Novel imaging biomarkers for metabolism and tissue function

Imaging biomarkers enable a comprehensive characterization of tissue by providing functional, physiological, metabolic, cellular, and molecular information beyond anatomical details. The research focus of our group lies on the development of sensitive hyperpolarized molecules for imaging metabolism and pH as well as on advancing diffusion MRI techniques.

Focus Group: Biomedical Magnetic Resonance

Prof. Franz Schilling (TUM University Hospital Rechts der Isar), Alumnus Rudolf Mößbauer Tenure Track Assistant Professor

Figure 1

Fig. 1, Simultaneous in vivo imaging of renal perfusion, filtration, and acid-base balance. a) Scheme for combined imaging of renal kidney perfusion and filtration in 3-D and renal pH in 2-D. b) Image time series of co-injected hyperpolarized [13C]urea. c) Average pH maps with high contrast between renal cortex and pelvis. d) Image time series of the C1-magnetization of hyperpolarized Z-OMPD. The higher SNR compared to [13C]urea together with the high spatial resolution allows better assessment of first-pass perfusion and renal filtration from the cortex (renal periphery) to the pelvis (renal center) when injected at identical concentrations. Hyperpolarized acquisitions are overlaid on an anatomical T2-weighted image (Reprinted with permission from M. Grashei et al. (2023)).

Overview of the Focus Group

For patients, specific non-invasive imaging strategies for early-stage disease detection, cellular phenotyping, and evaluation of response to therapy are not available at a satisfactory level, creating a pressing need for advanced technologies. In this Focus Group, we aimed to develop two novel non-invasive imaging techniques to measure extracellular pH and transmembrane water permeability. First, by using pH imaging, we characterized the handling of pH in the kidney and the acidic tumor microenvironment. Second, wetested whether imaging of transmembrane water permeability reports on early stages of cell death, thereby providing an early evaluation technology to detect whether a drug has hit its target before an anatomical reduction in tumor size is recognizable.

Simultaneous imaging of pH, perfusion, and filtration in kidneys and tumors

We have introduced [1,5-¹³C₂]Z-4-methyl-2-oxopent-3-enedioic acid (Z-OMPD) as a hyperpolarized extracellular pH and perfusion sensor for MRI. High pH sensitivity of up to 1.9 ppm per pH unit and suitability of using the C₁-label as internal frequency reference enable pH imaging in vivo of three pH compartments in healthy rat kidneys. Simultaneous imaging of perfusion and filtration in 3-D and pH in 2-D within one minute enables assessment of renal blood flow, glomerular filtration rates, and renal pH in healthy (Fig. 1) and hydronephrotic kidneys with superior sensitivity compared to routine clinical methods.

Z-OMPD has also been used in tumors to assess pH heterogeneity, resolving multiple pH compartments on a sub-voxel level (Fig. 2). The spectral readout provides an advantage compared to hyperpolarized ratiometric pH probes, such as ¹³C-bicarbonate, where only a mean pH per voxel can be retrieved.

Figure 2

Fig. 2, In vivo pH imaging in subcutaneous EL4 lymphoma tumors allows assessment of tumor heterogeneity. a) pH imaging in a subcutaneously implanted EL4 lymphoma (white ROI) reveals heterogeneous tumor acidification. The white square indicates the native image resolution. b) The majority of the tumor shows only one pH compartment being close to physiological pH (d), indicated by one OMPD-C5-peak (blue triangle, spectrum from white circle ROI in b). c) One or multiple subregions exhibit a second, acidic pH compartment, which is detectable as a second peak (e) for the OMPD-C5-resonance (red triangle) in the spectrum (spectrum from white circle ROI in c). f) Tumors generally show acidification of the mean pH (orange diamonds) compared to healthy muscle (blue squares) or blood pH (blue circles). Figure adapted, with permission, from M. Grashei et al. (2023).

