Martin J. Lizak

Detecting response of rat C6 glioma tumors to radiotherapy using hyperpolarized [1-13C]pyruvate and 13C magnetic resonance spectroscopic imaging

We show here that hyperpolarized [1‐13C]pyruvate can be used to detect treatment response in a glioma tumor model; a tumor type where detection of response with 18fluoro‐2‐deoxyglucose, using positron emission tomography, is limited by the high background signals from normal brain tissue. 13C chemical shift images acquired following intravenous injection of hyperpolarized [1‐13C]pyruvate into rats with implanted C6 gliomas showed significant labeling of lactate within the tumors but comparatively low levels in surrounding brain.Labeled pyruvate was observed at high levels in blood vessels above the brain and from other major vessels elsewhere but was detected at only low levels in tumor and brain.The ratio of hyperpolarized 13C label in tumor lactate compared to the maximum pyruvate signal in the blood vessels was decreased from 0.38 ± 0.16 to 0.23 ± 0.13, (a reduction of 34%) by 72 h following whole brain irradiation with 15 Gy. Magn Reson Med, 2011.

MR assessment of changes of tumor in response to hyperbaric oxygen treatment

Enhancement of image intensity, using the T1‐weighted spoiled gradient‐echo (SPGR) sequence, was measured in SCC tumor implanted in the flank of C3H mice while they were subjected to several types of oxygenation challenges inside a hyperbaric chamber designed and constructed to fit in an MRI resonator. The central portions of the tumor gave a positive enhancement, while the periphery showed signal reduction during both normobaric (NBO) and hyperbaric (HBO) oxygen challenges. In the contralateral normal leg, nearly 70% of the region showed a decrease in intensity, and the rest showed a positive enhancement. The positive signal enhancement was markedly greater under HBO compared to NBO. Calculated R1, R2, and M0 maps from multivariate fitting of images acquired by a multislice multiecho (MSME) sequence with variable TR before, during, and after HBO treatment confirm that the source of SPGR signal enhancement in the tumor is associated with shortening of T1. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Observation of microscopic diffusion anisotropy in the spinal cord using double-pulsed gradient spin echo MRI

A double‐pulsed gradient spin echo (d‐PGSE) filtered MRI sequence is proposed to detect microscopic diffusion anisotropy in heterogeneous specimen. The technique was developed, in particular, to characterize local microscopic anisotropy in specimens that are macroscopically isotropic, such as gray matter. In such samples, diffusion tensor MRI (DTI) produces an isotropic or nearly isotropic diffusion tensor despite the fact that the medium may be anisotropic at a microscopic length scale. Using d‐PGSE filtered MRI, microscopic anisotropy was observed in a “gray matter” phantom consisting of randomly oriented tubes filled with water, as well as in fixed pig spinal cord, within a range of b‐values that can be readily achieved on clinical and small animal MR scanners. These findings suggest a potential use for this new contrast mechanism in clinical studies and biological research applications. Magn Reson Med 59:803–809, 2008.

Study of Ocular Transport of Drugs Released from an Intravitreal Implant Using Magnetic Resonance Imaging

Ensuring optimum delivery of therapeutic agents in the eye requires detailed information about the transport mechanisms and elimination pathways available. This knowledge can guide the development of new drug delivery devices. In this study, we investigated the movement of a drug surrogate, Gd-DTPA (Magnevist®) released from a polymer-based implant in rabbit vitreous using T1-weighted magnetic resonance imaging (MRI). Intensity values in the MRI data were converted to concentration by comparison with calibration samples. Concentration profiles approaching pseudosteady state showed gradients from the implant toward the retinal surface, suggesting that diffusion was occurring into the retinal–choroidal–scleral (RCS) membrane. Gd-DTPA concentration varied from high values near the implant to lower values distal to the implant. Such regional concentration differences throughout the vitreous may have clinical significance when attempting to treat ubiquitous eye diseases using a single positional implant. We developed a finite element mathematical model of the rabbit eye and compared the MRI experimental concentration data with simulation concentration profiles. The model utilized a diffusion coefficient of Gd-DTPA in the vitreous of 2.8×10−6, cm2, s−1 and yielded a diffusion coefficient for Gd-DTPA through the simulated composite posterior membrane (representing the retina–choroid–sclera membrane) of 6.0×10−8, cm2, s−1. Since the model membrane was 0.03-cm thick, this resulted in an effective membrane permeability of 2.0×10−6, cm, s−1. Convective movement of Gd-DTPA was shown to have minimal effect on the concentration profiles since the Peclet number was 0.09 for this system.

EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia‐sensitive drug. The small molecule 3‐bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3‐bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3‐bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2 < 10 mmHg) in vivo using squamous cell carcinoma (SCCVII)‐bearing mouse model. Metabolic MRI studies using hyperpolarized 13C‐labeled pyruvate showed that monocarboxylate transporter‐1 is the major transporter for pyruvate and the analog 3‐bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter‐1 in vivo. Expression of monocarboxylate transporter‐1 was enhanced in moderately hypoxic (8–15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia‐activated drugs. Magn Reson Med, 2013.

MRI in ocular drug delivery

Conventional pharmacokinetic methods for studying ocular drug delivery are invasive and cannot be conveniently applied to humans. The advancement of MRI technology has provided new opportunities in ocular drug‐delivery research. MRI provides a means to non‐invasively and continuously monitor ocular drug‐delivery systems with a contrast agent or compound labeled with a contrast agent. It is a useful technique in pharmacokinetic studies, evaluation of drug‐delivery methods, and drug‐delivery device testing. Although the current status of the technology presents some major challenges to pharmaceutical research using MRI, it has a lot of potential. In the past decade, MRI has been used to examine ocular drug delivery via the subconjunctival route, intravitreal injection, intrascleral injection to the suprachoroidal space, episcleral and intravitreal implants, periocular injections, and ocular iontophoresis. In this review, the advantages and limitations of MRI in the study of ocular drug delivery are discussed. Different MR contrast agents and MRI techniques for ocular drug‐delivery research are compared. Ocular drug‐delivery studies using MRI are reviewed. Copyright

Aldose reductase, a key enzyme in the oxidative deamination of norepinephrine in rats.

The sympathoneural neurotransmitter norepinephrine (NE) is deaminated to 3,4-dihydroxymandelaldehyde (DHMAL) and subsequently converted to either 3,4-dihydroxymandelic acid (DHMA) or 3,4-dihydroxyphenylglycol (DHPG). In this study, we investigated the relative importance of aldose reductase versus aldehyde reductase in the formation of DHPG from DHMAL. The in vitro incubation of NE with aldose reductase in the presence of monoamine oxidase (MAO) resulted in the formation of DHPG, which was confirmed by mass spectrometry. Although aldehyde reductase also generated DHPG, its activity was much lower than that of aldose reductase. With northern blotting, the expression of both aldose reductase and aldehyde reductase was detected in rat superior cervical ganglia. However, with western blotting, only aldose reductase was immunologically detectable. Treatment of rats with aldose reductase inhibitors for 3 days increased the plasma level of DHMA. There was no correlation between the selectivity of inhibitors and effects on NE metabolite levels. A significant decrease in DHPG, however, was obtained only with an extremely high dose (9 mg/kg/day) of the nonselective inhibitor AL 1576. The present study confirmed that aldose reductase generates DHPG from NE in the presence of MAO. In rat sympathetic neurons, aldose reductase appears to be more important than aldehyde reductase for the formation of DHPG. However, when aldose reductase is inhibited, it appears that aldehyde reductase can compensate for the conversion of DHMAL to DHPG, indicating redundancy in the reduction pathway.

