Radiolabelled FGF-2 for Imaging Activated Fibroblasts in the Tumor Micro-Environment.

Tumor associated fibroblasts (TAFs) play a key role in tumor growth and metastatization. TAFs overexpress different biomarkers that are usually expressed at low levels in physiological conditions. Among them are the fibroblast growth factor receptors (FGFRs) that bind the fibroblast growth factors (FGFs). In particular, the overexpression of FGFR-2c in tumors has been associated with advanced clinical stages and increased metastatization. Here, we developed a non-invasive tool to evaluate, in vivo, the expression of FGFR-2c in metastatic cancer. This is based on 99mTc-labelled FGF-2. METHODS: 99mTc-FGF-2 was tested in vitro and in vivo in mice bearing allografts of sarcoma cells. Images of 99mTc-FGF-2 were acquired using a new portable high-resolution ultra-sensitive gamma camera for small animal imaging. RESULTS: FGF-2 was labeled with high specific activity but low labelling efficiency, thus requiring post-labeling purification by gel-filtration chromatography. In vitro binding to 2C human keratinocytes showed a Kd of 3.36 × 10-9 M. In mice bearing J774A.1 cell allografts, we observed high and rapid tumor uptake of 99mTc-FGF-2 with a high Tumor/Blood ratio at 24 h post-injection (26.1 %ID/g and 12.9 %ID) with low kidney activity and moderate liver activity. CONCLUSIONS: we labeled FGF-2 with 99mTc and showed nanomolar Kd in vitro with human keratinocytes expressing FGF-2 receptors. In mice, 99mTc-FGF-2 rapidly and efficiently accumulated in tumors expressing FGF-2 receptors. This new radiopharmaceutical could be used in humans to image TAFs.

BODIPY-Based Analogue of the TREM2-Binding Molecular Adjuvant Sulfavant A, a Chemical Tool for Imaging and Tracking Biological Systems.

Recently, we described synthetic sulfolipids named Sulfavants as a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. The members of this family, Sulfavant A (1), Sulfavant R (2), and Sulfavant S (3), showed important effects on triggering receptor expressed on myeloid cells 2 (TREM2)-induced differentiation and maturation of human dendritic cells (hDC), through a novel cell mechanism underlying the regulation of the immune response. As these molecules are involved in biological TREM2-mediated processes crucial for cell survival, here, we report the synthesis and application of a fluorescent analogue of Sulfavant A bearing the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene moiety (Me4-BODIPY). The fluorescent derivative, named PB-SULF A (4), preserving the biological activity of Sulfavants, opens the way to chemical biology and cell biology experiments to better understand the interactions with cellular and in vivo organ targets and to improve our comprehension of complex molecular mechanisms underlying the not fully understood ligand-induced TREM2 activity.

FMR1 deletion in rats induces hyperactivity with no changes in striatal dopamine transporter availability.

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder emerging in early life characterized by impairments in social interaction, poor verbal and non-verbal communication, and repetitive patterns of behaviors. Among the best-known genetic risk factors for ASD, there are mutations causing the loss of the Fragile X Messenger Ribonucleoprotein 1 (FMRP) leading to Fragile X syndrome (FXS), a common form of inherited intellectual disability and the leading monogenic cause of ASD. Being a pivotal regulator of motor activity, motivation, attention, and reward processing, dopaminergic neurotransmission has a key role in several neuropsychiatric disorders, including ASD. Fmr1 Δexon 8 rats have been validated as a genetic model of ASD based on FMR1 deletion, and they are also a rat model of FXS. Here, we performed behavioral, biochemical and in vivo SPECT neuroimaging experiments to investigate whether Fmr1 Δexon 8 rats display ASD-like repetitive behaviors associated with changes in striatal dopamine transporter (DAT) availability assessed through in vivo SPECT neuroimaging. At the behavioral level, Fmr1 Δexon 8 rats displayed hyperactivity in the open field test in the absence of repetitive behaviors in the hole board test. However, these behavioral alterations were not associated with changes in striatal DAT availability as assessed by non-invasive in vivo SPECT and Western blot analyses.

