Jerry D. Glickson

NMR study of in vivo RIF-1 tumors. Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1H and 13C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.

31P NMR spectroscopy of in vivo tumors

Abstract A probe, suitable for any wide-bore NMR spectrometer, was constructed for monitoring high-resolution spectra of in vivo subcutaneously implanted tumors in mice. Preliminary studies of a variety of murine tumors (MOPC 104E myeloma, Dunn osteosarcoma, colon-26, ovarian M5, and mammary adenocarcinoma as well as human colon, mammary, and lung tumors in athymic mice) indicate that the 31P NMR spectrum is a sensitive monitor of progressive metabolic changes that occur during untreated tumor growth and an early indicator of tumor response to chemotherapy, hyperthermia, and X radiation. Response to each of these therapeutic modalities is accompanied by distinctly different spectral changes.

Assessment of global and regional myocardial function in the mouse using cine and tagged MRI.

Mouse models are expected to play an important role in future investigations of human cardiac diseases. In the present report, MRI methods for determining global and regional cardiac function in the mouse are demonstrated. ECG‐gated cine images were acquired in five C57BL/6 mice at physiological temperatures (37°C) and heart rates of 500 ± 50 beats per minute. Left ventricular mass, ejection fraction, and cardiac output were estimated from the resulting images. Regional myocardial function was also determined in three animals by application of 2D SPAtial Modulation of Magnetization (SPAMM) in combination with the cine protocol. The quality of the tagged images was sufficient to allow mapping of myocardial strains and displacements. The results of the regional strain analysis were consistent with similar studies in larger animals. This work demonstrates the first characterization of regional myocardial function in the mouse via SPAMM techniques. Magn Reson Med 49:760–764, 2003.

Simultaneous measurement of arterial input function and tumor pharmacokinetics in mice by dynamic contrast enhanced imaging: Effects of transcytolemmal water exchange

A noninvasive technique for simultaneous measurement of the arterial input function (AIF) for gadodiamide (Omniscan) and its uptake in tumor was demonstrated in mice. Implantation of a tumor at a suitable location enabled its visualization in a cardiac short axis image. Sets of gated, low‐resolution saturation recovery images were acquired from each of five tumor‐bearing mice following intravenous administration of a bolus of contrast agent (CA). The AIF was extracted from the signal intensity changes in left ventricular blood using literature values of the CA relaxivity and a precontrast T1 map. The time‐dependent 1H2O relaxation rate constant (R1 = 1/T1) in the tumor was modeled using the BOLus Enhanced Relaxation Overview (BOLERO) method in two modes regarding the equilibrium transcytolemmal water exchange system: 1) constraining it exclusively to the fast exchange limit (FXL) (the conventional assumption), and 2) allowing its transient departure from FXL and access to the fast exchange regime (FXR), thus designated FXL/FXR. The FXL/FXR analysis yielded better fittings than the FXL‐constrained analysis for data from the tumor rims, whereas the results based on the two modes were indistinguishable for data from the tumor cores. For the tumor rims, the values of Ktrans (the rate constant for CA transfer from the vasculature to the interstitium) and ve (volume fraction of the tissue extracellular and extravascular space) returned from FXL/FXR analysis are consistently greater than those from the FXL‐constrained analysis by a factor of 1.5 or more corresponding to a CA dose of 0.05 mmole/kg. Magn Reson Med 52:248–257, 2004.

Diffusion imaging of human breast

It is shown that diffusion‐weighted imaging is possible in the human breast. Diffusion constants were measured in the breast parenchyma of four volunteers with no known breast lesions. The apparent diffusion constant of water measured in regions of interest chosen in normal human breast fibroglandular tissue was 1.64±0.19×10−5 cm2/s and that measured in the area of fatty breast tissue was 0.32±0.18×10−5 cm2/s. The resulting images indicate that fibroglandular tissue and fat can be clearly distinguished in diffusion‐weighted as well as in absolute diffusion images of the breast. Potential future applications of this technology for the study of breast pathologies are suggested.

Nuclear magnetic resonance studies of the denaturation of ubiquitin

The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.

Quantitative magnetic resonance and optical imaging biomarkers of melanoma metastatic potential

Noninvasive or minimally invasive prediction of tumor metastatic potential would facilitate individualized cancer management. Studies were performed on a panel of human melanoma xenografts that spanned the full range of metastatic potential measured by an in vivo lung colony assay and an in vitro membrane invasion culture system. Three imaging methods potentially transferable to the clinic [dynamic contrast-enhanced (DCE) MRI, T1ρ-MRI, and low-temperature fluorescence imaging (measurable on biopsy specimens)] distinguished between relatively less metastatic and more metastatic human melanoma xenografts in nude mice. DCE-MRI, analyzed with the shutter-speed relaxometric algorithm and using an arterial input function simultaneously measured in the left ventricle of the mouse heart, yielded a blood transfer rate constant, Ktrans, that measures vascular perfusion/permeability. Ktrans was significantly higher in the core of the least metastatic melanoma (A375P) than in the core of the most metastatic melanoma (C8161). C8161 melanoma had more blood vascular structures but fewer functional blood vessels than A375P melanoma. The A375P melanoma exhibited mean T1ρ values that were significantly higher than those of C8161 melanoma. Measurements of T1 and T2 relaxation times did not differ significantly between these 2 melanomas. The mitochondrial redox ratio, Fp/(Fp + NADH), where Fp and NADH are the fluorescences of oxidized flavoproteins and reduced pyridine nucleotides, respectively, varied linearly with the in vitro invasive potential of the 5 melanoma cell lines (A375P, A375M, A375P10, A375P5, and C8161). This study shows that a harsh microenvironment may promote melanoma metastasis and provides potential biomarkers of metastatic potential.

In vivo magnetic resonance spectroscopy of human brain tumors.

In vivo nuclear magnetic resonance (NMR) spectroscopy is a rapidly developing, noninvasive analytical technique that allows sequential studies of brain tumor metabolism. It can be implemented on most conventional high-field magnetic resonance imaging scanners. In animal tumor models. NMR spectroscopy has been used extensively to characterize the metabolic changes associated with tumor growth and response to therapy. Preliminary data in humans indicate the feasibility of recording phosphorus and proton spectra from focal lesions within the brain. It is hoped that the technique will provide useful prognostic information in terms of tumor growth behavior and prediction and detection of response to therapy. However, the technique suffers from relatively coarse spatial resolution, and the methodology is still under development. More studies with larger patient groups are required before the clinical utility of the technique can be fully evaluated.

Quantitative mitochondrial redox imaging of breast cancer metastatic potential

Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic (aggressive) MDA-MB-231 and less metastatic (indolent) MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp). The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp(NADH+Fp). We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.

Transcytolemmal water exchange in pharmacokinetic analysis of dynamic contrast‐enhanced MRI data in squamous cell carcinoma of the head and neck

To investigate the effect of transcytolemmal water exchange on the dynamic contrast‐enhanced (DCE) T1‐weighted MRI of human squamous cell carcinomas of the head and neck (HNSCC).