De-En Hu

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-13C]pyruvate that the lactate dehydrogenase–catalyzed flux of 13C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using 13C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H13CO3-) and 13CO2 following intravenous injection of hyperpolarized H13CO3-. The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.

Dynamic nuclear polarization of 13C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-13C2]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-13C2]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized 13C-labeled malate from [1,4-13C2]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.

Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose

In this study, we monitored glycolysis in mouse lymphoma and lung tumors by measuring the conversion of hyperpolarized [U-2H, U-13C]glucose to lactate using 13C magnetic resonance spectroscopy and spectroscopic imaging. We observed labeled lactate only in tumors and not in surrounding normal tissue or other tissues in the body and found that it was markedly decreased at 24 h after treatment with a chemotherapeutic drug. We also detected an increase in a resonance assigned to 6-phosphogluconate in the pentose phosphate pathway. This technique could provide a new way of detecting early evidence of tumor treatment response in the clinic and of monitoring tumor pentose phosphate pathway activity.

Hyperpolarized [1-13C]-Ascorbic and Dehydroascorbic Acid: Vitamin C as a Probe for Imaging Redox Status in Vivo

Dynamic nuclear polarization (DNP) of 13C-labeled metabolic substrates in vitro and their subsequent intravenous administration allow both the location of the hyperpolarized substrate and the dynamics of its subsequent conversion into other metabolic products to be detected in vivo. We report here the hyperpolarization of [1-13C]-ascorbic acid (AA) and [1-13C]-dehydroascorbic acid (DHA), the reduced and oxidized forms of vitamin C, respectively, and evaluate their performance as probes of tumor redox state. Solution-state polarization of 10.5 ± 1.3% was achieved for both forms at pH 3.2, whereas at pH 7.0, [1-13C]-AA retained polarization of 5.1 ± 0.6% and [1-13C]-DHA retained 8.2 ± 1.1%. The spin–lattice relaxation times (T1s) for these labeled nuclei are long at 9.4 T: 15.9 ± 0.7 s for AA and 20.5 ± 0.9 s for DHA. Extracellular oxidation of [1-13C]-AA and intracellular reduction of [1-13C]-DHA were observed in suspensions of murine lymphoma cells. The spontaneous reaction of DHA with the cellular antioxidant glutathione was monitored in vitro and was approximately 100-fold lower than the rate observed in cell suspensions, indicating enzymatic involvement in the intracellular reduction. [1-13C]-DHA reduction was also detected in lymphoma tumors in vivo. In contrast, no detectable oxidation of [1-13C]-AA was measured in the same tumors, consistent with the notion that tumors maintain a reduced microenvironment. This study demonstrates that hyperpolarized 13C-labeled vitamin C could be used as a noninvasive biomarker of redox status in vivo, which has the potential to translate to the clinic.

Uric Acid Promotes Tumor Immune Rejection

Uric acid released from dying cells has been shown recently to act as a danger signal for the immune system, stimulating dendritic cell maturation and enhancing T-cell responses to foreign antigens. Stimulation of dendritic cell maturation by uric acid has been proposed as a mechanism by which the immune system could generate responses against tumors. We show here that uric acid levels are elevated in tumors undergoing immune rejection and that the inhibition of uric acid production, by systemic administration of allopurinol, or the removal of uric acid, by administration of uricase, delayed tumor immune rejection, whereas subcutaneous administration of crystalline uric acid enhanced the rejection process.

Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate

Background:The recent introduction of a dynamic nuclear polarisation technique has permitted noninvasive imaging of tumour cell metabolism in vivo following intravenous administration of 13C-labelled cell substrates.Methods:Changes in hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate metabolism were evaluated in both MDA-MB-231 cells and in implanted MDA-MB-231 tumours following doxorubicin treatment.Results:Treatment of MDA-MB-231 cells resulted in the induction of apoptosis, which was accompanied by a decrease in hyperpolarised 13C label flux between [1-13C]pyruvate and lactate, which was correlated with a decrease in the cellular NAD(H) coenzyme pool. There was also an increase in the rate of fumarate conversion to malate, which accompanied the onset of cellular necrosis. In vivo, the decrease in 13C label exchange between pyruvate and lactate and the increased flux between fumarate and malate, following drug treatment, were shown to occur in the absence of any detectable change in tumour size.Conclusion:We show here that the early responses of a human breast adenocarcinoma tumour model to drug treatment can be followed by administration of both hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate. These techniques could be used, therefore, in the clinic to detect the early responses of breast tumours to treatment.

