Establishing Immortalized Brown and White Preadipocyte Cell Lines from Young and Aged Mice.

Studying adipogenesis and adipocyte biology requires the isolation of primary preadipocytes from adipose tissues. However, primary preadipocytes have a limited lifespan, can only undergo a finite number of divisions, and often lose their original biological characteristics before becoming senescent. The repeated isolation of fresh preadipocytes, particularly from young pups or aged animals, is costly and time consuming.

Fructose vs. glucose: modulating stem cell growth and function through sugar supplementation.

In multicellular organisms, stem cells are impacted by microenvironmental resources such as nutrient availability and oxygen tension for their survival, growth, and differentiation. However, the accessibility of these resources in the pericellular environment greatly varies from organ to organ. This divergence in resource availability leads to variations in the potency and differentiation potential of stem cells.

Ketohexokinase-mediated fructose metabolism is lost in hepatocellular carcinoma and can be leveraged for metabolic imaging.

The ability to break down fructose is dependent on ketohexokinase (KHK) that phosphorylates fructose to fructose-1-phosphate (F1P). We show that KHK expression is tightly controlled and limited to a small number of organs and is down-regulated in liver and intestinal cancer cells. Loss of fructose metabolism is also apparent in hepatocellular adenoma and carcinoma (HCC) patient samples.

Hyperpolarized MRI of Human Prostate Cancer Reveals Increased Lactate with Tumor Grade Driven by Monocarboxylate Transporter 1.

Metabolic imaging using hyperpolarized magnetic resonance can increase the sensitivity of MRI, though its ability to inform on relevant changes to biochemistry in humans remains unclear. In this work, we image pyruvate metabolism in patients, assessing the reproducibility of delivery and conversion in the setting of primary prostate cancer. We show that the time to max of pyruvate does not vary significantly within patients undergoing two separate injections or across patients.

Hyperpolarized MRI Visualizes Warburg Effects and Predicts Treatment Response to mTOR Inhibitors in Patient-Derived ccRCC Xenograft Models.

The ever-changing tumor microenvironment constantly challenges individual cancer cells to balance supply and demand, presenting tumor vulnerabilities and therapeutic opportunities. Everolimus and temsirolimus are inhibitors of mTOR (mTORi) approved for treating metastatic renal cell carcinoma (mRCC). However, treatment outcome varies greatly among patients.

Targeted AKT Inhibition in Prostate Cancer Cells and Spheroids Reduces Aerobic Glycolysis and Generation of Hyperpolarized [1-C] Lactate.

The PI3K/AKT/mTOR (PAM) signaling pathway is frequently mutated in prostate cancer. Specific AKT inhibitors are now in advanced clinical trials, and this study investigates the effect of MK2206, a non-ATP-competitive inhibitor, on the cellular metabolism of prostate cancer cells. We observed a reduction in cell motility and aerobic glycolysis in prostate cancer cells with treatment.

PKM2 activation sensitizes cancer cells to growth inhibition by 2-deoxy-D-glucose.

Cancer metabolism has emerged as an increasingly attractive target for interfering with tumor growth. Small molecule activators of pyruvate kinase isozyme M2 (PKM2) suppress tumor formation but have an unknown effect on established tumors. We demonstrate that TEPP-46, a PKM2 activator, results in increased glucose consumption, providing the rationale for combining PKM2 activators with the toxic glucose analog, 2-deoxy-D-glucose (2-DG).

Real-time quantitative analysis of metabolic flux in live cells using a hyperpolarized micromagnetic resonance spectrometer.

Metabolic reprogramming is widely considered a hallmark of cancer, and understanding metabolic dynamics described by the conversion rates or "fluxes" of metabolites can shed light onto biological processes of tumorigenesis and response to therapy. For real-time analysis of metabolic flux in intact cells or organisms, magnetic resonance (MR) spectroscopy and imaging methods have been developed in conjunction with hyperpolarization of nuclear spins. These approaches enable noninvasive monitoring of tumor progression and treatment efficacy and are being tested in multiple clinical trials.

High-Throughput Indirect Quantitation of C Enriched Metabolites Using H NMR.

Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics to perform quantitative profiling of low-molecular weight compounds from biological specimens. The measurement of endogenous metabolites using NMR has proven to be a powerful tool to identify new metabolic biomarkers in physiological and pathological conditions, and to study and evaluate treatment efficiency. In this study we present a rapid approach to indirectly quantify C enriched molecules using one-dimensional (1D) H NMR.

Sampling Hyperpolarized Molecules Utilizing a 1 Tesla Permanent Magnetic Field.

Hyperpolarized magnetic resonance spectroscopy (HP MRS) using dynamic nuclear polarization (DNP) is a technique that has greatly enhanced the sensitivity of detecting (13)C nuclei. However, the HP MRS polarization decays in the liquid state according to the spin-lattice relaxation time (T1) of the nucleus. Sampling of the signal also destroys polarization, resulting in a limited temporal ability to observe biologically interesting reactions.

(13) C magnetic resonance spectroscopy measurements with hyperpolarized [1-(13) C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo.

Purpose: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.

Novel Approaches to Imaging Tumor Metabolism.

The field of metabolism research has made a dramatic resurgence in recent years, fueled by a newfound appreciation of the interactions between metabolites and phenotype. Metabolic substrates and their products can be biomarkers of a wide range of pathologies, including cancer, but our understanding of their in vivo interactions and pathways has been hindered by the robustness of noninvasive imaging approaches. The past 3 decades have been flushed with the development of new techniques for the study of metabolism in vivo.

Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis.

Non-invasive detection of caspase-3/7 activity has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity . Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice.

Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo.

Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and already this has been used widely to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling the self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employ an optimized first-order bioorthogonal cyclization reaction to control the self-assembly of a fluorescent small molecule, and demonstrate its in vivo applicability by imaging caspase-3/7 activity in human tumour xenograft mouse models of chemotherapy.

Detection of transgene expression using hyperpolarized 13C urea and diffusion-weighted magnetic resonance spectroscopy.

Purpose: To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [(13) C]urea.

Methods: Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [(13) C]urea, a spin echo sequence was used to measure urea concentration, T1 , and diffusion in control and UTB-expressing tissue.

Hyperpolarized [1,4-(13)C]-diethylsuccinate: a potential DNP substrate for in vivo metabolic imaging.

The tricarboxylic acid (TCA) cycle performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non-invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4-(13)C]-diethylsuccinate, for hyperpolarized (13)C metabolic imaging of the TCA cycle was explored.

Dual-modality gene reporter for in vivo imaging.

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid).