The GEM-handle as convenient labeling strategy for bimodal single-domain antibody-based tracers carrying 99mTc and a near-infrared fluorescent dye for intra-operative decision-making.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99mTc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99mTc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

Safety assessment of fluorescently labeled anti-EGFR Nanobodies in healthy dogs.

Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient's quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer. Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration. Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies. Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFR-overexpressing spontaneous tumors.

Local and Nonlocal Transport Spectroscopy in Planar Josephson Junctions.

We report simultaneously acquired local and nonlocal transport spectroscopy in a phase-biased planar Josephson junction based on an epitaxial InAs-Al hybrid two-dimensional heterostructure. Quantum point contacts at the junction ends allow measurement of the 2×2 matrix of local and nonlocal tunneling conductances as a function of magnetic field along the junction, phase difference across the junction, and carrier density. A closing and reopening of a gap was observed in both the local and nonlocal tunneling spectra as a function of magnetic field. For particular tunings of junction density, gap reopenings were accompanied by zero-bias conductance peaks (ZBCPs) in local conductances. End-to-end correlation of gap reopening was strong, while correlation of local ZBCPs was weak. A model of the device, with disorder treated phenomenologically, shows comparable conductance matrix behavior associated with a topological phase transition. Phase dependence helps distinguish possible origins of the ZBCPs.

Improved Multimodal Tumor Necrosis Imaging with IRDye800CW-DOTA Conjugated to an Albumin-Binding Domain.

PURPOSE: To assess our improved NACA for the detection of tumor necrosis. METHODS: We increased the blood circulation time of our NACA by adding an albumin-binding domain to the molecular structure. We tested the necrosis avidity on dead or alive cultured cells and performed SPECT and fluorescence imaging of both spontaneous and treatment-induced necrosis in murine breast cancer models. We simultaneously recorded [18F]FDG-PET and bioluminescence images for complementary detection of tumor viability. RESULTS: We generated two albumin-binding IRDye800CW derivatives which were labeled with indium-111 with high radiochemical purity. Surprisingly, both albumin-binding NACAs had >10x higher in vitro binding towards dead cells. We selected [111In]3 for in vivo experiments which showed higher dead cell binding in vitro and in vivo stability. The doxorubicin-treated tumors showed increased [111In]3-uptake (1.74 ± 0.08%ID/g after saline treatment, 2.25 ± 0.16%ID/g after doxorubicin treatment, p = 0.044) and decreased [18F]FDG-uptake (3.02 ± 0.51%ID/g after saline treatment, 1.79 ± 0.11%ID/g after doxorubicin treatment, p = 0.040), indicating therapy efficacy. Moreover, we detected increased [111In]3-uptake and tumor necrosis in more rapidly growing EMT6 tumors. CONCLUSIONS: Our albumin-binding NACA based on IRDye800CW facilitates tumor-necrosis imaging for assessment of therapy efficacy and aggressiveness in solid tumors using both fluorescence and SPECT imaging.

Necrosis binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate: a consequence from cyanine-labeling of small molecules.

BACKGROUND: There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. RESULTS: Binding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. CONCLUSION: This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.

Evaluation of Ac-Lys0(IRDye800CW)Tyr3-octreotate as a novel tracer for SSTR2-targeted molecular fluorescence guided surgery in meningioma.

