Publications

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI).The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices.The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

Purpose: To investigate the parameters obtained with magnetic resonance (MR) diffusion-tensor imaging (DTI) of the breast throughout the menstrual cycle phases, during lactation, and after menopause, with and without hormone replacement therapy (HRT). Materials and Methods: All protocols were approved by the internal review board, and signed informed consent was obtained from all participants. Forty-five healthy volunteers underwent imaging by using T2-weighted and DTI MR sequences at 3 T. Premenopausal volunteers (n = 16) underwent imaging weekly, four times during one menstrual cycle. Postmenopausal volunteers (n = 19) and lactating volunteers (n = 10) underwent imaging once. The principal diffusion coefficients (lambda 1, lambda 2, and lambda 3), apparent diffusion coefficient (ADC), fractional anisotropy (FA), and maximal anisotropy (lambda 1-lambda 3) were calculated pixel by pixel for the fibroglandular tissue in the entire breast. Results: In all premenopausal volunteers, the DTI parameters exhibited high repeatability, remaining almost equal along the menstrual cycle, with a low mean within-subject coefficient of variance of lambda 1, lambda 2, lambda 3, and ADC (1%-2% for all) and FA (5%), as well as a high intraclass correlation of 0.92-0.98. The diffusion coefficients were significantly lower (a) in the group without HRT use as compared with the group with HRT use (P

The metabolic status of muscle changes according to the energetic demands of the organism. Two key regulators of these changes include exercise and insulin, with exercise eliciting catabolic expenditure within seconds and insulin enabling anabolic energy investment over minutes to hours. This study explores the potential of time-resolved hyperpolarized dynamic ¹³C spectroscopy to characterize the in vivo metabolic phenotype of muscle during functional and biochemical insulin-induced stimulation of muscle. Using [¹³C₁]pyruvic acid as a tracer, we find that despite the different time scales of these forms of stimulation, increases in pyruvate label transport and consumption and concomitant increases in initial rates of the tracer metabolism to lactate were observed for both stimuli. By contrast, rates of tracer metabolism to labeled alanine increased incrementally for insulin but remained unchanged following exercise-like muscle stimulation. Kinetic analysis revealed that branching of the hyperpolarized [¹³C]pyruvate tracer between lactate and alanine provides significant tissuespecific biomarkers that distinguish between anabolic and catabolic fates in vivo according to the routing of metabolites between glycolytic and amino acid pathways.

Objectives: To develop and evaluate a fast, objective and standardized method for image processing of dynamic contrast enhanced MRI of the prostate based on principal component analysis (PCA). Materials and Methods:The study was approved by the institutional internal review board; signed informed consent was obtained. MRI of the prostate at 3 Tesla was performed in 21 patients with biopsy proven cancers before radical prostatectomy. Seven 3-dimensional gradient echo datesets, 2 pre and 5 post-gadopentetate dimeglumine injection (0.1 mmol/kg), were acquired within 10.5 minutes at high spatial resolution. PCA of dynamic intensity-scaled (IS) and enhancement-scaled (ES) datasets and analysis by the 3-time points (3TP) method were applied using the latter method for adjusting the PCA eigenvectors. Results: PCA of 7 IS datasets and 6 ES datasets yielded their corresponding eigenvectors and eigenvalues. The first IS-eigenvector captured the major part of the signal variance because of a signal change between the precontrast and the first postcontrast arising from the inhomogeneous surface coil reception profile. The next 2 IS-eigenvectors and the 2 dominant ES-eigenvectors captured signal changes because of tissue contrast-enhancement, whereas the remaining eigenvectors captured noise changes. These eigenvectors were adjusted by rotation to reach congruence with the wash-in and wash-out kinetic parameters defined according to the 3TP method. The IS and ES-eigenvectors and rotation angles were highly reproducible across patients enabling the calculation of a general rotated eigenvector base that served to rapidly and objectively calculate diagnostically relevant projection coefficient maps for new cases. We found for the a priori selected prostate cancer patients that the projection coefficients of the IS-2nd eigenvector provided a higher accuracy for detecting biopsy proven cancers (94% sensitivity, 67% specificity, 80% ppv, and 89% npv) than the projection coefficients of the ES-2nd rotated and non rotated eigenvectors. Conclusions: PCA adjusted to correlate with physiological parameters selects a dominant eigenvector, free of the inhomogeneous radio-frequency field reception-profile and noise-components. Projection coefficient maps of this eigenvector provide a fast, objective, and standardized means for visualizing prostate cancer.

