Raphaël Boisgard

Evaluation of the PBR/TSPO radioligand [18F]DPA-714 in a rat model of focal cerebral ischemia

Focal cerebral ischemia leads to an inflammatory reaction involving an overexpression of the peripheral benzodiazepine receptor (PBR)/18-kDa translocator protein (TSPO) in the cerebral monocytic lineage (microglia and monocyte) and in astrocytes. Imaging of PBR/TSPO by positron emission tomography (PET) using radiolabeled ligands can document inflammatory processes induced by cerebral ischemia. We performed in vivo PET imaging with [18F]DPA-714 to determine the time course of PBR/TSPO expression over several days after induction of cerebral ischemia in rats. In vivo PET imaging showed significant increase in DPA (N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) uptake on the injured side compared with that in the contralateral area on days 7, 11, 15, and 21 after ischemia; the maximal binding value was reached 11 days after ischemia. In vitro autoradiography confirmed these in vivo results. In vivo and in vitro [18F]DPA-714 binding was displaced from the lesion by PK11195 and DPA-714. Immunohistochemistry showed increased PBR/TSPO expression, peaking at day 11 in cells expressing microglia/macrophage antigens in the ischemic area. At later times, a centripetal migration of astrocytes toward the lesion was observed, promoting the formation of an astrocytic scar. These results show that [18F]DPA-714 provides accurate quantitative information of the time course of PBR/TSPO expression in experimental stroke.

Imaging Microglial/Macrophage Activation in Spinal Cords of Experimental Autoimmune Encephalomyelitis Rats by Positron Emission Tomography Using the Mitochondrial 18 kDa Translocator Protein Radioligand [18F]DPA-714

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Activated microglia/macrophages play a key role in the immunopathogenesis of MS and its corresponding animal models, experimental autoimmune encephalomyelitis (EAE). Microglia activation begins at early stages of the disease and is associated with elevated expression of the 18 kDa mitochondrial translocator protein (TSPO). Thus, positron emission tomography (PET) imaging of microglial activation using TSPO-specific radioligands could be valuable for monitoring disease-associated neuroinflammatory processes. EAE was induced in rats using a fragment of myelin basic protein, yielding acute clinical disease that reflects extensive spinal cord inflammation. Enhanced TSPO expression in spinal cords of EAE rats versus those of controls was confirmed by Western blot and immunohistochemistry. Biodistribution studies in control and EAE rats were performed using the TSPO radioligand [18F]DPA-714 [N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide]. At 1 h after injection, almost fivefold higher levels of [18F]DPA-714 were measured in spinal cords of EAE rats versus controls. The specific binding of [18F]DPA-714 to TSPO in spinal cords was confirmed in competition studies, using unlabeled (R,S)-PK11195 [(R,S)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide)] or DPA-714 in excess. MicroPET studies affirm that this differential radioactivity uptake in spinal cords of EAE versus control rats could be detected and quantified. Using [18F]DPA-714, neuroinflammation in spinal cords of EAE-induced rats could be visualized by PET, offering a sensitive technique for monitoring neuroinflammatory lesions in the CNS and particularly in the spinal cord. In addition to current MRI protocols, this approach could provide molecular images of neuroinflammation for detection, monitoring, and research in MS.

Labelled Oligonucleotides as Radiopharmaceuticals: Pitfalls, Problems and Perspectives

The labelling of single-stranded oligonucleotides with a positron or single-photon emitter can result in valuable radiopharmaceuticals with promising applications for: (i) Imaging of specific mRNAs, i.e. visualisation of the expression of specific genes in vivo (ii) Monitoring of antisense chemotherapy, i.e. measuring the efficiency of efforts to block the expression of specific genes; (iii) Gene radiotherapy, i.e. the targeting of radiation damage to specific DNA sequences in order to destroy tumours; (iv) Imaging of protein targets by the use of aptamer oligonucleotides, i.e. oligonucleotide ligands obtained by in vitro evolution of selection-amplification steps, or selected for their interaction with nucleic acid-binding proteins; (v) Pre-targeting strategies based on the specificity of complementary sequence hybridisation. Nevertheless, oligonucleotides are intrinsically poor pharmaceuticals because of their large size, low stability, poor membrane passage and a number of undesirable and sometimes unpredictable side effects. As an alternative to the inherently unstable phosphodiester DNAs, chemically modified oligonucleotides such as phosphorothioate, methylphosphonate and peptide nucleic acid oligomers have been developed, and some are in clinical trials for the chemotherapy of several types of tumours. Imaging techniques could be useful in the development of such therapies. In addition, the potential of targeting virtually any disease or physiological process, by changing only the sequence of the oligomer, could provide a means to identify serious diseases in a very early stage, and be a highly specific modality to diagnose and differentiate various cancers. This has stimulated efforts to develop such radiopharmaceuticals in many laboratories, and encouraging results have been reported using technetium-99m, indium-111, carbon-11, fluorine-18, bromine-76 and iodine-125 labelled oligonucleotides.

