A Macrocyclic Hybrid PET/MRI Probe for Quantitative Perfusion Imaging In Vivo.

Perfusion dynamics play a vital role in delivering essential nutrients and oxygen to tissues while removing metabolic waste products. Imaging techniques such as magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET) use contrast agents to visualize perfusion and clearance patterns; however, each technique has specific limitations. Hybrid PET/MRI combines the quantitative power and sensitivity of PET with the high functional and anatomical detail of MRI and holds great promise for precision in molecular imaging. However, the development of dual PET/MRI probes has been hampered by challenging synthesis and radiolabeling. Here, we present a novel PET/MRI probe, [18F][Gd(FL1)], which exhibits excellent stability comparable to macrocyclic MRI contrast agents used in clinical practice. The unique molecular design of [18F][Gd(FL1)] allows selective and expeditious radiolabeling of the gadolinium chelate in the final synthetic step. Leveraging the strengths of MRI and PET signals, the probe enables quantitative in vivo mapping of perfusion and excretion dynamics through an innovative voxel-based analysis. The diagnostic capabilities of [18F][Gd(FL1)] were demonstrated in a pilot study on healthy mice, successfully detecting early cases of unilateral renal dysfunction. This study introduces a new approach for PET/MRI and emphasizes a streamlined probe design for improved diagnostic accuracy.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

Imaging Techniques in Pharmacological Precision Medicine.

Biomedical imaging is a powerful tool for medical diagnostics and personalized medicines. Examples of commonly used imaging modalities include Positron Emission Tomography (PET), Ultrasound (US), Single Photon Emission Computed Tomography (SPECT), and hybrid imaging. By combining these modalities, scientists can gain a comprehensive view and better understand physiology and pathology at the preclinical, clinical, and multiscale levels. This can aid in the accuracy of medical diagnoses and treatment decisions. Moreover, biomedical imaging allows for evaluating the metabolic, functional, and structural details of living tissues. This can be particularly useful for the early diagnosis of diseases such as cancer and for the application of personalized medicines. In the case of hybrid imaging, two or more modalities are combined to produce a high-resolution image with enhanced sensitivity and specificity. This can significantly improve the accuracy of diagnosis and offer more detailed treatment plans. In this book chapter, we showcase how continued advancements in biomedical imaging technology can potentially revolutionize medical diagnostics and personalized medicine.

Synchrotron Radiation X-ray Fluorescence Elemental Mapping in Healthy versus Malignant Prostate Tissues Provides New Insights into the Glucose-Stimulated Zinc Trafficking in the Prostate As Discovered by MRI.

Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (µSR-XRF). Quantitative µSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The µSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by µSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor.

Superoxide overproduction is known to occur in multiple disease states requiring critical care; yet, noninvasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by "click conjugating" paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced in vivo to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species-in particular, superoxide-and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for T1 contrast at low field (<3.0 T) and T2 contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for a "quenchless fluorescent" bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.

Enantiomeric Recognition of d- and l-Lactate by CEST with the Aid of a Paramagnetic Shift Reagent.

A previous report demonstrated that EuDO3A could be used as an NMR shift reagent for imaging extracellular lactate produced by cancer cells using CEST imaging. In this work, a series of heptadentate macrocyclic YbDO3A-trisamide complexes with δ-chiral carbons in the three pendant side-arms were examined as shift reagents for lactate detection. High resolution 1H NMR spectra and DFT calculations provided evidence for the formation of stereoselective lactate·YbDO3A-trisamide complexes each with a different CEST signature. This stereoselectivity allowed discrimination of d- versus l-lactate by both high-resolution NMR and CEST. This work demonstrates that lanthanide-based paramagnetic shift reagents can be designed to detect important metabolites by CEST MRI selectively.

Zinc as an Imaging Biomarker of Prostate Cancer.

Zinc has long been the focus of many biological investigations because of its essential role in biology including a catalytic role in many enzymes, a structural role in the many zinc finger proteins, and a physiological role in many secretory cell processes. Divalent zinc is known to be highly abundant in healthy prostate tissues and lower in prostate cancer (PCa). Given the need for newer diagnostic methods for detection of prostate cancer, zinc-responsive probes of various types have been considered as imaging tools for detecting tissue levels of zinc. Among them, recent zinc-responsive MRI probes show great promise for non-invasive detection of zinc ion secretion from the prostate and other tissues in vivo. In this review, we summarize the need for new diagnostic tools and demonstrate how responsive zinc probes and MRI could satisfy this unmet need.

Imaging Extracellular Lactate In Vitro and In Vivo Using CEST MRI and a Paramagnetic Shift Reagent.