PURPOSE: Meningioma recurrence rates can be reduced by optimizing surgical resection with the use of intraoperative molecular fluorescence guided surgery (MFGS). We evaluated the potential of the fluorescent tracer 800CW-TATE for MFGS using in vitro and in vivo models. It targets somatostatin receptor subtype 2 (SSTR2), which is overexpressed in all meningiomas. METHODS: Binding affinity of 800CW-TATE was evaluated using [177Lu] Lu-DOTA-Tyr3-octreotate displacement assays. Tumor uptake was determined by injecting 800CW-TATE in (SSTR2-positive) NCI-H69 or (SSTR2-negative) CH-157MN xenograft bearing mice and FMT2500 imaging. SSTR2-specific binding was measured by comparing tumor uptake in NCI-H69 and CH-157MN xenografts, blocking experiments and non-targeted IRDye800CW-carboxylate binding. Tracer distribution was analyzed ex vivo, and the tumor-to-background ratio (TBR) was calculated. SSTR2 expression was determined by immunohistochemistry (IHC). Lastly, 800CW-TATE was incubated on frozen and fresh meningioma specimens and analyzed by microscopy. RESULTS: 800CW-TATE binding affinity assays showed an IC50 value of 72 nM. NCI-H69 xenografted mice showed a TBR of 21.1. 800CW-TATE detection was reduced after co-administration of non-fluorescent DOTA-Tyr3-octreotate or administration of IRDye800CW. CH-157MN had no tumor specific tracer staining due to absence of SSTR2 expression, thereby serving as a negative control. The tracer bound specifically to SSTR2-positive meningioma tissues representing all WHO grades. CONCLUSION: 800CW-TATE demonstrated sufficient binding affinity, specific SSTR2-mediated tumor uptake, a favorable biodistribution, and high TBR. These features make this tracer very promising for use in MFGS and could potentially aid in safer and a more complete meningioma resection, especially in high-grade meningiomas or those at complex anatomical localizations.

Andreev Modes from Phase Winding in a Full-Shell Nanowire-Based Transmon.

We investigate transmon qubits made from semiconductor nanowires with a fully surrounding superconducting shell. In the regime of reentrant superconductivity associated with the destructive Little-Parks effect, numerous coherent transitions are observed in the first reentrant lobe, where the shell carries 2π winding of superconducting phase, and are absent in the zeroth lobe. As junction density was increased by gate voltage, qubit coherence was suppressed then lost in the first lobe. These observations and numerical simulations highlight the role of winding-induced Andreev states in the junction.

In Vivo Evaluation of Gallium-68-Labeled IRDye800CW as a Necrosis Avid Contrast Agent in Solid Tumors.

Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [68Ga]Ga-DOTA-PEG4-IRDye800CW ([68Ga]Ga-1) with a radiochemical purity of >96% (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [68Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [68Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [68Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.

Quantum Dot Parity Effects in Trivial and Topological Josephson Junctions.

An odd-occupied quantum dot in a Josephson junction can flip transmission phase, creating a π junction. When the junction couples topological superconductors, no phase flip is expected. We investigate this and related effects in a full-shell hybrid interferometer, using gate voltage to control dot-junction parity and axial magnetic flux to control the transition from trivial to topological superconductivity. Enhanced zero-bias conductance and critical current for odd parity in the topological phase reflects hybridization of the confined spin with zero-energy modes in the leads.

Parity-Protected Superconductor-Semiconductor Qubit.

Coherence of superconducting qubits can be improved by implementing designs that protect the parity of Cooper pairs on superconducting islands. Here, we introduce a parity-protected qubit based on voltage-controlled semiconductor nanowire Josephson junctions, taking advantage of the higher harmonic content in the energy-phase relation of few-channel junctions. A symmetric interferometer formed by two such junctions, gate-tuned into balance and frustrated by a half-quantum of applied flux, yields a cos(2φ) Josephson element, reflecting coherent transport of pairs of Cooper pairs. We demonstrate that relaxation of the qubit can be suppressed tenfold by tuning into the protected regime.

Suppressed Charge Dispersion via Resonant Tunneling in a Single-Channel Transmon.

We demonstrate strong suppression of charge dispersion in a semiconductor-based transmon qubit across Josephson resonances associated with a quantum dot in the junction. On resonance, dispersion is drastically reduced compared to conventional transmons with corresponding Josephson and charging energies. We develop a model of qubit dispersion for a single-channel resonance, which is in quantitative agreement with experimental data.

In Vivo Evaluation of Indium-111-Labeled 800CW as a Necrosis-Avid Contrast Agent.