Non-Invasive In Vivo Imaging of Calcium Signaling in Mice

Rapid and transient elevations of Ca2+ within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca2+ concentration ([Ca2+]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca2+-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca2+] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca2+] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca2+] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca2+ signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.

Proteomic analysis of β-catenin activation in mouse liver by DIGE analysis identifies glucose metabolism as a new target of the Wnt pathway

The Wnt/β‐catenin signaling pathway has been increasingly implicated in liver development and physiology. Aberrant activation of this pathway is one of the major genetic events observed during the process of human HCC development. To gain insight into the mechanism underlying β‐catenin action in the liver, we conducted a quantitative differential proteomic analysis using 2‐D DIGE combined with MS, in mice with liver‐specific deletion of Apc resulting in acute activation of β‐catenin signaling (ApcKOliv mice). We identified 94 protein spots showing differential expression between mutant ApcKOliv and control mice, corresponding to 56 individual proteins. Most of the proteins identified were associated with metabolic pathways, such as ammonia and glucose metabolism. Our analysis showed an increase in lactate dehydrogenase activity together with a downregulation of two mitochondrial ATPase subunits (ATP5a1 and ATP5b). These observations indicate that β‐catenin signaling may induce a shift in the glucose metabolism from oxidative phosphorylation to glycolysis, known as the “Warburg effect”. Imaging with 18F‐fluoro‐2‐deoxy‐D‐glucose‐positron emission tomography suggests that the specific metabolic reprogramming induced by β‐catenin in the liver does not imply the first step of glycolysis. This observation may explain why some HCCs are difficult to assess by fluoro‐2‐deoxy‐D‐glucose‐positron emission tomography imaging.

The translocator protein ligand [18F]DPA-714 images glioma and activated microglia in vivo

PurposeIn recent years there has been an increase in the development of radioligands targeting the 18-kDa translocator protein (TSPO). TSPO expression is well documented in activated microglia and serves as a biomarker for imaging neuroinflammation. In addition, TSPO has also been reported to be overexpressed in a number of cancer cell lines and human tumours including glioma. Here we investigated the use of [18F]DPA-714, a new TSPO positron emission tomography (PET) radioligand to image glioma in vivo.MethodsWe studied the uptake of [18F]DPA-714 in three different rat strains implanted with 9L rat glioma cells: Fischer (F), Wistar (W) and Sprague Dawley (SD) rats. Dynamic [18F]DPA-714 PET imaging, kinetic modelling of PET data and in vivo displacement studies using unlabelled DPA-714 and PK11195 were performed. Validation of TSPO expression in 9L glioma cell lines and intracranial 9L gliomas were investigated using Western blotting and immunohistochemistry of brain tissue sections.ResultsAll rats showed significant [18F]DPA-714 PET accumulation at the site of 9L tumour implantation compared to the contralateral brain hemisphere with a difference in uptake among the three strains (F > W > SD). The radiotracer showed high specificity for TSPO as demonstrated by the significant reduction of [18F]DPA-714 binding in the tumour after administration of unlabelled DPA-714 or PK11195. TSPO expression was confirmed by Western blotting in 9L cells in vitro and by immunohistochemistry ex vivo.ConclusionThe TSPO radioligand [18F]DPA-714 can be used for PET imaging of intracranial 9L glioma in different rat strains. This preclinical study demonstrates the feasibility of employing [18F]DPA-714 as an alternative radiotracer to image human glioma.

In vivo Tumor Targeting Using a Novel Intestinal Pathogen-Based Delivery Approach

Efficient methods for tumor targeting are eagerly awaited and must satisfy several challenges: molecular specificity, transport through physiologic barriers, and capacity to withstand extracellular or intracellular degradation and inactivation by the immune system. Through interaction with its hosts, the intestinal pathogen-produced Shiga toxin has evolved molecular properties that are of interest in this context. Its nontoxic B-subunit binds to the cellular toxin receptor, glycosphingolipid Gb3, which is highly expressed on human cancers and has recently been reported to be involved in the formation of metastasis in colorectal cancers. Its function as a target for cancer therapy has already been addressed in xenograft experiments. We here show that after oral or i.v. injections in mice, the B-subunit targets spontaneous digestive Gb3-expressing adenocarcinomas. The nontumoral mucosa is devoid of labeling, with the exception of rare enteroendocrine and CD11b-positive cells. As opposed to other delivery tools that are often degraded or recycled on cancer cells, the B-subunit stably associates with these cells due to its trafficking via the retrograde transport route. This can be exploited for the in vivo delivery of contrast agents to tumors, as exemplified using fibered confocal fluorescence endoscopy and positron emission tomography (PET) imaging. In conclusion, the data presented in this manuscript lay the groundwork for a novel delivery technology that, in addition to its use for molecular imaging applications such as noninvasive PET, could also be exploited for targeted tumor therapies.

