-
European Journal of Nuclear Medicine... Sep 2022Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor...
PURPOSE
Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies.
METHODS
FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent.
RESULTS
Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of Ga-FAP-2286, In-FAP-2286, and Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46.
CONCLUSION
In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of Ga-FAP-2286 for imaging and Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.
Topics: Adult; Animals; Cell Line, Tumor; Fibroblasts; Gallium Radioisotopes; HEK293 Cells; Humans; Mice; Radionuclide Imaging; Sarcoma; Tissue Distribution; Tumor Microenvironment
PubMed: 35608703
DOI: 10.1007/s00259-022-05842-5 -
Methods in Molecular Biology (Clifton,... 2024Autoradiography, the direct imaging of radioactive distribution in tissue sections, is a powerful technique that has several key advantages for the validation of PET...
Autoradiography, the direct imaging of radioactive distribution in tissue sections, is a powerful technique that has several key advantages for the validation of PET radiotracers. Using autoradiography, we can localize radiotracer uptake to neighbours of cells, and when multiplexed with additional radiotracers, fluorescent probes, or in situ tissue analysis, autoradiography can help to characterize the mechanism of radiotracer uptake and assess functional heterogeneity in tissue. In this chapter, the author outlines the basic ex vivo autoradiography protocol and shows how it can be multiplexed using dual radionuclides F and C. They also highlight where autoradiography can be combined with other technologies to provide synergistic information for interrogating spatial biology.
Topics: Positron-Emission Tomography; Autoradiography; Radioisotopes; Radiopharmaceuticals; Fluorescent Dyes; Fluorine Radioisotopes
PubMed: 38006510
DOI: 10.1007/978-1-0716-3499-8_24 -
Cold Spring Harbor Protocols Dec 2020Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces....
Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, β-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.
Topics: Autoradiography; Cloning, Molecular; DNA, Recombinant; Humans; Image Processing, Computer-Assisted; Phosphorus Radioisotopes; Reproducibility of Results; X-Ray Film
PubMed: 33262238
DOI: 10.1101/pdb.top100446 -
Journal of Nuclear Medicine : Official... Dec 2019Heart failure remains a major source of late morbidity and mortality after myocardial infarction (MI). The temporospatial presence of activated fibroblasts in the...
Heart failure remains a major source of late morbidity and mortality after myocardial infarction (MI). The temporospatial presence of activated fibroblasts in the injured myocardium predicts the quality of cardiac remodeling after MI. Therefore, monitoring of activated fibroblasts is of great interest for studying cardiac remodeling after MI. Fibroblast activation protein (FAP) expression is upregulated in activated fibroblasts. This study investigated the feasibility of imaging activated fibroblasts with a new Ga-labeled FAP inhibitor (Ga-FAPI-04) for PET imaging of fibroblast activation in a preclinical model of MI. MI and sham-operated rats were scanned with Ga-FAPI-04 PET/CT (1, 3, 6, 14, 23, and 30 d after MI) and with F-FDG (3 d after MI). Dynamic Ga-FAPI-04 PET and blocking studies were performed on MI rats 7 d after coronary ligation. After in vivo scans, the animals were euthanized and their hearts harvested for ex vivo analyses. Cryosections were prepared for autoradiography, hematoxylin and eosin (H&E), and immunofluorescence staining. Ga-FAPI-04 uptake in the injured myocardium peaked on day 6 after coronary ligation. The tracer accumulated intensely in the MI territory, as identified by decreased F-FDG uptake and confirmed by PET/MR and H&E staining. Autoradiography and H&E staining of cross-sections revealed that Ga-FAPI-04 accumulated mainly at the border zone of the infarcted myocardium. In contrast, there was only minimal uptake in the infarct of the blocked rats, comparable to the uptake in the remote noninfarcted myocardium (PET image-derived ratio of infarct uptake to remote uptake: 6 ± 2). Immunofluorescence staining confirmed the presence of FAP-positive myofibroblasts in the injured myocardium. Morphometric analysis of the whole-heart sections demonstrated 3- and 8-fold higher FAP-positive fibroblast density in the border zone than in the infarct center and remote area, respectively. Ga-FAPI-04 represents a promising radiotracer for in vivo imaging of post-MI fibroblast activation. Noninvasive imaging of activated fibroblasts may have significant diagnostic and prognostic value, which could aid clinical management of patients after MI.