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor α (ERα) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ERα and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

A wide range of dynamic-contrast-enhanced (DCE) sequences and protocols, image processing methods, and interpretation criteria have been developed and evaluated over the last 20 years. In particular, attempts have been made to better understand the origin of the contrast observed in breast lesions using physiological models that take into account the vascular and tissue-specific features that influence tracer perfusion. In addition, model-free algorithms to decompose enhancement patterns in order to segment and classify different breast tissue types have been developed. This review includes a description of the mechanism of contrast enhancement by gadolinium-based contrast agents, followed by the current status of the physiological models used to analyze breast DCE-MRI and related critical issues. We further describe more recent unsupervised and supervised methods that use a range of different common algorithms. The model-based and model-free methods strive to achieve scientific accuracy and high clinical performance - both important goals yet to be reached.

Rationale and Objectives: Neoadjuvant systemic therapy (NST) is the standard treatment for locally advanced breast cancer and a common option for primary operable disease. It is important to develop standardized imaging techniques that can monitor and quantify response to NST enabling treatment tailored to each individual patient, and facilitating surgical planning. Here we present a high spatial resolution, parametric method based on dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), which evaluates breast cancer response to NST. Materials and Methods: DCE-MRI examinations were performed twice on 17 breast cancer patients, before and after treatment. Seven sets of axial breast images were sequentially recorded at 1.5 Tesla applying a three-dimensional, gradient echo at a spatial resolution ∼2 × 1.2 × 0.6 mm³ and temporal resolution ∼2 minutes, using gadopentate dimeglumine (0.1 mmol/kg wt). Image analysis was based on a color-coded scheme related to physiologic perfusion parameters. Results: A high Pearson correlation coefficient of 0.96 (P

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFF) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis.

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immuodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers.

The clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure are currently limited. The maintenance of the corticomedullary sodium gradient, an indicator of normal kidney function, is presumably lost early in the course of ATN. Herein, sodium magnetic resonance imaging (²³Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN. Three-dimensional gradient echo sodium images were recorded at 4.7 T with high spatial resolution. ATN was produced by the administration of radiologic contrast medium, combined with inhibition of nitric oxide and prostaglandin synthesis. The sodium images revealed that the sham-controlled kidney exhibited a linear increase in sodium concentration along the corticomedullary axis of 30 ± 2 mmol/l/mm, resulting in an inner medulla to cortex sodium ratio of 4.3 ± 0.3 (n = 5). In the ATN kidney, however, the cortico-outer medullary sodium gradient was reduced by 21% (P 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney when morphologic tubular injury was still focal and very limited. MRI detection of corticomedullary sodium gradient abnormalities may serve to identify evolving ATN at its early phases.

The cholinergic system is an important modulatory neurotransmitter system in the brain. Changes in acetylcholine concentration have been previously determined directly in animal models and human brain biopsy specimens, and indirectly, by the effects of drugs, in living humans. Here, we developed a method for direct determination of acetylcholine synthesis in living brain tissue. The method is based on administration of choline, enriched with carbon-13 (stable isotope) in the two methylene positions, and detection of labeled acetylcholine and all other metabolic fates of choline, by carbon-13 magnetic resonance spectroscopy. We tested this method in rat brain slices and found it to be specific for acetylcholine synthesis in both the cortex and hippocampus. This method is potentially useful as a research tool for exploring the cholinergic system role in cognitive processes and memory storage as well as in diseases in which the malfunction of the cholinergic system has been implicated.

The parameters that characterize the intricate water diffusion in tumors may serve to reveal their distinct pathology. Specifically, the application of diffusion magnetic resonance imaging (MRI) can aid in characterizing breast cancer, as well as monitoring response to therapy. We present here a non-invasive, quantitative MRI investigation, at high spatial resolution, of water diffusion in hormonal dependent MCF7 breast tumors implanted orthotopically in immunodeticient mice. Distinctive MRI protocols were designed in this study, utilizing a broad range of diffusion times and diffusion gradient strengths. Application of these protocols allowed water diffusion in the tissue extracellular and intracellular compartments to be distinguished, and the effect of restricted diffusion and water exchange on the water diffusion in these compartments to be evaluated. Pixel-by-pixel analysis yielded parametric maps of the estimated volume fraction and apparent diffusion coefficient of each compartment. The diffusion of the water in the extracellular microenvironment was approximately two fold slower than that of free water, and in the intracellular compartment was about one order of magnitude slower than that of free water and demonstrated restriction of water diffusion at long diffusion times. Mapping of the water fraction in each compartment was further employed to monitor changes during tumor progression and to assess tumor response to hormonal manipulation with a new antiestrogenic drug, tamoxifen methiodide (TMI). It was found that, in parallel to the growth arrest by this drug, the volume fraction of the slowly diffusing water increased, suggesting a TMI-induced cell swelling. This study can serve as a basis for extending diffusion breast MRI in the clinical setting.

