Development of a bioluminescent nitroreductase probe for preclinical imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.publishedVersio

Novel Molecular Probes for Non-invasive Optical Imaging of Fatty Acid and Triglyceride Uptake in Living Animals

Molecular imaging allows noninvasive visualization of biological processes in their native environment within living systems. It can give a better understanding of fundamental biology, help in identifying disease mechanisms and visualizing pathological tissues, and enable monitoring disease progression and studying the drug efficacy in intact living organisms. Among various imaging modalities, bioluminescence and fluorescence imaging are powerful techniques that have become essential methods for non-invasive real-time studies of biological processes in vivo. The aim of my thesis was the development of novel fluorescent and bioluminescent probes for in vivo imaging of fatty acid and triglyceride uptake. The goal of my first project was the development of a near infrared fluorescent fatty acid probe for glioma imaging. Some tumors such as glioma may rely on the uptake of extracellular fatty acids. Fatty acids can therefore be considered as targeting molecules for these tumors and could be used to develop tumor-imaging probes. The probe design is based on a near-infrared fluorophore indocyanine green (ICG) that is conjugated to a long-chain fatty acid palmitic acid. The conjugation of ICG to a fatty acid (ICG-FA) was envisioned to improve cell permeability of the fluorophore and its accumulation in glioma. The ICG-FA probe was first evaluated for its ability to mimic the uptake of natural fatty acids in cells and was then applied for glioma imaging studies in vivo, where it showed significant tumor accumulation. As a proof-of-concept, the probe was tested for intraoperative image-guided surgery in a canine patent with mastocytoma, where it allowed intraoperative fluorescence tumor imaging and provided optical guidance for a surgeon during tumor resection. The aim of my second project was the development of bioluminescent triglyceride probes for real-time non-invasive imaging of triglyceride uptake in vivo. Applying an optimized stategy for generating conjugates with luciferin, I developed several bioluminescent triglyceride probes. These imaging tools were validated in cells and mice and were shown to mimic the absorption of natural triglycerides. The developed probes enabled sensitive non-invasive real-time imaging and quantification of triglyceride absorption in living mice. Futhermore, these probes were successfully used to demonstrate the effects of the anti-obesity drug orlistat and the influence of the gut microbiota on the intestinal absorption of triglycerides in vivo. Considering the high importance of triglycerides for human health and nutrition and their implication in several pathologies such as obesity, metabolic syndrome, type 2 diabetes and cancer, the bioluminescent triglyceride probes could offer valuable imaging tools for preclinical research

Measurement of Long-Chain Fatty Acid Uptake into Adipocytes

The ability of white and brown adipose tissue to efficiently take up long-chain fatty acids is key to their physiological functions in energy storage and thermogenesis, respectively. Several approaches have been taken to determine uptake rates by cultured cells and primary adipocytes including radio- and fluorescently labeled fatty acids. In addition, the recent description of activatable bioluminescent fatty acids has opened the possibility for expanding these in vitro approaches to real-time monitoring of fatty acid uptake kinetics by adipose depots in vivo. Here, we will describe some of the most useful experimental paradigms to quantitatively determine long-chain fatty acid uptake by adipocytes in vitro and provide the reader with detailed instruction on how bioluminescent probes for in vivo imaging can be synthesized and used in living mice

Chapter Seven Measurement of Long-Chain Fatty Acid Uptake into Adipocytes

The ability of white and brown adipose tissue to efficiently take up long-chain fatty acids is key to their physiological functions in energy storage and thermogenesis, respectively. Several approaches have been taken to determine uptake rates by cultured cells and primary adipocytes including radio- and fluorescently labeled fatty acids. In addition, the recent description of activatable bioluminescent fatty acids has opened the possibility for expanding these in vitro approaches to real-time monitoring of fatty acid uptake kinetics by adipose depots in vivo. Here, we will describe some of the most useful experimental paradigms to quantitatively determine long-chain fatty acid uptake by adipocytes in vitro and provide the reader with detailed instruction on how bioluminescent probes for in vivo imaging can be synthesized and used in living mice

