Monitoring anti-angiogenic therapy in colorectal cancer murine model using dynamic contrast-enhanced MRI: comparing pixel-by-pixel with region of interest analysis.

Sorafenib is a multi-kinase inhibitor that blocks cell proliferation and angiogenesis. It is currently approved for advanced hepatocellular and renal cell carcinomas in humans, where its major mechanism of action is thought to be through inhibition of vascular endothelial growth factor and platelet-derived growth factor receptors. The purpose of this study was to determine whether pixel-by-pixel analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is better able to capture the heterogeneous response of Sorafenib in a murine model of colorectal tumor xenografts (as compared with region of interest analysis). MRI was performed on a 9.4 T pre-clinical scanner on the initial treatment day. Then either vehicle or drug were gavaged daily (3 days) up to the final image. Four days later, the mice were again imaged. The two-compartment model and reference tissue method of DCE-MRI were used to analyze the data. The results demonstrated that the contrast agent distribution rate constant (K(trans)) were significantly reduced (p < 0.005) at day-4 of Sorafenib treatment. In addition, the K(trans) of nearby muscle was also reduced after Sorafenib treatment. The pixel-by-pixel analysis (compared to region of interest analysis) was better able to capture the heterogeneity of the tumor and the decrease in K(trans) four days after treatment. For both methods, the volume of the extravascular extracellular space did not change significantly after treatment. These results confirm that parameters such as K(trans), could provide a non-invasive biomarker to assess the response to anti-angiogenic therapies such as Sorafenib, but that the heterogeneity of response across a tumor requires a more detailed analysis than has typically been undertaken.

Selective depletion of tumor ATP by 2-deoxyglucose and insulin, detected by 31P magnetic resonance spectroscopy.

The purpose of this study was to investigate whether substrate deprivation acutely and selectively decreases ATP concentration in an experimental sarcoma. Two methods of substrate deprivation were examined: glycolysis was inhibited using 2-deoxyglucose (2DG), and plasma substrate levels were reduced using insulin. The effects of treatment on tumor ATP, inorganic phosphate, and pH were studied by 31P nuclear magnetic resonance spectroscopy. 2DG (2 g/kg) was administered i.p. to rats bearing s.c. methylcholanthrene-induced sarcomas. Inhibition of glycolysis by 2DG caused a 52 +/- 13% (SE) decrease in the tumor ATP to inorganic phosphate ratio, associated with a decrease in pH of 0.38 +/- 0.10 unit. The same dose of 2DG caused no significant change in the ratio of phosphocreatine to ATP in brain. Insulin (125 units/kg, i.p.) caused a 68% decline in plasma glucose and a 71% decline in betahydroxybutyrate compared to saline-treated animals. Concomitantly, 31P nuclear magnetic resonance spectroscopy detected a 48 +/- 13% decrease in sarcoma ATP, with a reciprocal elevation of inorganic phosphate in insulin-treated animals. In contrast, the brain phosphocratine/ATP ratio was unaffected by insulin. These results suggest that large tumors are acutely sensitive to inhibition of glycolysis and reductions in plasma levels of substrates for oxidative phosphorylation and glycolysis, while the brain is unaffected. In addition, this work provides support for the use of 31P nuclear magnetic resonance spectroscopy to monitor tumor response to therapy.

Image-guided 31P magnetic resonance spectroscopy of normal and transplanted human kidneys.

