99mTc­labeled single-domain antibody for SPECT/CT assessment of HER2 expression in diverse cancer types.

The expression status of human epidermal growth factor receptor 2 (HER2) in cancer predicts response to HER2-targeted therapy. Therefore, its accurate determination is of utmost importance. In recent years, there has been an increase in research on noninvasive techniques for molecular imaging, as this method offers the advantages of a more accurate determination of HER2 status without the need for multiple biopsies. The technetium-labeled single-domain antibody RAD201, previously known as 99mTc-NM-02, has been shown to be safe for use in breast cancer imaging with reasonable radiation doses, favorable biodistribution, and imaging characteristics. METHODS: A total of six HER2-positive, heavily pretreated patients with different cancer types aged between 42 and 69 years (5 women and 1 man; the median age of 55.5) have been examined. In six of seven scans, the patients were administered 500 ml of Gelofusine® solution (40 mg/ml) for radiation protection before the tracer injection (434 ± 42 MBq). Planar scans were acquired with the patient supine at 10 min, 60 min, 160 min, 20 h, and 24 h after injection. A CT scan was acquired at 95 min, followed by local tomographic SPECT imaging. RESULTS: One patient was scanned twice with RAD201, 3 months apart, resulting in a total of seven scans for six patients. Here, we show that the use of RAD201 in our patient group shows the same favorable biodistribution as in a previous study with RAD201 (NCT04040686) and that the radiation dose to the critical organ kidney can be reduced by the application of the plasma expander Gelofusine® by almost 50%. CONCLUSION: RAD201 appears safe for use in humans and is a promising noninvasive tool for discriminating HER2 status in metastatic (breast) cancer, regardless of ongoing HER2-targeted antibody treatment.

Improved protocol for the radiosynthesis of [18 F]FTC-146: A potent and selective sigma-1 receptor radioligand.

[18 F]FTC-146 was introduced as a very potent and selective sigma-1 receptor radioligand, which has shown promising application as an imaging agent for neuropathic pain with positron emission tomography. In line with a multi-laboratory project on animal welfare, we chose this radioligand to investigate its potential for detecting neuropathic pain and tissue damage in tumor-bearing animals. However, the radiochemical yield (RCY) of around 4-7% was not satisfactory to us, and efforts were made to improve it. Herein, we describe an improved approach for the radiosynthesis of [18 F]FTC-146 resulting in a RCY, which is sevenfold higher than that previously reported. A tosylate precursor was synthesized and radio-fluorination experiments were performed via aliphatic nucleophilic substitution reactions using either K[18 F]F-Kryptofix®222 (K2.2.2 )-carbonate system or tetra-n-butylammonium [18 F]fluoride ([18 F]TBAF). Several parameters affecting the radiolabeling reaction such as solvent, 18 F-fluorination agent with the corresponding amount of base, labeling time, and temperature were investigated. Best labeling reaction conditions were found to be [18 F]TBAF and acetonitrile as solvent at 100°C. The new protocol was then translated to an automated procedure using a FX2 N synthesis module. Finally, the radiotracer reproducibly obtained with RCYs of 41.7 ± 4.4% in high radiochemical purity (>98%) and molar activities up to 171 GBq/µmol.

[68 Ga]Ga-FAPI uptake correlates with the state of chronic kidney disease.

PURPOSE: Kidney fibrosis leads to a progressive reduction in kidney function ultimately resulting in kidney failure. Diagnostic tools to detect kidney fibrosis are all invasive in nature requiring kidney biopsies with subsequent histological validation. In this retrospective study, the diagnostic value of three different radiotracers for the noninvasive prediction of kidney fibrosis was analyzed, taking into account the glomerular filtration rate (GFR) and the intra-renal parenchymal radiotracer uptake. METHODS: In 81 patients receiving either one of the following molecular imaging probes, [68 Ga]Ga-FAPI, [68 Ga]Ga-PSMA, or [68 Ga]Ga-DOTATOC, kidney function parameters were correlated with SUVmax and SUVmean of the renal parenchyma and background activity measured in lung parenchyma, myocardium, gluteal muscle, and the abdominal aorta. Patients were clustered according to their grade of chronic kidney disease (CKD), and a regression analysis and one-way ANOVA were conducted in this retrospective analysis. RESULTS: We found a negative correlation between GFR and [68 Ga]Ga-FAPI uptake for both SUVmax and SUVmean values, whereas background activity showed no correlation with GFR. [68 Ga]Ga-DOTATOC and [68 Ga]Ga-PSMA did not correlate between CKD stage and intra-renal parenchymal radiotracer uptake. Only [68 Ga]Ga-PSMA background activity exhibited a positive correlation with GFR suggesting an unspecific binding/retention potentially due to longer circulation times. CONCLUSION: There is a significant negative correlation between renal parenchymal [68 Ga]Ga-FAPI uptake and GFR, which was not the case for [68 Ga]Ga-DOTATOC and [68 Ga]Ga-PSMA. This correlation suggests a specific binding of FAPI rather than a potential unspecific retention in the renal parenchyma, underlining the potential value of [68 Ga]Ga-FAPI for the noninvasive quantitative evaluation of kidney fibrosis.

Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

Structural basis of PI(4,5)P2-dependent regulation of GluA1 by phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP5K2A).

Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P2, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P2 effect by performing an alanine scan that suggested PI(4,5)P2 binding to this area. A PIP strip assay revealed PI(4,5)P2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.

Monitoring of post-translational modification dynamics with genetically encoded fluorescent reporters.

Post-translational modifications (PTMs) of proteins are essential mechanisms for virtually all dynamic processes within cellular signaling networks. Genetically encoded reporters based on fluorescent proteins (FPs) are powerful tools for spatiotemporal visualization of cellular parameters. Consequently, commonly used modular biosensor designs have been adapted to generate several protein-based indicators for monitoring various PTMs or the activity of corresponding enzymes in living cells, providing new biological insights into dynamics and regulatory functions of individual PTMs. In this review, we describe the application of general design strategies focusing on PTMs and discuss important considerations for engineering feasible indicators depending on the purpose. Moreover, we present developments and enhancements of PTM biosensors from selected studies and give an outlook on future perspectives of this versatile approach.

Deep learning based identification of bone scintigraphies containing metastatic bone disease foci.

PURPOSE: Metastatic bone disease (MBD) is the most common form of metastases, most frequently deriving from prostate cancer. MBD is screened with bone scintigraphy (BS), which have high sensitivity but low specificity for the diagnosis of MBD, often requiring further investigations. Deep learning (DL) - a machine learning technique designed to mimic human neuronal interactions- has shown promise in the field of medical imaging analysis for different purposes, including segmentation and classification of lesions. In this study, we aim to develop a DL algorithm that can classify areas of increased uptake on bone scintigraphy scans. METHODS: We collected 2365 BS from three European medical centres. The model was trained and validated on 1203 and 164 BS scans respectively. Furthermore we evaluated its performance on an external testing set composed of 998 BS scans. We further aimed to enhance the explainability of our developed algorithm, using activation maps. We compared the performance of our algorithm to that of 6 nuclear medicine physicians. RESULTS: The developed DL based algorithm is able to detect MBD on BSs, with high specificity and sensitivity (0.80 and 0.82 respectively on the external test set), in a shorter time compared to the nuclear medicine physicians (2.5 min for AI and 30 min for nuclear medicine physicians to classify 134 BSs). Further prospective validation is required before the algorithm can be used in the clinic.

Fluorescent Biosensors for Multiplexed Imaging of Phosphoinositide Dynamics.

Phosphoinositides constitute a critical family of lipids that regulate numerous cellular processes. Phosphatidylinositol 4,5-bisphosphate (PIP2) is arguably the most important plasma membrane phosphoinositide and is involved in regulating diverse processes. It is also the precursor of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is critical for growth factor signaling, as well as membrane polarization and dynamics. Studying these lipids remains challenging, because of their compartmentalized activities and location-dependent signaling profiles. Here, we introduce several new genetically encoded fluorescent biosensors, including FRET-based and dimerization-dependent fluorescent protein (ddFP)-based biosensors, that enable real-time monitoring of PIP2 levels in live cells. In addition, we developed a red fluorescent biosensor for 3-phosphoinositides that can be co-imaged with the green PIP2 indicator. Simultaneous visualization of the dynamics of PIP2 and 3-phosphoinositides in the same cell shows that plasma membrane PIP3 formation upon EGF stimulation is coupled to a decrease in the local pool of PIP2.

Targeted Palliative Radionuclide Therapy for Metastatic Bone Pain.