Magnetization transfer measurements of exchange between hyperpolarized [1-13C]pyruvate and [1-13C]lactate in a murine lymphoma

Measurements of the conversion of hyperpolarized [1‐13C]pyruvate into lactate, in the reaction catalyzed by lactate dehydrogenase, have shown promise as a metabolic marker for the presence of disease and response to treatment. However, it is unclear whether this represents net flux of label from pyruvate to lactate or exchange of isotope between metabolites that are close to chemical equilibrium. Using saturation and inversion transfer experiments, we show that there is significant exchange of label between lactate and pyruvate in a murine lymphoma in vivo. The rate constants estimated from the magnetization transfer experiments, at specific points during the time course of label exchange, were similar to those obtained by fitting the changes in peak intensities during the entire exchange time course to a kinetic model for two‐site exchange. These magnetization transfer experiments may therefore provide an alternative and more rapid way of estimating flux between pyruvate and lactate to serial measurements of pyruvate and lactate 13C peak intensities following injection of hyperpolarized [1‐13C]pyruvate. Magn Reson Med 63:872–880, 2010.

Detection of Cell Death in Tumors by Using MR Imaging and a Gadolinium-based Targeted Contrast Agent

PURPOSE To prospectively determine in an animal model whether an ionic gadolinium (Gd(3+)) chelate conjugate of the C2A domain of synaptotagmin I can be used with magnetic resonance (MR) imaging to detect tumor cell death noninvasively in vivo. MATERIALS AND METHODS Animal experiments were approved by a local ethics review committee. Gd(3+) chelates and fluorescent probes were attached to the lysine epsilon-amino groups of a glutathione-S-transferase-C2A fusion protein. Binding to phosphatidylserine (PS) was characterized by using surface plasmon resonance, and binding to dying cells in vitro was characterized by using flow cytometry and MR imaging. Binding to dying tumor cells in vivo was detected with T1 mapping and T1-weighted MR imaging and compared in drug-treated animals (n = 10); in animals injected with a site-directed mutant, which was inactive in PS binding (PS inactive) and which showed lesser binding to dying cells (n = 6); and in untreated animals injected with PS-active (n = 6) and PS-inactive (n = 6) contrast agents. Among groups, differences that were significant were analyzed by using analysis of variance and Dunnett post hoc analysis. RESULTS The contrast agent had a relatively high affinity for PS (dissociation constant = 333 nmol/L +/- 85 [mean +/- standard error of the mean]; n = 3) and bound to apoptotic and necrotic, but not viable, cells in vitro. There was a greater tumor accumulation of the PS-active contrast agent compared with the PS-inactive contrast agent in drug-treated animals (P < .05) and compared with untreated animals injected with the PS-active and PS-inactive contrast agents (P < .01 for both). CONCLUSION A relatively small (approximately 100 kDa) Gd(3+)-based contrast agent, which gives positive contrast on MR images, can be used to detect tumor cell death in vivo, and future derivatives of it may be used to assess early tumor responses to treatment.

Detection of Tumor Response to a Vascular Disrupting Agent by Hyperpolarized 13C Magnetic Resonance Spectroscopy

Nuclear spin hyperpolarization can dramatically increase the sensitivity of the 13C magnetic resonance experiment, allowing dynamic measurements of the metabolism of hyperpolarized 13C-labeled substrates in vivo. Here, we report a preclinical study of the response of lymphoma tumors to the vascular disrupting agent (VDA), combretastatin-A4-phosphate (CA4P), as detected by measuring changes in tumor metabolism of hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate. These measurements were compared with dynamic contrast agent–enhanced magnetic resonance imaging (DCE-MRI) measurements of tumor vascular function and diffusion-weighted MRI (DW-MRI) measurements of the tumor cell necrosis that resulted from subsequent loss of tumor perfusion. The rate constant describing flux of hyperpolarized 13C label between [1-13C]pyruvate and lactate was decreased by 34% within 6 hours of CA4P treatment and remained at this lower level at 24 hours. The rate constant describing production of labeled malate from hyperpolarized [1,4-13C2]fumarate increased 1.6-fold and 2.5-fold at 6 and 24 hours after treatment, respectively, and correlated with the degree of necrosis detected in histologic sections. Although DCE-MRI measurements showed a substantial reduction in perfusion at 6 hours after treatment, which had recovered by 24 hours, DW-MRI showed no change in the apparent diffusion coefficient of tumor water at 6 hours after treatment, although there was a 32% increase at 24 hours (P < 0.02) when regions of extensive necrosis were observed by histology. Measurements of hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate metabolism may provide, therefore, a more sustained and sensitive indicator of response to a VDA than DCE-MRI or DW-MRI, respectively. Mol Cancer Ther; 9(12); 3278–88. ©2010 AACR.