Image-guided 31-phosphorus magnetic resonance spectroscopy (MRS) was used to obtain spatially localized 31P spectra of good quality from healthy normal human kidneys and from well-functioning renal allografts. A surface coil of 14 cm diameter was used for acquiring phosphorus signals solely from a volume-of-interest located within the kidney. To determine the effects of kidney transplantation on renal metabolism, patients with well functioning allografts were studied. Little or no phosphocreatine in all spectra verifies the absence of muscle contamination, and is consistent with proper volume localization. The intensity ratio of phosphomonoesters (PME) to adenosine triphosphate (ATP) resonances in transplanted kidneys (PME/ATP = 1.1 +/- 0.4) was slightly elevated (P = 0.2) compared to that of healthy normal kidneys (PME/ATP = 0.8 +/- 0.3). The inorganic phosphate (Pi) to ATP ratio was similar in the two groups (Pi/ATP = 1.1 +/- 0.1 in transplanted kidneys vs. 1.2 +/- 0.6 in normal kidneys). Acid/base status, as evidenced from the chemical shift of Pi, was the same in both normal controls and transplanted kidneys. Despite the practical problems produced by organ depth, respiratory movement, and tissue heterogeneity, these results demonstrate that image-guided 31P MR spectra can reliably be obtained from human kidneys.

Non-invasive quantitation of human liver metabolites using image-guided 31P magnetic resonance spectroscopy.

Phosphorus-containing metabolites in normal human liver have been quantitated non-invasively with 31P magnetic resonance spectroscopy using surface coils. The location of the volume of interest (VOI) was defined by 1H magnetic resonance imaging. Subsequently, a modified three-dimensional localization technique (ISIS) was used to acquire 31P magnetic resonance spectra from the VOI. To account for partial saturation produced by rapid signal averaging, the spin/lattice relaxation times (T1) of all hepatic phosphorus resonances were measured. The corrected resonance integrals were used to derive absolute molar concentrations for the following hepatic metabolites (mmol/kg wet weight): ATP, 2.0; inorganic phosphate, 2.1; phosphodiesters, 5.4; and phosphomonoesters, 0.9. These values are compared with previously reported values for humans using freeze-clamping techniques, and provide a basis for comparison with studies of hepatic disease in this laboratory.

Comparison of 31P MRS and 1H MRI at 1.5 and 2.0 T.

The goals of this study were to compare 31P magnetic resonance spectroscopy (MRS) and 1H magnetic resonance imaging (MRI) of human subjects and phantoms at 1.5 and 2.0 T. The 31P signal-to-noise (S/N) ratios in phantom standards and in localized volumes in human brain and liver were compared at 1.5 and 2.0 T. In addition, T1 values for 31P resonances in human brain, 31P linewidths of metabolites in human brain and liver, 1H S/N in a phantom standard, and MR image quality in human head and body were compared at the two field strengths. The results of our study showed that at the higher strength field, (1) in vivo 31P MRS studies benefited from up to 32% improvement in S/N; (2) in vivo 31P MRS studies also benefited from increased spectral dispersion; (3) the quality of MR head images remained comparable; and (4) body images showed some decrease in image quality due to increased chemical shift, and flow and motion artifacts.

Response of tumors to therapy studied by 31P magnetic resonance spectroscopy.

Magnetic resonance (MR) methods have been used to study the metabolic and vascular response of model tumors to tumor necrosis factor (TNF). Magnetic resonance measurements demonstrated acute reductions in tumor blood flow, measured from tumor uptake of D2O, and in tumor adenosine triphosphate (ATP), measured by 31P magnetic resonance spectroscopy (MRS) following administration of TNF. The decrease in ATP generally followed reduction in tumor blood flow, and therefore was probably due to ischemia caused by damage to tumor vasculature. Superficial human tumors have been studied by MRS to characterize their 31P spectra, and to measure metabolic changes during therapy. The ratio of the intensities of the phosphomonoester (PME) and ATP resonances (PME/ATP) was much higher in tumors than in the normal tissue displaced by the tumors. During therapy, decreases in PME/ATP were detected that paralleled, but did not anticipate, decreases in tumor size. In some cases, a transient increase in PME/ATP was detected during therapy, which did not correlate with changes in tumor size, and which may reflect stimulation of cell growth in some tumor zones.

Abnormalities of the liver evaluated by 31P MRS.

