High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8⁺ cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine ([¹⁸F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [¹⁸F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer.

Dynamic nuclear polarization of ¹³ C-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- ¹³ C ₂ ]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- ¹³ C ₂ ]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized ¹³ C-labeled malate from [1,4- ¹³ C ₂ ]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.

Measurements of the kinetics of hyperpolarized (13)C label exchange between [1-(13)C]pyruvate and lactate in suspensions of intact and lysed murine lymphoma cells, and in cells in which lactate dehydrogenase expression had been modulated by inhibition of the PI3K pathway, were used to determine quantitatively the role of enzyme activity and membrane transport in controlling isotope flux. Both steps were shown to share in the control of isotope flux in these cells. The kinetics of label exchange were well described by a kinetic model that employed rate constants for the lactate dehydrogenase reaction that had been determined previously from steady state kinetic studies. The enzyme showed pyruvate inhibition in steady state kinetic measurements, which the kinetic model predicted should also be observed in the isotope exchange measurements. However, no such pyruvate inhibition was observed in either intact cells or cell lysates and this could be explained by the much higher enzyme concentrations present in the isotope exchange experiments. The kinetic analysis presented here shows how lactate dehydrogenase activity can be determined from the isotope exchange measurements. The kinetic model should be useful for modeling the exchange reaction in vivo, particularly as this technique progresses to the clinic.

Background: Methods: Results: Conclusion:

Acute kidney injury (AKI) is a common and important medical problem, affecting 10% of hospitalized patients, and it is associated with significant morbidity and mortality. The most frequent cause of AKI is acute tubular necrosis (ATN). Current imaging techniques and biomarkers do not allow ATN to be reliably differentiated from important differential diagnoses, such as acute glomerulonephritis (GN). We investigated whether ¹³ C magnetic resonance spectroscopic imaging (MRSI) might allow the noninvasive diagnosis of ATN. ¹³ C MRSI of hyperpolarized [1,4- ¹³ C ₂ ]fumarate and pyruvate was used in murine models of ATN and acute GN (NZM2410 mice with lupus nephritis). A significant increase in [1,4- ¹³ C ₂ ]malate signal was identified in the kidneys of mice with ATN early in the disease course before the onset of severe histological changes. No such increase in renal [1,4- ¹³ C ₂ ]malate was observed in mice with acute GN. The kidney [1- ¹³ C]pyruvate/[1- ¹³ C]lactate ratio showed substantial variability and was not significantly decreased in animals with ATN or increased in animals with GN. In conclusion, MRSI of hyperpolarized [1,4- ¹³ C ₂ ]fumarate allows the detection of early tubular necrosis and its distinction from glomerular inflammation in murine models. This technique may have the potential to identify a window of therapeutic opportunity in which emerging therapies might be applied to patients with ATN, reducing the need for acute dialysis with its attendant morbidity and cost.

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.