Topics: Animals; Feasibility Studies; Fibroblasts; Gallium Radioisotopes; Isotope Labeling; Male; Membrane Proteins; Myocardial Infarction; Positron Emission Tomography Computed Tomography; Quinolines; Rats; Rats, Wistar
PubMed: 31405922
DOI: 10.2967/jnumed.119.226993 -
The Journal of Headache and Pain May 2023Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative...
BACKGROUND AND AIMS
Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative electrophysiological event underlying migraine aura and a headache trigger. Migraine is three times more prevalent in women than men, linked to circulating female hormones. High estrogen levels or estrogen withdrawal may be a migraine trigger for many women. We, therefore, aimed to examine whether sex, gonadectomy, and female hormone supplementation and withdrawal affect the susceptibility to CSD.
METHODS
To determine CSD susceptibility, we recorded the frequency of CSDs triggered during 2-h topical KCl application in intact or gonadectomized female and male rats, without or with estradiol or progesterone supplementation via daily intraperitoneal injections. Estrogen or progesterone treatment followed by withdrawal was studied in a separate cohort. To take the first step towards identifying potential mechanisms, we studied glutamate and GABA receptor binding using autoradiography.
RESULTS
The CSD frequency in intact female rats was higher than intact male and ovariectomized rats. We did not detect a change in CSD frequency during different stages of the estrous cycle in intact females. Daily estrogen injections for three weeks did not change CSD frequency. However, one-week estrogen withdrawal after two weeks of treatment significantly increased CSD frequency compared with the vehicle group in gonadectomized females. The same protocol of estrogen treatment and withdrawal was ineffective in gonadectomized males. In contrast to estrogen, daily progesterone injections for three weeks elevated CSD susceptibility, and one-week withdrawal after two weeks of treatment partially normalized this effect. Autoradiography did not reveal significant changes in glutamate or GABA receptor binding density after estrogen treatment and withdrawal.
CONCLUSIONS
These data suggest that females are more susceptible to CSD, and sexual dimorphism is abrogated by gonadectomy. Moreover, estrogen withdrawal after prolonged daily treatment enhances CSD susceptibility. These findings may have implications for estrogen-withdrawal migraine, although the latter tends to be without aura.
Topics: Rats; Female; Male; Animals; Cortical Spreading Depression; Progesterone; Receptors, GABA-A; Estrogens; Migraine Disorders; Glutamates
PubMed: 37237336
DOI: 10.1186/s10194-023-01598-x -
Annual Review of Pharmacology and... Jan 2020Here, I recount some adventures that I and my colleagues have had over some 60 years since 1957 studying the effects of drugs and neurotransmitters on neuronal...
Here, I recount some adventures that I and my colleagues have had over some 60 years since 1957 studying the effects of drugs and neurotransmitters on neuronal excitability and ion channel function, largely, but not exclusively, using sympathetic neurons as test objects. Studies include effects of centrally active drugs on sympathetic transmission; neuronal action and neuroglial uptake of GABA in the ganglia and brain; the action of muscarinic agonists on sympathetic neurons; the action of bradykinin on neuroblastoma-derived cells; and the identification of M-current as a target for muscarinic action, including experiments to determine its distribution, molecular composition, neurotransmitter sensitivity, and intracellular regulation by phospholipids and their hydrolysis products. Techniques used include electrophysiological recording (extracellular, intracellular microelectrode, whole-cell, and single-channel patch-clamp), autoradiography, messenger RNA and complementary DNA expression, antibody injection, antisense knockdown, and membrane-targeted lipidated peptides. I finish with some recollections about my scientific career, funding, and changes in laboratory life and pharmacology research over the past 60 years.
Topics: Animals; Humans; Ion Channels; Neurons; Neurotransmitter Agents; Pharmaceutical Preparations; Pharmacology
PubMed: 31914894
DOI: 10.1146/annurev-pharmtox-010919-023755 -
Molecular Biology of the Cell Aug 2021With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation...
With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.
