Kayvan R. Keshari: Projects

The projects in our lab center around two main themes and below are examples of the directions we have gone in.

HP MR Probes for Translation

Many probes have been recently developed to study cancer metabolism and response to therapy, including HP pyruvate, fructose, bicarbonate, and fumarate. These probes take advantage of long T1 relaxation times, which govern the lifetime of the probe, making it useful for imaging. In order to develop new HP probes, we utilize a combination of biochemical and biophysical approaches, with a specific metabolic aberration of interest.

Recently, we have been very interested in oxidative stress and the effects of reactive oxygen species (ROS) on the cancer cell. Reducing and oxidizing (Redox) state is a balance that is tightly regulated in the cell, maintained by a wide range of enzymatic and signaling pathways. At the center of this interplay is our predominant antioxidant glutathione, vitamin C, and other redox species such as NADPH.

With the increased generation of ROS, the redox state of the cell is stressed and this can affect a huge number of pathways in cancer. With this in mind we have developed an endogenous HP redox sensor, [1-13C] dehydroascorbate (DHA), which is the oxidized form of vitamin C. HP DHA can rapidly enter the cell and be reduced to vitamin C(1) (Figure 1). This rate of reduction is indicative of the cells capacity to reduce substrates and allows DHA to inform on the cellular redox network.

We’ve found that while some cells overall become oxidized, the internal concentration of reductant is still dramatically increased, providing an environment where cells can continue to rapidly reduce substrates(2). Broadly, this suggests a prognostic role for this new redox sensor in determining the vulnerability of both normal and abnormal tissues to ROS. In the case of cancer, this could impact many of our chemotherapeutics and radiation regiments, where the cancer cell can use this mechanism to survive therapy.

Figure 1 Figure 1 Biochemical mechanism, hyperpolarisation, and reduction of [1-13C] DHA. (A) Relationship between redox pairs NADPH/NADP, GSSG/GSH and vitamin C/DHA with associated enzymes. (B) Reversible reduction of labeled DHA to vitamin C demonstrating the position of the hyperpolarized carbon. (C) Sequential coronal T2-weighted images and corresponding 13C 3D MRSI demonstrating distribution of hyperpolarized DHA and vitamin C (VitC) in a transgenic adenocarcinoma model of prostate (TRAMP) mouse post–intravenous injection of 350uL of 15mM hyperpolarized [1-13C] DHA. This data set was collected using a variable tip-angle scheme initiated 25s following this injection, with individual voxels describing the relative reduction of [1-13C] DHA to [1-13C] VitC at the same time point. The liver and kidneys are best seen in (top image) and prostate tumor in (bottom image), but both imaging slices contain significant amounts of liver, kidney, and tumor tissue. Regions of liver, kidney and prostate tumor are segmented and superimposed on the spectral grid (color-coded dashed lines). Differences in metabolite ratios are seen between normal organs and between prostate tumor and normal surrounding tissues. Representative 13C spectra from liver, kidney, and prostate tumor in a TRAMP mouse are shown to the right. (D) Axial T2-weighted images and corresponding color overlays of hyperpolarized DHA and VitC signal in a normal rat brain.

HP Metabolic Dynamics

Contrary to popular belief, before gene transcription and proteins, metabolism is acutely sensitive to change. In some cases, rapid changes in substrate availability can then become a signal to initiate signaling cascades or gene transcription. Since metabolism is dynamic and the probes we develop require a relevant system, we utilize custom-designed MR-compatible bioreactors to investigate changes in the metabolism of our HP probes and compare them to other multimodality approaches.

Recently we’ve shown that the cell’s ability to transport lactate, a by-product of increased glycolytic metabolism, could inform on renal cell carcinoma aggressiveness(3). This we could visualize in an MR-compatible bioreactor system with HP pyruvate (Figure 2). We can then develop schemes to translate this phenomenon in vivo, for instance by using diffusion(4).

While changes in the metabolism of immortal cells and animal models have been used as the basis for probe and drug discovery, it has been difficult to translate these mechanisms to the clinic. This is possibly due to the deviation between the metabolism of these cells and the actual human condition. For this reason, we’ve begun to explore the use of human tissue slice cultures (TSCs) with our hyperpolarized probes for translation. In some recent work (5), we show that the metabolism of primary human prostate tissue slices are very different from that of immortal cells and show hyperpolarized lactate as a biomarker for prostate cancer (Figure 3). We are in the process of developing new probes for study in TSCs and their translation to the clinic.

Figure 2 Enlarge Image Figure 2 (A) Scheme of 13C-labeled carbon atom transitions used to detect C1-labeled pyruvate metabolism during the hyperpolarized experiment. (B) Fitted pyruvate-to-lactate flux and representative spectra (inset) of 13C pyruvate and lactate in the UMRC6 cells. (C) Schematic illustrating the relationship between flow rates and observed HP pyruvate-to-lactate flux in the bioreactor. At high flow rates, the extracellular lactate is more likely to flow out of the NMR coil’s sensitive volume and not contribute to the MR signal, thereby decreasing the observed pyruvate-to-lactate flux. The dotted square represents NMR sensitive region. О denotes encapsulated microspheres containing cells. █ denotes extracellular lactate. (D) HP pyruvate-to-lactate flux of UOK262 and UMRC6 cells at three different flow rates (N=5 for each). There is a decreasing trend in observed pyruvate-to-lactate flux with increasing flow rate for UOK262 cells, which transport lactate out via MCT4 at significantly higher rates (approximately 30% decrease in flux). All values are reported as mean ± std. err. * denotes significance (p<0.05). Figure 3 Enlarge Image Figure 3 Representative 31P MR spectra of living benign and malignant TSCs (A) with accompanying histology after perfusion in the bioreactor (B). These resonances represent quantitative differences in bioenergetics (E). Hyperpolarized dynamics (C, D) demonstrate the increased production of labeled HP lactate as well as the total area under the curve (AUC, F). These are correlated to changes in LDH activity (G) as well as to expression of LDHA and relevant monocarboxylate transporters (MCT1 and 4, H). All values are reported as mean ± standard error of the mean.