Angiogenic activity and formation of a vascular network facilitate tumor perfusion and play a critical role in tumor growth and metastasis. Tumor vasculature may be visualized by means of parametric imaging of specific morphological and physiological characteristics that collectively describe its properties. In this review, we describe advanced magnetic resonance imaging (MRI) techniques that have been developed in order to image and quantify the distribution of tumor vasculature throughout the tumor and characterize its function. These techniques have been used to monitor changes in the magnetic resonance signal intensity of tissue water hydrogens generated by intrinsic effects, as well as by exogenous contrast agents administered into the blood circulation. We further describe specific applications of magnetic resonance imaging using a contrast agent, gadolinium diethylene triamine penta-acetic acid (GdDTPA), which has long been approved for clinical use. Examples include studies of the vascular properties of breast cancer tumors and metastases in animal models, as well as of breast cancer vasculature in patients. We also discuss the use of MRI to improve breast cancer diagnosis in humans by quantifying the permeability of the tumor vasculature. By maximizing the spatial resolution of the images in both animal and human studies, the capacity of magnetic resonance imaging to enhance our understanding of the processes regulating tumor angiogenesis, and improve the diagnosis of cancer, could be clearly demonstrated.

Enhanced glycolysis represents a striking feature of cancers and can therefore serve to indicate a malignant transformation. We have developed a noninvasive, quantitative method to characterize tumor glycolysis by monitoring ¹³C-labeled glucose and lactate with magnetic resonance spectroscopy. This method was applied in MCF7 human breast cancer implanted in the mammary gland of female CD1-NU mice and was further employed to assess tumor response to hormonal manipulation with the antiestrogen tamoxifen. Analysis of the kinetic data based on a unique physiological-metabolic model yielded the rate parameters of glycolysis, glucose perfusion, and lactate clearance in the tumor, as well as glucose pharmacokinetics in the plasma. Treatment with tamoxifen induced a twofold reduction in the rate of glycolysis and of lactate clearance but did not affect the other parameters. This metabolic monitoring can thus serve to evaluate the efficacy of new selective estrogen receptor modulators and may be further extended to improve diagnosis and prognosis of breast cancer.

Purpose: To quantitatively evaluate the spatial distribution of flow- and permeability-limited perfusion in MCF7 human breast cancer tumors orthotopically implanted in CD1-NU mice. Materials and Methods: Flow-limited perfusion was derived from ²H-MRI recorded before and after infusion of deuterated water. Permeability-limited perfusion was evaluated from GdDTPA-enhanced ¹H-MRI. Results: The dominant processes in tumor perfusion, namely blood flow and capillary permeability, were mapped in orthotopically implanted MCF7 human breast cancer tumors. The dynamic data were processed according to physiological models, yielding parametric maps of intravascular volume fraction, water perfusion rate, GdDTPA permeability rate constant, and extracellular volume fraction accessible to GdDTPA. The maps exhibited the heterogeneous distribution of each perfusion parameter. Most of the tumor tissue (≥95%) was perfused with HDO, while Gd-DTPA was perfused in only about 50% of it. In most loci the perfusion rate was limited by capillary permeability to Gd-DTPA. Conclusion: The results demonstrated the instructive value of tracers with different properties used in conjunction to achieve a deeper understanding of tumor perfusion capacity. This study offers tools for the accurate, noninvasive evaluation of drug delivery efficacy.

The present investigation was performed to elucidate the role of purine nucleotides as potential indicators of chemosensitivity of malignant tumors. Drug-sensitive (s) and -resistant (r) tumor cell lines grown as monolayers (s: T47D, MCF-7 wild-type; r: NCI/ADR-RES, MCF-7/MDR) or as multicellular spheroids (T47D; NCI/ADR-RES) were exposed to 0.1, 1.0, and 10.0 microM Doxorubicin for up to 24 h. Purine nucleotides were assayed using HPLC and with some selected spheroids using imaging bioluminescence. The data show that in the time frame of the experiments reproducible and statistically significant changes in the nucleotides only occur at the highest drug concentration investigated. Under these conditions and using monolayer cultures, Doxorubicin caused a significant increase in ATP and GTP in sensitive but not in resistant cancer cells. Consequently, this differential change may be exploited for drug sensitivity testing in vitro. Doxorubicin exposure to spheroids was associated with significant increases in ATP and GTP in both sensitive and resistant variants. However, the kinetic of the changes in GTP was largely different between T47D and NCI/ADR-RES spheroids with a long-lasting, almost 3-fold elevation and a smaller, relatively short transient increase in GTP, respectively. Supplementing experiments with Doxorubicin treatment under inhibition of oxidative phosphorylation with Oligomycin abolished the drug-induced ATP and GTP peaks at persistent increases in ADP and AMP. Assuming that the spheroids may represent the in vivo situation to a better degree than monolayer cultures, experimental in vivo studies should clarify whether kinetic changes in GTP could be used as differential markers for the chemosensitivity of solid tumors. The experiments using Oligomycin support the hypothesis that purine nucleotides may be recycled from DNA fragments that result from the interaction of the drug with the DNA strands.

