Southwestern In Vivo Cellular and Molecular Imaging Program

Core 1
Magnetic Resonance
(Director Ralph P. Mason, Ph.D.)

Nuclear Magnetic Resonance (NMR) is a non-invasive technique using radiofrequency waves to investigate the nuclei of atoms in a magnetic field. NMR spectroscopy primarily provides information regarding chemical structures while MRI provides spatial information. Since the techniques are non-invasive each individual, tumor or location within a tumor can serve as its own control and reveal diagnostic changes with respect to an intervention or therapy.

Small Animal MR | Clinical MR

¹H MRI of tissue water can provide exquisite anatomical detail. While MRI is itself a relatively new technique, it has become the modality of choice for many clinical investigations. However, technical developments in terms of magnetic field strength, gradient quality and pulse waveform programmability now allow many variations on the basic sequence of Excitation, Evolution and Detection. Indeed, solvent suppression allows detection of millimolar metabolites (e.g., lactate, N-acetyl aspartate, choline, creatine) in the face of 10⁵ fold dynamic range with respect to tissue water. Localization provides access to small tumor nodules revealing heterogeneity and avoiding contamination from surrounding tissues. Echo planar and potentially echo volume techniques together with appropriate computing power offer real time images of dynamic variations in water characteristics reflecting perfusion, diffusion, oxygenation and flow. Multiple quantum filters may also be applied to detect specific resonances. Progressively, many of the methods designed for chemical analysis are becoming feasible in small animals and man. In addition to hardware developments, contrast agents may highlight specific lesions or reveal dynamic heterogeneity indicative of vascular development. Based on molecular size, weight, lipophilicity and tissue affinity new classes of agent are becoming available for diagnosis. In addition, heteronuclear capabilities, particularly ¹⁹F NMR provide insight into specific physiological parameters such as pO₂, pH, metal ions and potentially gene expression using specific reporter molecules. These methods open new opportunities to investigate tumor physiology and response to therapy,e.g., anti vascular agents, chemotherapeutic agents and gene therapy. The extensive availability of MR for both small animal research and clinical investigations ensures that technique development and pre-clinical studies are feasible prior to translation to the clinic. Moreover, novel clinical observations may be rigorously pursued in complementary animal, cellular and ex vivo studies.

Small Animal MR
(Director Ralph P. Mason, Ph.D.)

This research will primarily use the facilities of the Mary Nell and Ralph B. Rogers NMR Center.

Supported by The Whitaker Foundation, The American Heart Association, and more recently, The American Cancer Society, NCI and DOD Breast Cancer Initiative we have been developing non-invasive methods for monitoring tissue physiology. Particular emphasis has been on quantitative measurement of tumor oxygenation: pO₂ quantitation and dynamics. We routinely apply various ¹H and ¹⁹F imaging methods to observe anatomy and physiological function in tumors. Methods to be applied in the foreseen Development Projects include BOLD, FAIR, Arterial Spin Tagging, fMRI based on contrast agent uptake and clearance curves, and ¹⁹F MR oximetry and pH measurements. These methods may be interleaved to provide multiparametric information reflecting dynamic changes in response to an intervention. It has been suggested that the kinetic curves of contrast agent uptake may be diagnostic for detecting tumor malignancy. We propose to investigate whether contrast changes induced by inhaling oxygen may provide a simpler approach, characteristic of cellular metabolic activity and blood flow.

We have pioneered a unique approach to assess dynamic changes in regional tumor oxygenation. ¹⁹F EPI Oximetry protocol (FREDOM: Fluorocarbon Relaxometry for Dynamic Oxygen Mapping). Hexafluorobenzene (HFB) is injected directly into selected areas of tumors as a reporter molecule. HFB has many virtues including high sensitivity to changes in pO₂, minimal response to temperature and a single sharp ¹⁹F NMR signal. HFB is also cheap, readily available commercially and exhibits minimal toxicity (LD₅₀ > 25 g/kg). Direct intra tumoral injection provides measurements analogous to the Eppendorf Histograph, which has been found to provide clinically relevant baseline pO₂ data. Specifically, it allows immediate interrogation of regions of interest. The great advantage of the NMR approach, however, is the ability to monitor dynamic changes in regional pO₂ at multiple locations, simultaneously, without reintroducing a needle. By applying a single-spin-echo EPI sequence with "blipped" phase encoding (MBEST), a pulse burst saturation recovery (PBSR) pulse train and a new data acquisition protocol, ARDVARC (Alternated Relaxation Delays with Variable Acquisitions to Reduce Clearance effects), we are able to achieve a typical precision ~ + 2-3 torr for 20 - 50 voxels in 7 mins within a tumor. Specific regions may be followed for a period of hours with respect to intervention. Comparing results with the traditional polarographic technique (Eppendorf Histograph) and show that the two methods provided very similar results. MR having the advantage of providing multiple repeat measurements without the need to reintroduce a needle. 

This Core will primarily use an Omega 4.7 T CSI 40 cm horizontal MR system shortly to be upgraded. This imaging and spectroscopy system has actively shielded gradients (5 G/cm) and waveform generators on all RF and gradient channels capable of multinuclear in vivo investigations of small animals. Imaging methods, which are used for a variety of multinuclear applications include EPI, multi-slice/multi-echo, gradient echo, multiple-quantum-filtered, cardiac- and respiratory-gating, FLASH, and STIR. Numerous ¹H/¹⁹F tunable surface coils and whole body coils are available, optimized for volume for interest. In addition, a vertical bore 9.4 T/8.9 cm microscopic imaging and (gradient-enhanced) spectroscopy system with actively shielded gradients (120 G/cm) is available for performing multinuclear investigations of mice and perfused organs, as well as high resolution spectroscopy of tissue extracts. Shared equipment is available for physiological monitoring, e.g., blood gas analyzer, Luxtron optical fiber temperature system, small animal anesthesia unit and Nonin fiber optic pulse oximeter compatible with MRI studies and modified with software to monitor rat physiology (heart rate and blood gases).