Bone metastasis develops in multiple malignancies with a wide range of incidence. The presence of multiple bone metastases, leading to a multitude of complications and poorer prognosis. The corresponding refractory bone pain is still a challenging issue managed through multidisciplinary approaches to enhance the quality of life. Radiopharmaceuticals are mainly used in the latest courses of the disease. Bone-pain palliation with easy-to-administer radionuclides offers advantages, including simultaneous treatment of multiple metastatic foci, the repeatability and also the combination with other therapies. Several ߯- and α-emitters as well as pharmaceuticals, from the very first [89Sr]strontium-dichloride to recently introduced [223Ra]radium-dichloride, are investigated to identify an optimum agent. In addition, the combination of bone-seeking radiopharmaceuticals with chemotherapy or radiotherapy has been employed to enhance the outcome. Radiopharmaceuticals demonstrate an acceptable response rate in pain relief. Nevertheless, survival benefits have been documented in only a limited number of studies. In this review, we provide an overview of bone-seeking radiopharmaceuticals used for bone-pain palliation, their effectiveness and toxicity, as well as the results of the combination with other therapies. Bone-pain palliation with radiopharmaceuticals has been employed for eight decades. However, there are still new aspects yet to be established.

Observing the Assembly of Protein Complexes in Living Eukaryotic Cells in Super-Resolution Using refSOFI.

Few approaches are currently available that allow the detection of protein-protein interactions (PPIs) in super-resolution, and the observation of the assembly of protein complexes in living cells has been particularly challenging. We developed reconstituted fluorescence-based stochastic optical fluctuation imaging (refSOFI), which is based on bimolecular fluorescence complementation (BiFC) and SOFI, allowing us to detect protein complex assembly 30 min after the induction of complex formation. Here we describe how to use refSOFI to map the assembly of two proteins of interest into a complex within living cells at super-resolution.

RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells.

It has become increasingly clear that protein-protein interactions (PPIs) are compartmentalized in nanoscale domains that define the biochemical architecture of the cell. Despite tremendous advances in super-resolution imaging, strategies to observe PPIs at sufficient resolution to discern their organization are just emerging. Here we describe a strategy in which PPIs induce reconstitution of fluorescent proteins (FPs) that are capable of exhibiting single-molecule fluctuations suitable for stochastic optical fluctuation imaging (SOFI). Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells. Using this strategy, termed reconstituted fluorescence-based SOFI (refSOFI), we investigated the interaction between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the pore-forming channel subunit ORAI1, a crucial process in store-operated Ca(2+) entry (SOCE). Stimulating SOCE does not appear to change the size of existing STIM1/ORAI1 interaction puncta at the ER-plasma membrane junctions, but results in an apparent increase in the number of interaction puncta.

Differential effects of genetically-encoded Gßγ scavengers on receptor-activated and basal Kir3.1/Kir3.4 channel current in rat atrial myocytes.

Opening of G-protein-activated inward-rectifying K(+) (GIRK, Kir3) channels is regulated by interaction with ßγ-subunits of Pertussis-toxin-sensitive G proteins upon activation of appropriate GPCRs. In atrial and neuronal cells agonist-independent activity (I(basal)) contributes to the background K(+) conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that I(basal) requires free G(ßγ). In cells with intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two G(ßγ)-binding proteins (myristoylated phosducin - mPhos - and G(αi1)) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt G(ßγ)-signaling. Agonist-induced and basal currents were recorded using whole cell voltage-clamp. Expression of mPhos and G(αi1) reduced activation of Kir3 current via muscarinic M(2) receptors (IK(ACh)). Inhibition of IK(ACh) by mPhos consisted of an irreversible component and an agonist-dependent reversible component. Reduction in density of IK(ACh) by overexpressed Gαi1, in contrast to mPhos, was paralleled by substantial slowing of activation, suggesting a reduction in density of functional M2 receptors, rather than G(ßγ)-scavenging as underlying mechanism. In line with this notion, current density and activation kinetics were rescued by fusing the αi1-subunit to an Adenosine A(1) receptor. Neither mPhos nor G(αi1) had a significant effect on I(basal), defined by the inhibitory peptide tertiapin-Q. These data demonstrate that basal Kir3 current in a native environment is unrelated to G-protein signaling or agonist-independent free G(ßγ). Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist.

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

BACKGROUND: Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P(2) in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane(.) For controlled and reversible depletion of PtdIns(4,5)P(2), voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes. METHODS AND RESULTS: Expression and correct targeting of ECFP-PH-PLCδ1(,) EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P(2) in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K(+) (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P(2) was identified. CONCLUSIONS: Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.