Vascular endothelial growth factor (VEGF) mRNA and protein levels were higher in MCF7 breast cancer cells and orthotopic tumors after treatment with tamoxifen, as compared with treatment with estrogen. Accordingly, tumor vascular permeability, evaluated in vivo from contrastenhanced magnetic resonance imaging, was elevated during treatment with tamoxifen. The results indicate that estrogen regulates angiogenesis in MCF7 tumors by maintaining VEGF at levels sufficient for the generation of functional microcapillaries and consequently facilitates tumor growth. During tamoxifen treatment, estrogen regulation is removed, and VEGF increases to levels that enhance markedly vascular permeability and reduce their perfusion function, leading to inhibition of tumor progression.

The purpose of this study was to develop, standardize, and test reproducibility of a lexicon for reporting contrast-enhanced breast magnetic resonance imaging (MRI) examinations. To standardize breast MRI lesion description and reporting, seven radiologists with extensive breast MRI experience developed consensus on technical detail, clinical history, and terminology reporting to describe kinetic and architectural features of lesions detected on contrast-enhanced breast MR images. This lexicon adapted American College of Radiology Breast Imaging and Data Reporting System terminology for breast MRI reporting, including recommendations for reporting clinical history, technical parameters for breast MRI, descriptions for general breast composition, morphologic and kinetic characteristics of mass lesions or regions of abnormal enhancement, and overall impression and management recommendations. To test morphology reproducibility, seven radiologists assessed morphology characteristics of 85 contrast-enhanced breast MRI studies. Data from each independent reader were used to compute weighted and unweighted kappa (κ) statistics for interobserver agreement among readers. The MR lexicon differentiates two lesion types, mass and non-mass-like enhancement based on morphology and geographical distribution, with descriptors of shape, margin, and internal enhancement, Lexicon testing showed substantial agreement for breast density (κ =0.63) and moderate agreement for lesion type (κ =0.57), mass margins (κ =0.55), and mass shape (κ =0.42). Agreement was fair for internal enhancement characteristics. Unweighted kappa statistics showed highest agreement for the terms dense in the breast composition category, mass in lesion type, spiculated and smooth in mass margins, irregular in mass shape, and both dark septations and rim enhancement for internal enhancement characteristics within a mass. The newly developed breast MR lexicon demonstrated moderate interobserver agreement. While breast density and lesion type appear reproducible, other terms require further refinement and testing to lead to a uniform standard language and reporting system for breast MRI.

Early metabolic events in Escherichia coli exposed to nalidixic acid, a topoisomerase II inhibitor and an inducer of the SOS system, were investigated by in vivo NMR spectroscopy, a technique that permits monitoring of bacteria under controlled physiological conditions. The energetics of AB1157 (wild type) and of its isogenic, SOS-defective mutants, rεcBC, lexA, and ΔrecA, were studied by ³¹P and ¹⁹F NMR before, during, and after exposure to nalidixic acid. The content of the NTP in E. coli embedded in agarose beads and perfused at 36°C was found to be 4.3 ± 1.1 x 10^-18 mol/cell, yielding a concentration of -2.7 ± 0.7 mM. Nalidixic acid induced in the wild type and mutants a rapid 2-fold increase in the content of the NTP, predominantly ATP. This induction did not involve synthesis of uracil derivatives or breakdown of RNA and caused cell proliferation to stop. Removal of nalidixic acid after 40 min of treatment rescued the cells and resulted in a decrease of ATP to control levels and resumption of proliferation. However, in ΔrecA cells, which were more sensitive to the activity of the drug, ATP elevation could not be reversed, and ATP content continued to increase faster than in control cells. The results ruled out association between the elevation of ATP and the induction of the SOS system and suggested involvement of a process reminiscent of apoptosis in the stimulation of ATP synthesis. Thus, the presence of the RecA protein was found to be essential for reversing the ATP increase and cell rescue, possibly by its function in repair of DNA damage.

Variations in the cellular volume fraction and in the microvascular permeability of MCF7 human breast tumors were used to assess response to tamoxifen. These pathophysiological features were mapped by applying the three-time-point, contrast-enhanced, high resolution magnetic resonance imaging method (H. Degani et at., Nat. Med., 3: 780-782, 1997). Short-term treatment with tamoxifen caused a highly significant increase in the fraction of pixels displaying intermediate contrast agent clearance pattern and a significant increase in the fraction of pixels displaying high rate of contrast agent entrance. These changes resulted from a marked rise in the extracellular volume fraction, indicating increased necrosis, and from an augmentation in the microvascular permeability, predominantly in the vicinity of the high extracellular volume fraction areas, as a result of stress- induced angiogenesis.

Tumor necrosis factor α (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G₀+G₁/S, accompanied by apoptosis. ³¹P and ¹³C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and ²H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52±6%. Later (~15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis. Copyright (C) 1998 Elsevier Science B.V.

A modified Prony method (MPM) was applied to analyze the main signals present in spatially resolved ³¹P NMR spectra of MCF7 breast tumors implanted in nude mice. First, the method was tested on synthetic data to establish its limits of reliability. Its performance with respect to peak identification and quantification of signal intensities was then exploited on data from three implanted tumors during hormonal manipulation with estrogen and the antiestrogenic drug tamoxifen. The phosphomonoester peak was resolved into phosphocholine (PC) and phosphoethanolamine (PE). Treatment with tamoxifen led to a significant reduction in the PE to PE+PC peak amplitude ratio in the tumors under consideration. MPM analysis also revealed the presence of two different inorganic phosphate pools: a larger acidic pool and a smaller alkaline pool during estrogen-induced growth and the reverse during tumor regression.

Magnetic resonance imaging (MRI) is a noninvasive method that reveals anatomical details in vivo and detects lesions for diagnosis. Although standard breast MRI cannot clearly delineate breast cancer, contrast-enhanced MRI enables the detection of breast masses with high sensitivity. Dynamic studies demonstrated that malignant lesions were characterized by a faster signal enhancement rate than benign ones. Dynamic MRI of human breast cancer in mice revealed high heterogeneity in the distribution of contrast-enhanced curves and derived pathophysiological features, indicating the importance of high spatial resolution. With clinical MRI, it is difficult to achieve simultaneously high spatial and temporal resolution. In previous dynamic studies, the emphasis was on high temporal resolution and mainly empiric analyses. We describe here a new model based method that optimizes spatial resolution by using only three time points, and yet characterizes tumor heterogeneity in terms of microvascular permeability and extracellular fraction. Mapping these pathophysiological features may aid diagnosis and prognosis assessment, while the high spatial resolution may improve the capacity to detect smaller lesions. The method was tested in human breast tumors implanted in mice and in a limited number of benign and malignant breast lesions of patients.

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space. Correlation of these maps with T₂-weighted spin echo images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that surrounded necrotic loci. The high leakage from the intratumoral permeable capillaries indicated an induction of a specific angiogenic process associated with stress conditions that cause necrosis. This induction was augmented in tumors responding to tamoxifen treatment. Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.

By comparing the metabolism of human mammary epithelial cells and human breast cancer cells (MCF7 and T47D), proliferating at approximately the same rate, it was possible to isolate the effect of malignancy alone on the energetics and phospholipid metabolism of cancer cells. ³¹p NMR of perfused mammary cells and of water soluble extracts of these cells showed that the levels of phosphocholine, phosphoethanolamine, and glycerol derivatives of these metabolites were very low and significantly less than that in the cancer cells, suggesting an association of malignancy with induction of phospholipid biosynthesis and breakdown. The level of the high energy phosphates and the rates of glucose consumption and aerobic glycolysis did not reveal distinct differences between normal and cancer cells. The comparable energetic appear to be related to the similarity of proliferating capacity in culture of the normal and cancer cells.