PURPOSE: Current clinical measurements for tumor treatment efficiency rely often on changes in tumor volume measured as shrinkage by CT or MRI, which become apparent after multiple lines of treatment and pose a physical and psychological burden on the patient. Detection of therapy-induced cell death in the tumor can be a fast measure for treatment efficiency. However, there are no reliable clinical tools for detection of tumor necrosis. Previously, we studied the necrosis avidity of cyanine-based fluorescent dyes, which suffered long circulation times before tumor necrosis could be imaged due to low hydrophilicity. We now present the application of radiolabeled 800CW, a commercially available cyanine with high hydrophilicity, to image tumor necrosis in a mouse model. PROCEDURES: We conjugated 800CW to DOTA via a PEG linker, for labeling with single-photon emission-computed tomography isotope indium-111, yielding [111In]In-DOTA-PEG4-800CW. We then investigated specific [111In]In-DOTA-PEG4-800CW uptake by dead cells in vitro, using both fluorescence and radioactivity as detection modalities. Finally, we investigated [111In]In-DOTA-PEG4-800CW uptake into necrotic tumor regions of a 4T1 breast tumor model in mice. RESULTS: We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific [111In]In-DOTA-PEG4-800CW uptake by dead cells, using both fluorescence and radioactivity. Albeit with a tumor uptake of only 0.37%ID/g at 6 h post injection, we were able to image tumor necrosis with a tumor to background ratio of 7:4. Fluorescence and radioactivity in cryosections from the dissected tumors were colocalized with tumor necrosis, confirmed by TUNEL staining. CONCLUSIONS: [111In]In-DOTA-PEG4-800CW can be used to image tumor necrosis in vitro and in vivo. Further research will elucidate the application of [111In]In-DOTA-PEG4-800CW or other radiolabeled hydrophilic cyanines for the detection of necrosis caused by chemotherapy or other anti-cancer therapies. This can provide valuable prognostic information in treatment of solid tumors.

Coherent transport through a Majorana island in an Aharonov-Bohm interferometer.

Majorana zero modes are leading candidates for topological quantum computation due to non-local qubit encoding and non-abelian exchange statistics. Spatially separated Majorana modes are expected to allow phase-coherent single-electron transport through a topological superconducting island via a mechanism referred to as teleportation. Here we experimentally investigate such a system by patterning an elongated epitaxial InAs-Al island embedded in an Aharonov-Bohm interferometer. With increasing parallel magnetic field, a discrete sub-gap state in the island is lowered to zero energy yielding persistent 1e-periodic Coulomb blockade conductance peaks (e is the elementary charge). In this condition, conductance through the interferometer is observed to oscillate in a perpendicular magnetic field with a flux period of h/e (h is Planck's constant), indicating coherent transport of single electrons through the islands, a signature of electron teleportation via Majorana modes.

Relating Andreev Bound States and Supercurrents in Hybrid Josephson Junctions.

We demonstrate concomitant measurement of phase-dependent critical current and Andreev bound state spectrum in a highly transmissive InAs Josephson junction embedded in a dc superconducting quantum interference device (SQUID). Tunneling spectroscopy reveals Andreev bound states with near unity transmission probability. A nonsinusoidal current-phase relation is derived from the Andreev spectrum, showing excellent agreement with the one extracted from the SQUID critical current. Both measurements are reconciled within a short junction model where multiple Andreev bound states, with various transmission probabilities, contribute to the entire supercurrent flowing in the junction.

Flux-induced topological superconductivity in full-shell nanowires.

Hybrid semiconductor-superconductor nanowires have emerged as a promising platform for realizing topological superconductivity (TSC). Here, we present a route to TSC using magnetic flux applied to a full superconducting shell surrounding a semiconducting nanowire core. Tunneling into the core reveals a hard induced gap near zero applied flux, corresponding to zero phase winding, and a gapped region with a discrete zero-energy state around one applied flux quantum, corresponding to 2π phase winding. Theoretical analysis indicates that the winding of the superconducting phase can induce a transition to a topological phase supporting Majorana zero modes. Measured Coulomb blockade peak spacing around one flux quantum shows a length dependence that is consistent with the existence of Majorana modes at the ends of the nanowire.