Overproduction of lactate is a hallmark of cancer, yet a method to quantitatively measure lactate production by cancer cells is not straight-forward. Chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can potentially be used to image lactate but the small difference in chemical shift of the lactate -OH proton and water proton resonances make it challenging. Like other spectroscopic methods, CEST MRI cannot discriminate intracellular lactate from extracellular lactate. Herein, we demonstrate a relatively simple way to shift the lactate -OH proton resonance far away from water by addition of the paramagnetic shift reagent, EuDO3A, while retaining the CEST properties of lactate itself. The potential of the method was demonstrated by imaging extracellular lactate excreted from lung cancer cells in tissue culture without interference from other components in the culture media and by imaging excess lactate excreted into the bladder of a mouse.

Gallium-68 Complexes Conjugated to Pittsburgh Compound B: Radiolabeling and Biological Evaluation.

PURPOSE: The aim of this work is to develop an efficient and fully automated radiosynthesis of three derivatives of the Pittsburgh compound B labeled with gallium-68 for the detection of amyloid plaques. PROCEDURES: The radiolabeling of the precursors and purification of the radiolabeled agents by high pressure liquid chromatography has been studied prior to their in vitro and in vivo evaluations. RESULTS: The complete process led, in 50 min, to pure Ga-68 products in a 12-38 % yield and with appreciable specific radioactivity (SRA, 85-168 GBq/µmol) which enabled us to demonstrate a considerable in vivo stability of the products. Unfortunately, this result was associated with a poor blood-brain barrier (BBB) permeability and a limited uptake of our compounds by amyloid deposits was observed by in vitro autoradiography. CONCLUSION: Although we have not yet identified a compound able to significantly mark cerebral amyloidosis, this present investigation will likely contribute to the development of more successful Ga-68 radiotracers.

Basic MR relaxation mechanisms and contrast agent design.

The diagnostic capabilities of magnetic resonance imaging (MRI) have undergone continuous and substantial evolution by virtue of hardware and software innovations and the development and implementation of exogenous contrast media. Thirty years since the first MRI contrast agent was approved for clinical use, a reliance on MR contrast media persists, largely to improve image quality with higher contrast resolution and to provide additional functional characterization of normal and abnormal tissues. Further development of MR contrast media is an important component in the quest for continued augmentation of diagnostic capabilities. In this review we detail the many important considerations when pursuing the design and use of MR contrast media. We offer a perspective on the importance of chemical stability, particularly kinetic stability, and how this influences one's thinking about the safety of metal-ligand-based contrast agents. We discuss the mechanisms involved in MR relaxation in the context of probe design strategies. A brief description of currently available contrast agents is accompanied by an in-depth discussion that highlights promising MRI contrast agents in the development of future clinical and research applications. Our intention is to give a diverse audience an improved understanding of the factors involved in developing new types of safe and highly efficient MR contrast agents and, at the same time, provide an appreciation of the insights into physiology and disease that newer types of responsive agents can provide.

A biophysical approach to menadione membrane interactions: relevance for menadione-induced mitochondria dysfunction and related deleterious/therapeutic effects.

Menadione (MEN), a polycyclic aromatic ketone, was shown to promote cell injury by imposing massive oxidative stress and has been proposed as a promising chemotherapeutic agent for the treatment of cancer diseases. The mechanisms underlying MEN-induced mitochondrial dysfunction and cell death are not yet fully understood. In this work, a systematic study was performed to unveil the effects of MEN on membrane lipid organization, using models mimicking mitochondrial membranes and native mitochondrial membranes. MEN was found to readily incorporate in membrane systems composed of a single phospholipid (phosphatidylcholine) or the lipids dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and tetraoleoylcardiolipin at 1:1:1 molar ratio, as well as in mitochondrial membranes. Increased permeability in both membrane models, monitored by calcein release, seemed to correlate with the extent of MEN incorporation into membranes. MEN perturbed the physical properties of vesicles composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine plus tetraoleoylcardiolipin (at 7:3 molar ratio), as reflected by the downshift of the lipid phase transition temperature and the emergence of a new transition peak in the mixed lipid system, detected by DSC. (31)P NMR studies revealed that MEN favored the formation of non-lamellar structures. Also, quenching studies with the fluorescent probes DPH and TMA-DPH showed that MEN distributed across the bilayer thickness in both model and native mitochondrial membranes. MEN's ability to promote alterations of membrane lipid organization was related with its reported mitochondrial toxicity and promotion of apoptosis, predictably involved in its anti-carcinogenic activity.