Clinical phosphorus-31 magnetic resonance spectroscopy (31P MRS) of the liver requires the use of whole-body magnets and of spectroscopy techniques that acquire signal from defined volumes-of-interest within the liver. Such localization techniques and recent clinical studies are briefly reviewed. These studies indicate that (1) high phosphomonoester levels are present in liver diseases involving structural damage, and (2) that MRS of liver tumors may provide a sensitive and rapid indication of response to cancer therapy. Abnormalities of the liver such as alcoholic liver disease, viral hepatitis, and metastasis were analyzed to determine hepatic acid/base status (pH) and to derive absolute molar concentrations of hepatic phosphorus metabolites rather than metabolite ratios. These parameters allow diagnosis and differentiation of several liver pathologies, suggesting an increasing future role of MRS in medical investigation, clinical diagnosis, and patient treatment.

Regulation of hepatic inorganic phosphate and ATP in response to fructose loading: an in vivo 31P-NMR study.

Fructose loading results in hepatic accumulation of fructose 1-phosphate (Fru1 P). The goals of the present experiments were: first, to distinguish between ATP, intracellular inorganic phosphate (Pi), and extracellular Pi as sources of phosphate for the phosphorylation of fructose, and second, to examine the influence of ATP and Fru1 P on movement of phosphate into and out of these three pools. To achieve these goals, 31P-NMR was used to monitor the response of hepatic ATP, Pi and Fru1 P to two consecutive injections of fructose. The first was administered with ATP at the control level, and the second, 1 h after the first, with ATP at 65% of the control level. Changes in intra- and extracellular Pi were distinguished by correlating measurements of total NMR-detectable phosphorus and NMR-detectable Pi with measurements of plasma Pi. The initial fructose injection resulted in rapid accumulation of Fru1 P, small decreases in plasma and NMR-detectable Pi and a dramatic decrease in ATP. Total NMR-detectable phosphorus did not change, suggesting that phosphate did not enter or leave the liver. Therefore, accumulation of Fru1 P was initially balanced by an equivalent decrease in ATP, without large changes in Pi. Following the second injection, when ATP was at 65% of control. Fru1 P accumulated at approximately the same rate and to the same level as achieved following the first injection. There was little further change in ATP and a marked decrease in NMR-detectable Pi, while plasma Pi was higher than after the first injection. Therefore the greater decrease in NMR-detectable Pi following the second injection represented a significant decrease in intracellular Pi. Return of Fru1 P to control coincided with a dramatic increase in plasma Pi, and a decrease in total NMR-detectable phosphate. This suggests that phosphate released from Fru1 P entered the extracellular space. These data suggest the mechanisms by which intracellular Pi is regulated. When sufficient ATP is available, ATP hydrolysis supplies phosphate for the synthesis of Fru1 P, and prevents a significant decrease in intracellular Pi. When ATP is reduced, accumulation of Fru1 P depletes intracellular Pi. Therefore, decreased availability of ATP correlates with increased utilization of intracellular Pi. When Fru1 P returns to control, the increase in intracellular Pi is limited by release of Pi into the plasma.

A 31P NMR study of the GI tract: effect of fructose loading and measurement of transverse relaxation times.

The effect of fructose loading on high-energy phosphates in the jejunum, ileum, and large intestine of rats was studied using 31P NMR. Following fructose loading, an increase in the intensity of the PME resonance was observed in the jejunum, indicating an accumulation of fructose-1-phosphate. There were no significant changes in ATP or Pi. This demonstrates that the activity of fructokinase in the jejunum can be monitored by 31P NMR. Fructose loading had no detectable effect on metabolite levels in the ileum and large intestine. Resolution of intestinal spectra was poor due to unusually large linewidths and the presence of broad underlying signals. To study the mechanism of line broadening, the T2's of the phosphorus resonances were measured using a solenoidal coil. The T2's of the ATP, Pi, PME, and PCr resonances were much longer than the T2's, suggesting that the linewidths of these resonances are primarily due to susceptibility gradients and/or compartmentation of metabolites. Other signals, particularly in the PDE region, were homogeneously broadened and had very short T2's. Spin echoes obtained with evolution times of 1 to 4 ms suppressed these broad components, with little loss of intensity in the inhomogeneously broadened resonances; as a result, resolution was improved.