Mapping Prefrontal Circuits In Vivo with Manganese-Enhanced Magnetic Resonance Imaging in Monkeys

Manganese-enhanced magnetic resonance imaging (MEMRI) provides a powerful tool to study multisynaptic circuits in vivo and thereby to link information about neural structure and function within individual subjects. Making the best use of MEMRI in monkeys requires minimizing manganese-associated neurotoxicity, maintaining sensitivity to manganese-dependent signal changes and mapping transport throughout the brain without a priori anatomical hypotheses. Here, we performed intracortical injections of isotonic MnCl2, comparisons of preinjection and postinjection scans, and voxelwise statistical mapping. Isotonic MnCl2 did not cause cell death at the injection site, damage to downstream targets of manganese transport, behavioral deficits, or changes in neuronal responsiveness. We detected and mapped manganese transport throughout cortical–subcortical circuits by using voxelwise statistical comparisons of at least 10 preinjection and two postinjection scans. We were able to differentiate between focal and diffuse projection fields and to distinguish between the topography of striatal projections from orbitofrontal and anterior cingulate cortex in a single animal. This MEMRI approach provides a basis for combining circuit-based anatomical analyses with simultaneous single-unit recordings and/or functional magnetic resonance imaging in individual monkeys. Such studies will enhance our interpretations of functional data and our understanding of how neuronal activity is transformed as it propagates through a circuit.

13C-MR Spectroscopic Imaging with Hyperpolarized [1-13C]pyruvate Detects Early Response to Radiotherapy in SCC Tumors and HT-29 Tumors

Purpose: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized 13C-MRI enabled detecting metabolic changes in tumors using a tracer [1-13C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized 13C-MRI. Experimental Design: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-13C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and 13C-MR signals were acquired using a 4.7 T scanner. Results: [1-13C]pyruvate and [1-13C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-13C]pyruvate administration. The [1-13C]lactate to [1-13C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on 13C-MRI. Conclusions: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized 13C-MRI could be useful for the evaluation of early response to radiotherapy. Clin Cancer Res; 21(22); 5073–81. ©2015 AACR. See related commentary by Lai et al., p. 4996

In vivo imaging of tumor physiological, metabolic, and redox changes in response to the anti-angiogenic agent sunitinib: longitudinal assessment to identify transient vascular renormalization.

AIMS The tumor microenvironment is characterized by a highly reducing redox status, a low pH, and hypoxia. Anti-angiogenic therapies for solid tumors frequently function in two steps: the transient normalization of structurally and functionally aberrant tumor blood vessels with increased blood perfusion, followed by the pruning of tumor blood vessels and the resultant cessation of nutrients and oxygen delivery required for tumor growth. Conventional anatomic or vascular imaging is impractical or insufficient to distinguish between the two steps of tumor response to anti-angiogenic therapies. Here, we investigated whether the noninvasive imaging of the tumor redox state and energy metabolism could be used to characterize anti-angiogenic drug-induced transient vascular normalization. RESULTS Daily treatment of squamous cell carcinoma (SCCVII) tumor-bearing mice with the multi-tyrosine kinase inhibitor sunitinib resulted in a rapid decrease in tumor microvessel density and the suppression of tumor growth. Tumor pO2 imaging by electron paramagnetic resonance imaging showed a transient increase in tumor oxygenation after 2-4 days of sunitinib treatment, implying improved tumor perfusion. During this window of vascular normalization, magnetic resonance imaging of the redox status using an exogenously administered nitroxide probe and hyperpolarized (13)C MRI of the metabolic flux of pyruvate/lactate couple revealed an oxidative shift in tumor redox status. INNOVATION Redox-sensitive metabolic couples can serve as noninvasive surrogate markers to identify the vascular normalization window in tumors with imaging techniques. CONCLUSION A multimodal imaging approach to characterize physiological, metabolic, and redox changes in tumors is useful to distinguish between the different stages of anti-angiogenic treatment.