Role of Nuclear Imaging to Understand the Neural Substrates of Brain Disorders in Laboratory Animals: Current Status and Future Prospects.

Molecular imaging, which allows the real-time visualization, characterization and measurement of biological processes, is becoming increasingly used in neuroscience research. Scintigraphy techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) provide qualitative and quantitative measurement of brain activity in both physiological and pathological states. Laboratory animals, and rodents in particular, are essential in neuroscience research, providing plenty of models of brain disorders. The development of innovative high-resolution small animal imaging systems together with their radiotracers pave the way to the study of brain functioning and neurotransmitter release during behavioral tasks in rodents. The assessment of local changes in the release of neurotransmitters associated with the performance of a given behavioral task is a turning point for the development of new potential drugs for psychiatric and neurological disorders. This review addresses the role of SPECT and PET small animal imaging systems for a better understanding of brain functioning in health and disease states. Brain imaging in rodent models faces a series of challenges since it acts within the boundaries of current imaging in terms of sensitivity and spatial resolution. Several topics are discussed, including technical considerations regarding the strengths and weaknesses of both technologies. Moreover, the application of some of the radioligands developed for small animal nuclear imaging studies is discussed. Then, we examine the changes in metabolic and neurotransmitter activity in various brain areas during task-induced neural activation with special regard to the imaging of opioid, dopaminergic and cannabinoid receptors. Finally, we discuss the current status providing future perspectives on the most innovative imaging techniques in small laboratory animals. The challenges and solutions discussed here might be useful to better understand brain functioning allowing the translation of preclinical results into clinical applications.

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea.

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6-/- COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1-/- with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods.

Directional probe for radio-guided surgery: A pilot study.

PURPOSE: The sentinel lymph node (SLN) biopsy technique has highly evolved during the last 20 yr. Consequently, the intraoperative use of Gamma Probes (GPs) for SLN mapping is increased. This preliminary study evaluates a novel directional GP prototype. This proof-of-concept prototype is designed to identify the direction of radiopharmaceuticals uptakes, by combining the information from multiple detectors. The purpose of this work is to develop a tool able to effectively guide the surgeon reducing the surgery time. METHODS: The proposed prototype consists of three CsI(Tl) scintillation crystals, each coupled with an S10931 silicon photomultiplier (Hamamatsu Photonics K.K., Hamamatsu, JP). The three detectors lie on the same plane with an angle of 30° between them. The central detector is placed as in a common GP, so it can be used to pinpoint the target tissue. Meanwhile, the lateral sensors provide a broader view of the surgical field. A dedicated data acquisition system digitizes and processes the signals from the front-end electronics. Finally, an embedded system, based on ARM processor, calculates and displays the acquired count rates. In order to assess the prototype behavior, the isosensitivity curves for the three detectors were measured. Meanwhile, for the central one, the main quality criteria measurements were also performed (i.e., sensitivity, radial sensitivity, and spatial resolution). RESULTS: For the central detector, the measured sensitivity at the tip of the probe is better than 5 cps/kBq. The full width at half maximum (FWHM) of the radial sensitivity is less than 30° and the FWHM of the lateral sensitivity (spatial resolution) is about 7.2 mm. The central detector measured isosensitivity distribution shows a narrow profile in agreement with the spatial resolution measured. On the contrary, the two lateral detectors exhibit widespread isosensitivity distributions that mean a larger field of view. The system had shown satisfactory performance and reliability, meeting the minimal requirements of gamma probe systems. CONCLUSIONS: The prototype presented in this paper allows a rapid localization by the use of the whole system, while the sole central detector can be used to pinpoint the target source. This device, unlike common GPs, allows localizing simultaneously different areas of radiopharmaceuticals uptake, thus precisely guiding the surgeon to the region of interest. These preliminary results encourage to develop a further prototype for intraoperative validation.