Bioluminescence imaging of small biomolecules

The invention relates to a technique to detect small molecules using Bioluminescence imaging (BLI) to image and quantify non-invasively, in vitro and in vivo,intracellular metabolite fluxes and which can be applied to azido-modified compounds, such as azido-modified biomolecules

A System for In Vivo Imaging of Hepatic Free Fatty Acid Uptake

Alterations in hepatic free fatty acid (FFA) uptake and metabolism contribute to the development of prevalent liver disorders such as hepatosteatosis. However, detecting dynamic changes in FFA uptake by the liver in live model organisms has proven difficult. To enable noninvasive real-time imaging of FFA flux in the liver, we generated transgenic mice with liver-specific expression of luciferase and performed bioluminescence imaging with an FFA probe. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. By using this method, we detected a decrease in FFA accumulation in the liver after mice were given injections of deoxycholic acid and an increase after they were fed fenofibrate. In addition, we observed diurnal regulation of FFA hepatic uptake in living mice. Our imaging system appears to be a useful and reliable tool for studying the dynamic changes in hepatic FFA flux in models of liver disease

A System for In Vivo Imaging of Hepatic Free Fatty Acid Uptake

Alterations in hepatic free fatty acid (FFA) uptake and metabolism contribute to the development of prevalent liver disorders such as hepatosteatosis. However, detecting dynamic changes in FFA uptake by the liver in live model organisms has proven difficult. To enable noninvasive real-time imaging of FFA flux in the liver, we generated transgenic mice with liver-specific expression of luciferase and performed bioluminescence imaging with an FFA probe. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. By using this method, we detected a decrease in FFA accumulation in the liver after mice were given injections of deoxycholic acid and an increase after they were fed fenofibrate. In addition, we observed diurnal regulation of FFA hepatic uptake in living mice. Our imaging system appears to be a useful and reliable tool for studying the dynamic changes in hepatic FFA flux in models of liver disease

Matrix-Assisted Transplantation of Functional Beige Adipose Tissue

Novel, clinically relevant, approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion of brown adipose tissues that express uncoupling protein (UCP) 1 and thus can uncouple mitochondrial respiration from ATP synthesis. While expansion of UCP1-expressing adipose depots may be achieved in rodents via genetic and pharmacological manipulations or the transplantation of brown fat depots, these methods are difficult to use for human clinical intervention. We present a novel cell scaffold technology optimized to establish functional brown fat-like depots in vivo. We adapted the biophysical properties of hyaluronic acid-based hydrogels to support the differentiation of white adipose tissue-derived multipotent stem cells (ADMSCs) into lipid-accumulating, UCP1-expressing beige adipose tissue. Subcutaneous implantation of ADMSCs within optimized hydrogels resulted in the establishment of distinct UCP1-expressing implants that successfully attracted host vasculature and persisted for several weeks. Importantly, implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain, demonstrating the therapeutic merit of this highly translatable approach. This novel approach is the first truly clinically translatable system to unlock the therapeutic potential of brown fat-like tissue expansion

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important

Imaging of NTR activity in cells and in <i>in vivo</i> cancer model with NCL.

<p>(A) Concentration-dependent uncaging of NCL in MDA-MB-231 NTR+luc+ cancer cells in comparison with luciferin. (B) Selectivity of NTR imaging by NCL in the same cells in comparison with NTR-luc+ cells. The dashed line indicates background (cells only), *P = 0.0001. (C) <i>In vivo</i> imaging of NTR activity in subcutaneous NTR+ and NTR- xenografts (n = 5). Total luminescence over 1 h from IP injection of luciferin (1.5 mg) and NCL (1.9 mg). d) Representative images of mice 15 min post injection of luciferin or NCL.</p