Glucose metabolism in control and estrogen stimulated rat uteri was investigated using ¹³C NMR spectroscopy. By employing an NMR adapted perifusion system, and developing protocols and methods based on the application of [1¹³C]glucose labeling, it was possible to measure, with a temporal resolution of 10 min, the kinetics of glucose consumption, lactate production and ¹³C incorporation into glutamate, alanine and glycogen. In control immature rat uteri, under aerobic conditions, the rates (±SEM) of [1¹³C]glucose consumption and [3¹³C]lactate production were 24±2 and 7.5±0.5mmol/g wet weight/h. The rates of synthesis of [4¹³C]glutamate, [3¹³C]alanine and ¹³C labeling of glycogen at C₁ of its glucose moieties were significantly lower and were in the range 0.30.6 mmol/g wet weight/h. Thus, ca 35% of the labeled glucose was accounted for by the glycolytic and other observed pathways. In vitro stimulation of the uteri by estrogen was found to increase significantly, within 1 h, glucose consumption by 80%, lactate production by 150% and glutamate and glycogen synthesis by 150%, in parallel to a rapid hormonal induction of mRNA for the brain type isozyme of creatine kinase.

Alterations in the energy state and glucose metabolism of hippocampal slice exposed to high extracellular K⁺ ([K⁺]_o) were monitored using ³¹P and ¹³C NMR spectroscopy. Slices were perfused (37°C) continously within the NMR spectrometer and tissue viability and metabolic activity were maintained for at least 18 h. ³¹P spectra showed that upon exposure to 40 mM [K⁺]_o, there was a rapid compromise in tissue energetics where, by 15 min of exposure, the ratio of phosphocreatine and of nucleoside triphosphates to inorganic phosphate (extra- and intracellular) decreased 30-50% relative to pre-exposure values. This was accompanied by a pH decrease of ≈0.3 units in both the intra and extracellular environments. A lower but stable energy state was reached at ≈15 min of exposure and full recovery was observed by 30 min following the removal of high [K⁺]_o. Utilizing ¹³C NMR in the presence of [1-¹³C]glucose, an immediate and dramatic acceleration in tissue glycolysis was observed when slices were exposed to 40 mM [K⁺]_o: the rates of both [1-¹³C]glucose consumption and [3-¹³C]lactate synthesis increased by ≈ 20 fold. By 60 min following the removal of high-[K⁺]_o, pre-exposure rates of tissue glycolysis were restored. The results indicated that the rapid and dramatic induction of energy production via glycolysis probably accounts for the ability of hippocampal slices to maintain viability and recuperate from brief but intense depolarizing conditions which are remiscent of seizure states in vivo.

Keywords: Endocrinology & Metabolism; Obstetrics & Gynecology

³¹P and ³¹C NMR spectroscopy of lipid extracts of T47D human breast cancer spheroids and the use of ¹³Clabeled lipid precursors [3¹³C ] serine, [1,2¹³C] ethanolamine, and [1,2¹³C]choline enabled us to determine the rate of ¹³C incorporation into the major phospholipids and to show that the synthesis of phosphatidylethanolarnine in T47D cells is via both the CDPethanolamine pathway and serine decarboxylation, with the extent of each depending on the concentration of ethanolamine in the medium. In the presence of low ethanolamine (3.4 μ M), both pathways contribute in equal proportions, while in the presence of high ethanolamine, the CDPethanolamine pathway predominates. © 1992 Academic Press, Inc.

³¹P- and ¹³C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 μm) spheroids. Spheroids were perfused inside the spectrometer with 1,2-¹³C-labelled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4±1 fmol/cell compared to 8±1 fmol/cell in small (150 μm) proliferating spheroids (P -1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2-0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 μM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells (P -) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2-0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.

Phosphorous 31 (³¹P) nuclear magnetic resonance (NMR) spectra were recorded from perchloric acid extracts of benign and malignant breast tumors. The spectra were correlated with the histopathologic diagnosis and the steroid receptor status of the tumor. Higher relative content of the lipidderived metabolite glycerolphosphoethanolamine (GPE), the highenergy nucleoside phosphates (nucleosidediphosphate [NDP], nucleosidetriphosphate [NTP]), and sugar esters of uridine diphosphate (UDPS) appeared in the carcinomas. Malignant tumors also showed a lower ratio of phosphoethanolamine to phosphocholine (PE/PC) than benign conditions. Lower content of the lipidderived metabolite glycerolphosphocholine (GPC) and high content of the highenergy compound phosphocreatine (PCr) were associated with malignant tumors having high content of estrogen receptors (ER). High PCr content was also associated in the carcinomas with high progesterone receptors (PgR) content. In the benign tumors NDP and NTP were higher in tumors with high PgR content. The authors suggest that ³¹P magnetic resonance spectroscopy (MRS) of the breast can provide additional variables to diagnose malignancy, and when combined with magnetic resonance imaging (MRI), invasive procedures may be avoided. It also seems that levels of PCr and GPC obtained from the spectra can serve as markers to hormonal receptor status of breast carcinomas, and may be used in addition to the ER and PgR content to improve prediction of the response to hormonal therapy. Additional development requires in situ MRI and MRS combined studies.