Metabolic imaging

Hyperpolarized ¹³C MRI provides a unique insight into metabolism and tissue function. [1] It utilizes molecular transformations as a mechanism for imaging contrast. The most successful hyperpolarized ¹³C MRI probe so far is [1-¹³C]pyruvate. Pyruvate is a key metabolite in energy metabolism and, as the end product of glycolysis, it is further metabolized to lactate, alanine, or acetyl-CoA (after cleavage of ¹³CO₂), which then enters the tricarboxylic acid (TCA) cycle. Quantifying pyruvate’s downstream metabolism enables the differentiation of glycolytic tissue from tissue dominated by aerobic respiration. Besides dissolution dynamic nuclear polarization (dDNP), which operates at liquid helium temperature close to absolute zero, other methods for generating hyperpolarized ¹³C-labeled probes are based on polarization transfer from parahydrogen and require substantially less technological effort and cost. In a recent collaborative effort, we have demonstrated preclinical evidence for safe and reliable polarization and in vivo administration of [1-¹³C]pyruvate using parahydrogen. We aim to further translate this approach to the clinic, with preparations for the first patients being scanned at TUM University Hospital in 2027 already ongoing.

Imaging early cell death in vitro, in vivo, and in patients using transmembrane water exchange imaging

Cell death plays a key role in cancer staging and treatment strategy. Various mechanisms of cell death in cancer are known, including apoptosis, necrosis, and necroptosis. The integrity of the cell membrane is often compromised after the onset of cell death. In yeast cells, we have demonstrated that AXR is sensitive in changes to membrane permeabilization that cannot be detected by changes in ADC.

In addition, preclinical data in acute myeloid leukemia (AML) and subcutaneous EL4 lymphoma cells show that transmembrane water exchange rate is an early biomarker of cell death when quantified by apparent exchange rate (AXR) calculated by filter exchange spectroscopy (FEXSY) or imaging (FEXI). AXR correlated with apparent diffusion coefficient (ADC) and in vitro flow cytometry using fluorescent cell death markers. AXR measurements in human patients with uterine fibroids show that they are sensitive to changes after treatment shortly after embolization and underline the clinical applicability of our proposed method.

Conclusion

In summary, we have developed novel non-invasive magnetic resonance (MR) imaging bio-markers of unprecedented sensitivity for the characterization of tissue physiology, metabolism, and response to therapy. Previously unexplored pH-sensitive hyperpolarized molecules, optimized acquisition strategies for metabolic imaging, and advanced diffusion MRI techniques were introduced that provide novel information currently not accessible with existing methods.

In collaboration with doctoral candidates Athanasia Kaika and Martin Grashei.

Selected publications

M. Grashei et al., “Simultaneous magnetic resonance imaging of pH, perfusion and renal filtration using hyperpolarized ¹³C-labelled Z-OMPD”, Nat. Commun., vol. 14, no. 1, p. 5060, 2023, doi: 10.1038/s41467-023-40747-3.
A. Kaika et al., “Early Detection of Cell Death Using Transmembrane Water Exchange Magnetic Resonance Imaging”, Adv. Sci., in press, doi: 10.1002/advs.202513317
L. Nagel et al., “Parahydrogen-Polarized [1-¹³C] Pyruvate For Reliable and Fast Preclinical Metabolic Magnetic Resonance Imaging”, Adv. Sci., vol. 10, no. 30, p. 2303441, 2023, doi: 10.1002/advs.202303441.
J. G. Skinner et al., “Spectrally selective bSSFP using off-resonant RF excitations permits high spatiotemporal resolution 3D metabolic imaging of hyperpolarized [1-¹³C] Pyruvate-to-[1-¹³C] lactate conversion”, Magn. Reson. in Med., vol. 90, no. 3, pp. 894–909, 2023, doi: 10.1002/mrm.29676.
A. Kaika, G. J. Topping, L. Nagel, and F. Schilling, “Filter-exchange spectroscopy is sensitive to gradual cell membrane degradation”, NMR Biomed., p. e5202, 2024, doi: 10.1002/nbm.5202