Radiolabeled humanized anti-CD3 monoclonal antibody visilizumab for imaging human T-lymphocytes.

UNLABELLED: Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-FcgammaR binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a radiolabeled anti-CD3 antibody might serve as a diagnostic tool for imaging T-cell traffic and lymphocytic infiltration of tissues and organs affected by autoimmune diseases. Here we describe the results of in vitro and animal experiments with (99m)Tc-succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH)-visilizumab. METHODS: For mAb labeling, we used a 2-step method with a heterobifunctional linker SHNH. Several titrations were performed to obtain the best labeling efficiency. In vitro quality controls included stability assay, cysteine challenge, sodium dodecyl sulfate polyacrylamide gel electrophoresis, binding assay, and immunoreactivity assay. In vivo studies by high-resolution images were performed at 6 and 24 h after the injection of (99m)Tc-SHNH-visilizumab. These included cell-targeting experiments in BALB/c mice xenografted subcutaneously with an increasing number of HuT78 cells in the leg and displaced with an excess of cold antibody. We also studied irradiated severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells (hPBMCs) and injected with (99m)Tc-labeled visilizumab or control mAb. After dynamic imaging for 3 h, major organs were removed, counted, and processed for immunohistologic examination. RESULTS: Visilizumab was labeled with HYNIC with high labeling efficiency (>90%) and high specific activity (SA; 10,360-11,100 MBq/mg), with retained biochemical integrity and in vitro binding activity to CD3-positive cells. The in vivo targeting experiment showed a proportional increase of specific uptake with the number of injected cells, both at 6 and at 24 h, and the in vivo competition study demonstrated more than 60% decreased uptake after an excess of unlabeled antibody. In SCID mice, hPBMCs in different tissues were detected by (99m)Tc-labeled visilizumab and confirmed by histology. CONCLUSION: Visilizumab can be efficiently labeled with (99m)Tc with high efficiency and SA and could be a valuable tool for the study of human T-lymphocyte trafficking and lymphocytic infiltration of tissues and organs.

Thyroid cancer imaging in vivo by targeting the anti-apoptotic molecule galectin-3.

BACKGROUND: The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. METHODS: The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific (99m)Tc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 microCi of (99m)Tc-labeled mAb to galectin-3 (30 microg protein/in 100 microl saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. FINDINGS: Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. CONCLUSIONS: These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed.

High-resolution, hand-held camera for sentinel-node detection.

BACKGROUND: The imaging probe (IP) is a high-resolution (HR), 1-in(2) field-of-view hand-held gamma camera. We used it to detect breast cancer sentinel node (SN). PATIENTS AND METHODS: We divided 120 T1 breast cancer patients, who underwent Anger camera lymphoscintigraphy (ACL), in two subgroups of 60 patients who were age, body mass index, and cancer size matched: subgroup A (SA) and B (SB). SN was detected with a common gamma probe (GP) in SA, with IP plus GP in SB. RESULTS: Surgeons removed radioactive nodes without exceeding four nodes. Eighty-two (82) SNs were taken off in SA and 105 in SB (p<0.01). Of SA, 22 of 60 patients and 36 of 60 patients of SB showed more than 1 node, and 3 of them showed 3 nodes and 1 showed 4 nodes. Thirteen (13) patients resulted N(+) (21.6%) in SA. Ten (10) patients of SA showed an invasion on the hottest nodes and 3 on the second nodes. In the SB, 18 patients (25%) showed invasion. Sixteen (16) invasions were on hot, 4 on second, and 1 on the third node. Withdrawal time of SN was 11.25+/-4.7 minutes for SA and 7.4+/-2.8 minutes for SB (p<0.025). CONCLUSIONS: SN biopsy with IP is fast and discovers more SNs and more invasions than ACL.