Magnetic resonance H-1-imaging and P-31-localized spectroscopy were utilized to monitor, noninvasively, MCF7 human breast cancer tumors implanted in immunodeficient mice. The tumors were followed during estrogen dependent growth and tamoxifen induced remission. Early after tamoxifen administration enhanced necrosis developed, extending to most of the tumor volume. This was followed by growth of repair tissue along with tumor regression. The short-term tamoxifen treatment also modified the content of the phosphate metabolites, increasing the nucleoside triphosphate to inorganic phosphate ratio from 0.41 +/- 0.15 (n = 14) before treatment to 1.10 +/- 0.70 (n = 8, P

The in vivo ³¹P NMR spectrum of T47D human breast cancer cells grown as spheroids shows changes in the phosphomonoester lipid precursors as a function of spheroid size. The ratio of phosphorylethanolamine (PE) to phosphorylcholine (PC) was 1.0±0.3 for 3-day-old, 150 μm spheroids. This ratio increased to 2.4±0.4 for spheroids 7 days and older and which were at least 300 μm in diameter. To investigate the phosphatidylethanolamine to phosphatidylcholine (PdyIE/Pdy1C) ratio in the membranes, chloroform/methanol extracts of spheroids were performed. The ³¹P spectrum of these extracts showed no change with spheroid size, namely the PdyIE/Pdy1C ratio was 0.5±0.06 for spheroids of all ages suggesting that membrane composition is strongly regulated at the precursor level.

A technique for the entrapment of the ünicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steadystate conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMRvisible polyphosphates decreased as well, presumably due to the production of longer, NMRinvisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.

The thermodynamic and kinetic parameters of the reaction catalyzed by creatine kinase (CK) were measured in vitro in the temperature range 13 to 35°C, using ³¹P NMR spectroscopy, including magnetization transfer methods. The apparent equilibrium constant of the reaction and the associated enthalpy for the formation of ATP at 35°C, pH 8.2, and excess [Mg²⁺] were 3.5 × 10⁹ M⁻¹ and −2.4 ± 0.5 kcal/mol, respectively. The rates at equilibrium at 35°C catalyzed by 1 unit/ml CK were 12.4 and 10.7 μM /s at pH 7 and 8, respectively. The rate constants per 1 unit CK/ 1 ml at 35°C, pH 7, were 1.3 × 10⁸ s⁻¹M⁻² and 9.9 × s⁻¹ M⁻¹ in the direction of ATP and PCr formation, respectively. The activation energies in both directions were similar and corresponded to 15 ± 2 kcal/mol at pH 7 and 17.5 ± 1.5 kcal/mol at pH 8. Comparison of in vivo results with the above in vitro data may provide information regarding the activity and kinetics of the CK reaction.

Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with ³¹P and ¹³C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. The content of phosphocholine had increased by 10% to 30% within the first hour of estrogen stimulation, but the content of the other observed phosphate metabolites as well as the pH remained unchanged. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.

Two methods for growing anchoragedependent cells were adapted for nuclear magnetic resonance (NMR) measurements: growing cells on agarose polyacrolein microsphere beads and on \u201cfilters\u201d made of nonwoven polyester fabric. Both were found to be convenient and most suitable for NMR studies in any conventional spectrometer without probe modification. These methods were employed in studies of human breast cancer T47DA11 cells, using scanning electron microscopy and 31P NMR spectroscopy. The results show that the contents per cell of phosphorylcholine, phosphorylethanolamine, and their glycerol derivatives depend on the mode of cell assembly and decrease gradually with the increase in cellcell interaction along the growth curve.

³¹P nuclear magnetic resonance (NMR) was used to study the effects of 17β-estradiol on the content of phosphates and on the flux catalyzed by creatine kinase in immature rat uteri. Perifusion with oxygenated medium at 36 C maintained the uteri in a viable state for at least 10 h in vitro during ³¹P NMR measurements. In vitro administration of 17β-estradiol to the perifused uteri induced changes in the concentration of the high energy phosphates similar to those found after in vivo stimulation: a rapid fall in the concentrations of ATP, phosphocreatine, and the phosphomonoesters during the first 2 h, followed by a slower return to initial concentrations by approximately 6 h. Analysis of the time course of this modulation indicated that after estrogen stimulation, the energy utilization rate was about twice the production rate. The flux through the creatine kinase (CK) reaction was measured independently using ³¹P magnetization transfer techniques; it was found to increase in uteri 24 h after estradiol injection by the same extent (65%) as the specific activity of CK measured by a spectrophotometric assay. The congruence between the results of these two techniques (in the absence of increased substrate concentrations) provides evidence that the early stimulation of brain-type CK synthesis by estrogen results in a net increase in the concentration of this enzyme.