Topics: Autoradiography; Chromatography, Gel; Ethylene Glycol; Osmotic Pressure; Phosphorylation; Polyethylene Glycols; Protein Conformation; Protein Kinases; Protein Multimerization; Scattering, Small Angle; WNK Lysine-Deficient Protein Kinase 1; Water; X-Ray Diffraction
PubMed: 33689398
DOI: 10.1091/mbc.E20-01-0089 -
Molecular Imaging and Biology Feb 2021Molecular imaging agents targeting butyrylcholinesterase (BChE) have shown promise in other neurodegenerative disorders and may have utility in detecting changes to...
PURPOSE
Molecular imaging agents targeting butyrylcholinesterase (BChE) have shown promise in other neurodegenerative disorders and may have utility in detecting changes to normal appearing white matter in multiple sclerosis (MS). BChE activity is present in white matter and localizes to activated microglia associated with MS lesions. The purpose of this study was to further characterize changes in the cholinergic system in MS pathology, and to explore the utility of BChE radioligands as potential diagnostic and treatment monitoring agents in MS.
PROCEDURE
Cortical and white matter lesions were identified using myelin staining, and lesions were classified based on microglial activation patterns. Adjacent brain sections were used for cholinesterase histochemistry and in vitro autoradiography using phenyl 4-[I]-iodophenylcarbamate (I-PIP), a previously described small-molecule cholinesterase-binding radioligand.
RESULTS
BChE activity is positively correlated with microglial activation in white matter MS lesions. There is no alteration in cholinesterase activity in cortical MS lesions. I-PIP autoradiography revealed uptake of radioactivity in normal white matter, absence of radioactivity within demyelinated MS lesions, and variable uptake of radioactivity in adjacent normal-appearing white matter.
CONCLUSIONS
BChE imaging agents have the potential to detect MS lesions and subtle pathology in normal-appearing white matter in postmortem MS brain tissue. The possibility of BChE imaging agents serving to supplement current diagnostic and treatment monitoring strategies should be evaluated.
Topics: Acetylcholinesterase; Aged; Autoradiography; Butyrylcholinesterase; Case-Control Studies; Female; Gray Matter; Humans; Male; Middle Aged; Molecular Imaging; Multiple Sclerosis; Phenylcarbamates; White Matter
PubMed: 32926288
DOI: 10.1007/s11307-020-01540-6 -
BioRxiv : the Preprint Server For... Sep 2023Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain...
Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain tissue with elevated tau burden, purified filaments, and determined the structure of second-generation high avidity PET ligand MK-6240 at 2.31 Å resolution, which bound at a 1:1 ratio within the cleft of tau paired-helical filament (PHF), engaging with glutamine 351, lysine K353, and isoleucine 360. This information elucidates the basis of MK-6240 PET in quantifying PHF deposits in AD and may facilitate the structure-based design of superior ligands against tau amyloids.
PubMed: 37790438
DOI: 10.1101/2023.09.22.558671 -
Methods in Molecular Biology (Clifton,... 2020Unbound, single-stranded RNA can be digested by RNase (A or T1) to ribonucleotides, whereas double-stranded RNA is not digested by RNase. Based on this principle, the...
Unbound, single-stranded RNA can be digested by RNase (A or T1) to ribonucleotides, whereas double-stranded RNA is not digested by RNase. Based on this principle, the RNase Protection Assay (RPA) is used to validate chimeric RNAs. Importantly, this assay does not employ reverse transcription (RT), thus avoiding potential false-positive results which could occur during RT such as template-switching. We first generate RNA probes with phosphate (P) or biotin that are complementary to the predicted nucleotide sequence of the chimeric RNA, then hybridize them to RNA samples. The labeled RNA probes can bind specifically with the target chimeric RNA in order to form double-stranded RNA. This newly formed RNA is resistant to digestion by RNase and therefore can be identified by high-resolution, denaturing polyacrylamide gel electrophoresis.
Topics: Autoradiography; Binding Sites; Electrophoresis, Polyacrylamide Gel; Isotope Labeling; Molecular Probes; Nucleic Acid Hybridization; Protein Binding; RNA; RNA, Double-Stranded; RNA-Binding Proteins; Ribonucleases
PubMed: 31728965
DOI: 10.1007/978-1-4939-9904-0_8