine normalized by the content of nucleotide triphosphates in a homogeneous sample of 32 NHL patients studied by in vivo (31)P MRS showed good reproducibility among different institutions. No statistical differences were found between the institution with the largest number of cases accrued and the rest of the multi-institutional NHL data (2.28 +/- 0.64, mean +/- standard error; n = 17, vs 2.08 +/- 0.14, n = 15). The preliminary data reported demonstrate that the institutions involved in this trial are obtaining reproducible 31P MR spectroscopic data non-invasively from human tumors. This is a fundamental prerequisite for the international cooperative group to be able to demonstrate the clinical value of the normalized determination of phosphoethanolamine plus phosphocholine by 31P MRS as predictor for treatment response in cancer patients.

The assessment of antiangiogenic and antivascular therapies in early-stage clinical trials using magnetic resonance imaging: issues and recommendations.

Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used.

Key factors in the acquisition of contrast kinetic data for oncology.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has recently emerged as a promising method for both diagnosis and prognosis of cancer despite considerable variation in both the methods of data acquisition and analysis. Both to facilitate integration of results from multiple institutions and to ensure that the data reflect the underlying physiology as accurately as possible, several aspects of data acquisition should be taken into account when developing protocols for DCE-MRI regardless of how the data are analyzed. Among the relevant issues are the relationship between signal enhancement and contrast agent concentration, intra- or inter-patient variation in the blood contrast agent concentration as a function of time, requirements for spatial and temporal resolution, the impact of tumor heterogeneity, and the impact of patient motion during the study. This review considers these factors and, when possible, makes specific recommendations for addressing them experimentally.

In vivo 19F nuclear magnetic resonance spectroscopy: a potential monitor of 5-fluorouracil pharmacokinetics and metabolism.

In vivo 19F nuclear magnetic resonance (NMR) spectroscopy has the potential to non-invasively measure the concentration of 5-fluorouracil (FUra) and some of its metabolites in humans. Such a measure could be useful in predicting and optimizing the response of individual patients treated with FUra. The ability of 19F NMR to monitor FUra metabolism in situ in rodent tumors and liver and in human liver has been demonstrated. However, the potential impact of this technique as a predictor of FUra response in individual patients is limited by both the sensitivity (i.e., limit of detection) and the resolution (i.e., ability to distinguish among magnetically similar metabolites) of NMR. To date, the ability of in vivo 19F NMR spectroscopy to provide information that can distinguish FUra-sensitive from FUra-insensitive tumors has not been established. This crucial point should be addressed in the immediate future in studies using the best of experimental conditions (i.e., optimum sensitivity and resolution in well-defined rodent tumor models with NMR methodology appropriate for measurement of absolute metabolite concentrations). The information gained from such studies and any new technical developments to enhance in vivo NMR sensitivity should be directly applicable to any future application of 19F NMR spectroscopy in clinical FUra therapy.