The kinetics of the phosphate exchange by creatine kinase (CK) was studied in solution and in the Langendorff-perfused rat heart at 37 °C. 31P inversion-transfer (IT) and saturation-transfer (ST) methods were applied. The kinetic parameters obtained by the two magnetization transfer methods were the same, whether in solution or in the perfused heart. Inversion transfer is the more efficient method, yielding the kinetic constants for the exchange and the relaxation rates of the transferred phosphate in both substrates, in one experiment. In solution the forward (kF) and reverse (kR) pseudo-first-order rate constants for the CK reaction (kF = kx [MgADP] [H+]; kK = k.x[creatine]) as well as the concentrations of phosphocreatine (PCr), MgATP, and creatine (Cr) remained constant between pH 6.9 and pH 7.8. Equilibrium at this pH region is therefore maintained by compensating changes in the concentration of MgADP. The forward and reverse fluxes in the perfused heart were equal with an average flux ratio (fluxF/fluxR) of 0.975 ± 0.065 obtained by both methods. Average values of kF and kR were 0.725 ± 0.077 and 1.12 ± 0.14 s-1, respectively. These results clearly indicate that the CK reaction in the Langendorff-perfused heart is in equilibrium and its rate is not limited by the diffusion of substrates between different locations of the enzyme. There is therefore no indication of compartmentation of substrates of the CK reaction.

The exchange rate and enthalpy and entropy of activation of the diffusion of the first five n-alkylamines across egg phosphatidylcholine vesicles has been measured by ¹H-NMR spectroscopy employing the 1:2 Gd³⁺-EDTA complex as a relaxation reagent. The permeability determined from the exchange rate of the ethyl through the pentyl derivatives increased sequentially with increasing chain length from 710^-7 to 410^-4 cm/s, respectively, at 25°C. The permeability of methylamine was similar to that of ethylamine (110^-6 cm/s at 25°C) and exhibited a relatively smaller entropy increase. The enthalpy of activation for the transfer reaction was high for all amine derivatives (20 kcal/mol). The entropy of activation increased with increasing chain length. The results indicate that the rate of diffusion is dominated by the partition into the membrane. Methylamine, being the smallest molecule in this series, can probably diffuse also through vacancies formed by the internal motions of the lipid chains.

15N NMR spectra have been obtained for anion I. In the deprotonated form N2O32- (pH 13), resonances occur at 355.2 and 339.5 ppm for N(1) and N(2), respectively, referred to NH3(1). The N(2) resonance position remains unchanged in the monoprotonated form HN2O3- (pH 7.8), while the N(l) resonance undergoes a 24-ppm upfield shift (pH 8), indicating protonation at N(1). This conclusion is corroborated by an observed NOE = -2.1 for N(l) at pH 8. The coupling constant 1J15N-15N = 16.6 Hz for N2O32-. Both 15N resonances exhibit broadening with decreasing pH, to an extent that is substantial in the case of N(2), which also shows a small upfield shift between pH 7.8 and pH 6.8. Implications of these results for interpretation of the protonation-induced destabilization of the anion are discussed.

Nuclear relaxation measurements of ¹H and ¹⁷O of water have been applied to study the kinetics of water diffusion across vesicular lipid membranes. Differentiation between the intra- and extravesicular media was achieved by entrapping Mn²⁺ inside the vesicles. The water permeability of egg phosphatidylcholine vesicles was found to be 2.9 · 10⁻³ cm/s at 25°C, with an activation energy of 10.5 kcal/mol which remains constant through the temperature range 065°C. The water permeability across vesicular bilayers of L-α-dipalmitoyl phosphatidylcholine exhibited a sharp change through the lipid phase transition. The permeability in the lipid crystalline phase (45°C) was found to be 7.2 · 10⁻³ cm/s with an activation energy of 7.2 kcal/mol. Below the transition at the gel phase (35°C) a permeability of 1.0 · 10⁻³ cm/s was determined. The results indicate that water diffuses through lipid membranes in the liquid crystalline phase in a similar fashion to its diffusion in hydrocarbon liquids. However, when the lipids undergo a phase transition to the gel state, this similarity does not hold any more and water diffusion becomes much more restricted than in hydrocarbon liquids. The change in water permeability through the phase transition was correlated with the changes observed in the lipid segmental motion determined from ¹³C T₁ measurements.