Imaging of perfusion, angiogenesis, and tissue elasticity after stroke.

Blood flow interruption in a cerebral artery causes brain ischemia and induces dramatic changes of perfusion and metabolism in the corresponding territory. We performed in parallel positron emission tomography (PET) with [15O]H2O, single photon emission computed tomography (SPECT) with [99mTc]hexamethylpropylene-amino-oxime ([99mTc]HMPAO) and ultrasonic ultrafast shear wave imaging (SWI) during, immediately after, and 1, 2, 4, and 7 days after middle cerebral artery occlusion (MCAO) in rats. Positron emission tomography and SPECT showed initial hypoperfusion followed by recovery at immediate reperfusion, hypoperfusion at day 1, and hyperperfusion at days 4 to 7. Hyperperfusion interested the whole brain, including nonischemic areas. Immunohistochemical analysis indicated active angiogenesis at days 2 to 7, strongly suggestive that hyperperfusion was supported by an increase in microvessel density in both brain hemispheres after ischemia. The SWI detected elastic changes of cerebral tissue in the ischemic area as early as day 1 after MCAO appearing as a softening of cerebral tissue whose local internal elasticity decreased continuously from day 1 to 7. Taken together, these results suggest that hyperperfusion after cerebral ischemia is due to formation of neovessels, and indicate that brain softening is an early and continuous process. The SWI is a promising novel imaging method for monitoring the evolution of cerebral ischemia over time in animals.

Differential expression of the 18 kDa translocator protein (TSPO) by neoplastic and inflammatory cells in mouse tumors of breast cancer.

Tumor-associated inflammation has been linked to angiogenesis, metastasis and poor prognosis. The 18 kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is expressed in activated immune cells such as macrophages, but also in a number of cancer cell lines such as those of breast cancer. There is an increasing clinical interest in TSPO expression as it has been proposed as a poor prognostic factor for survival in lymph-node negative breast cancer patients. This study aims to assess of the presence of neoplastic cell-associated TSPO and tumor macrophage-associated TSPO in mouse xenografts generated from the MDA-MB-231 and the MCF-7 breast cancer cell lines, as well as 25 different breast tumors originally derived from patient-tissue but propagated in mice using two antibodies, each specific to either the human or the murine form of TSPO. Autoradiography with the TSPO ligand [¹⁸F]DPA-714 and immunohistochemistry were also performed on the excised tumor tissues from the MDA-MB-231, MCF-7 and one of the patient-derived xenografts (HBCx-12B). High TSPO expression (either cancer or stromal cell-associated, or both) was measured in 20/25 (80%) of the patient-derived breast cancer xenografts. [¹⁸F]DPA-714 showed displaceable binding to both the human and murine TSPO on tumor tissue sections. Immunohistochemistry demonstrated that a significant portion of the tumor stromal TSPO expression colocalized with F4/80 positive macrophages cells. This study constitutes a first report of the tumor TSPO expression by mixed cell populations, and it may have important implications for cancer biology as well as for the development of imaging and therapeutic ligands targeted to TSPO.

Internal Radiotherapy of Liver Cancer with Rat Hepatocarcinoma-Intestine-Pancreas Gene as a Liver Tumor-Specific Promoter

The hepatocarcinoma-intestine-pancreas (HIP) gene, also called pancreatitis-associated protein-1 (PAP1) or Reg IIIalpha, is activated in most human hepatocellular carcinomas (HCCs) but not in normal liver, which suggests that HIP regulatory sequence could be used as efficient liver tumor-specific promoters to express a therapeutic polynucleotide in liver cancer. The sodium iodide symporter (NIS), which has recognized therapeutic and reporter gene properties, is appropriate to evaluate the transcriptional strength and specificity of the HIP promoter in HCC. For this purpose, we constructed a recombinant rat HIP-NIS adenoviral vector (AdrHIP-NIS), and evaluated its performance as a mediator of selective radioiodide uptake in tumor hepatocytes. Western blot, immunofluorescence, and iodide uptake assays were performed in AdrHIP-NIS-infected primary hepatocytes and transformed hepatic and nonhepatic cells. Nuclear imaging, tissue counting and immunohistochemistry were performed in normal and HCC-bearing Wistar rats infected with AdrHIP-NIS intratumorally or via the hepatic artery. In AdrHIP-NIS-infected transformed hepatic cells, functional NIS was strongly expressed, as in cells infected with a cytomegalovirus-NIS vector. No NIS expression was found in AdrHIP-NIS-infected normal hepatocytes or transformed nonhepatic cells. In rats bearing multinodular HCC, AdrHIP-NIS triggered functional NIS expression that was preferential in tumor hepatocytes. Administration of 18 mCi of (131)I resulted in the destruction of AdrHIP-NIS-injected nodules. This study has identified the rHIP regulatory sequence as a potent liver tumor-specific promoter for the transfer of therapeutic genes, and AdrHIP-NIS-mediated (131)I therapy as a valuable option for the treatment of multinodular HCC.