Controlled dc Monitoring of a Superconducting Qubit.

Creating a transmon qubit using semiconductor-superconductor hybrid materials not only provides electrostatic control of the qubit frequency, it also allows parts of the circuit to be electrically connected and disconnected in situ by operating a semiconductor region of the device as a field-effect transistor. Here, we exploit this feature to compare in the same device characteristics of the qubit, such as frequency and relaxation time, with related transport properties such as critical supercurrent and normal-state resistance. Gradually opening the field-effect transistor to the monitoring circuit allows the influence of weak-to-strong dc monitoring of a "live" qubit to be measured. A model of this influence yields excellent agreement with experiment, demonstrating a relaxation rate mediated by a gate-controlled environmental coupling.

Conductance-Matrix Symmetries of a Three-Terminal Hybrid Device.

We present conductance-matrix measurements of a three-terminal superconductor-semiconductor hybrid device consisting of two normal leads and one superconducting lead. Using a symmetry decomposition of the conductance, we find that antisymmetric components of pairs of local and nonlocal conductances qualitatively match at energies below the superconducting gap, and we compare this finding with symmetry relations based on a noninteracting scattering matrix approach. Further, the local charge character of Andreev bound states is extracted from the symmetry-decomposed conductance data and is found to be similar at both ends of the device and tunable with gate voltage. Finally, we measure the conductance matrix as a function of magnetic field and identify correlated splittings in low-energy features, demonstrating how conductance-matrix measurements can complement traditional single-probe measurements in the search for Majorana zero modes.

Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth.

Cancer cells sustain growth by altering their metabolism to accelerated aerobic glycolysis accompanied by increased glucose demand and employ glutamine as additional nutrient source. This metabolic adaptation induces upregulation of glucose transporters GLUT-1 and -3, and simultaneous targeting of both transporters and of glutamine metabolism may offer a promising approach to inhibit cancer cell growth. We describe the discovery of the very potent glucose uptake inhibitor Glutor, which targets glucose transporters GLUT-1, -2, and -3, attenuates glycolytic flux and potently and selectively suppresses growth of a variety of cancer cell lines. Co-treatment of colon cancer cells with Glutor and glutaminase inhibitor CB-839 very potently and synergistically inhibits cancer cell growth. Such a dual inhibition promises to be particularly effective because it targets the metabolic plasticity as well as metabolic rescue mechanisms in cancer cells.

Is the use of Cannabis associated with periodontitis? A systematic review and meta-analysis.

Recent studies have shown that there is also biological plausibility for a possible relationship between periodontal disease and Cannabis use, thus the aim of this study was to investigate whether the use of Cannabis is associated with periodontitis. Electronic searches were performed in PubMed, Scopus, ISI-Web of Science, BVS-Virtual health library and Scielo without restrictions. Search strategy was performed using relevant keywords considering the structure of each database. Longitudinal and cross-sectional studies that investigated the association between the use of Cannabis and periodontal disease were included. Meta-analyses and sensitivity analysis were conducted. A total of 143 records were found in the initial searches and five articles were included in the systematic review, being four studies included in the meta-analysis. Overall, 13 491 individuals were included, of which 49.5% were males. Three of included studies investigated the relationship between cannabis and periodontal disease in adults and the other two studies were performed in adolescents. A positive association was observed between the use of cannabis and periodontitis (PR 1.12 CI 95% [1.06-1.19]) with 19.0% of heterogeneity. The analysis of sensibility showed that none study influenced the results enough to change the pooled estimate. Regarding to the quality assessment, all studies presented high quality. The results of systematic review and meta-analyses demonstrate that the use of Cannabis is associated with a higher prevalence of periodontitis.