Comparison with nuclear imaging methods will allow us to follow uptake and delivery of labeled agents (e.g. antibodies) and monitor their efficacy (physiological MRI) in a single tumor. This Core will also be critical to the assessment of the novel gene reporter molecules being developed by Core 4.

Clinical MR Techniques
(Director: Pam Nurenberg, M.D.)

Clinical MR will use the facilities of the Mary Nell and Ralph B. Rogers NMR Center and the Meadows Center. Our modern clinical MR systems allow rapid acquisition of high resolution spectra and images. MR spectroscopy allows qualitative and semi-quantitative evaluation of metabolites. It can localize and characterize tumors as well as follow disease progression, monitor treatment, localize non-necrotic regions within the tumor for biopsy, and evaluate post surgical/post radiation zones for recurrence. One can overlay anatomic contours from routine MR imaging onto the spectroscopic image (e.g., Figure). Kurhanewicz et al. demonstrated that proton NMR spectroscopy of cancerous prostate has much lower citrate levels and higher choline levels than normal prostate. (Choline + creatine)/citrate ratios > 0.86 were highly specific for prostate cancer (3 standard deviations above mean), with only a slight overlap with benign prostatic hypertrophy. This ratio is particularly helpful for locating cancer in patients with rising Prostate Specific Antigen levels highly suspicious for cancer, but with multiple negative biopsies. We propose to undertake such studies for several patient populations by adding MRS to routine clinical evaluations for patients with breast, prostate and brain cancer. Correlative biopsy data to be evaluated through the SPORE- genetic signature program should lead to new diagnostic criteria. Relevant studies can be undertaken with respect to Development Projects 3 (Fink, Neuro oncology), 4 (Hsieh, Urology), and in conjunction with the ongoing clinical trials of Dr. Weatherall (breast cancer). This Figure shows MSRI from the same patient as these figures (1, 2). Dr. Nurenberg is the PI for an NCI R21 high impact grant to study renal masses with MRS in patients suspected of having Tuberous Sclerosis in cooperation with the Tuberous Sclerosis (TS) Society: MR spectroscopy of renal masses in patients with TS may distinguish benign angiomyolipomas that do not have visible fat on routine imaging studies from malignant tumors.

In addition to spectroscopy clinical MR is developing new imaging approaches, particularly related to vasculature, angiogenesis and oxygenation based on such measurements as blood flow (e.g. AST), perfusion/diffusion, hemoglobin saturation (BOLD Blood Oxygen Level Dependant) and contrast agent kinetics. Hawighorst et al. demonstrated that contrast enhancement detected by MRI, as indicative of vascularity corresponded well with vessel density and VEGF expression determined histologically and may thus be a significant marker of prognosis. Steady state gadolinium enhanced MR images show tumor enhancement based on the status of blood brain barrier. Dynamic susceptibility contrast studies have high temporal and spatial resolution and provide information on relative cerebral blood volume (rCBV). Dynamic susceptibility contrast images are obtained using fast T2* or T2 weighted imaging sequences during susceptibility contrast injection. Changes in the tissue transverse relaxation rate (DR2 or DR2*) induced by susceptibility contrast agents are linearly dependent on the tissue concentration of the agent. The DR2* calculated for perfusion are a measure of the maximum amount of contrast agent that enters the voxel during the first passage of contrast. The first passage of the contrast bolus can be detected by tracking the signal reduction due to the contrast agent as a concentration versus time curve. The maximum value of DR2* is related to the perfusion and the area under the R2* curve represents the rCBV when blood brain barrier is intact. Absolute blood flow or volume measurements (cc/min) are not feasible with this technique, but relative changes in rCBV between intact brain tissue and lesions can be documented. We have the equipment and capabilities to employ all these techniques at our institution in animal research as well as human clinical studies. We can evaluate patients being treated with antiangiogenic drugs as well as monitor gene therapy or aid in the detection of chromosomal aberrations. Dr. Yetkin will be involved very closely in performing, analyzing, and interpreting perfusion and oxygenation studies. Core 7 (Dr. McColl) will be critical in terms of data manipulation, storage, presentation and integration with other modalities.

Clinical MRI equipment includes two 1.5 T MRI GE Signa LX capable of acquiring single-shot EPI in arbitrary planes, a Philips NT and a new Intera scanner each with Turbo Spectroscopic imaging capabilities are available for research at specified times. Clinical “power-injectors are available for dynamic contrast enhancement studies to assess perfusion and evaluate tumors using first pass contrast agent uptake. Clinical and research studies using the BOLD phenomenon have been ongoing at UT Southwestern since 1994, starting with an experimental echo-planar gradient insert in a hybrid Picker HPQ/Edge system. Real-time image reconstruction allows the user to both direct scans from a remote workstation, and to monitor the course of a functional experiment via on-the-fly statistical analysis as the scan progresses.

The patient presented in Figs. 1, 2 and 3 had previously received radiation therapy and two types of chemotherapy. He received gamma knife radiosurgery to a nodule of enhancement two months before the MRI scan (Figure), and MR spectroscopy (Figure) done at that time confirms probable high grade tumor recurrence rather than radiation necrosis. This was confirmed with an ¹⁸FDG PET scan done elsewhere (Baylor), which showed hypermetabolic tumor tissue located deep at the site of MR Gd-enhancement. UT-Southwestern now has it's own clinical PET (Positron Emmission Tomography) facility.

For Further Information Contact: RALPH  P. MASON, Ph.D.
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