Imaging Studies In Mice Research Articles

Characterization of Luciferase from Photorhabdus kayaii and its Application for In vivo Imaging Studies in Mice.

Bioluminescence, or the production of light by luciferases, is the basis of a well-known reporter technology. A quick way to study the efficacy of antimicrobial drugs and vaccines is in vivo bioluminescence imaging (BLI). Photorhabdus spp. represent the only terrestrial group of bioluminescent bacteria. The luciferase obtained from Photorhabdus luminescence has been widely used in BLI studies. However, little information is available about the functions of luciferases obtained from other members of this genera. This study aimed to evaluate the applicability of the luciferase obtained from Photorhabdus kayaii for BLI studies. P. kayaii starE, an Iranian isolate of P. kayaii, was cultivated on NBTA agar plates. The resulting colonies were cultured on McConkey agar to determine the bacterial phase. Bioluminescence emission was measured using a multimode reader. The luciferase genes of this bacterium were sequenced following the PCR amplification, and the corresponding amino acid sequences were determined. The luciferase tertiary structure was then obtained from the TACOS web server and compared to that of P. luminescence in CE software. The lux operon encoding the luciferase (luxA and luxB genes) and substrate synthesis complex was cloned and expressed in Escherichia coli BL21 (DE3) using the pBBR1MCS2_START vector. The luminescence emission during the growth was examined. Moreover, the effects of pH and sodium deoxycholate (bile salt) on bioluminescence emission were investigated. Appropriate conditions for the use of bioluminescent E. coli for BLI studies in mice were demonstrated in terms of cell numbers and injection routes. The bacterium was luminescent and in phase I. Its luciferase monomers (α and β) shared 100% amino acid homology with P. kayaii M-HU2 and more than 92% with P. luminescence. Tertiary structures of the luciferase monomers were 93%- 95% identical to those of P. luminescence. The lux operon was expressed in E. coli, and the maximum bioluminescence signal was observed during the decelerating phase of growth. The bioluminescence at different pH values correlated with the cell survival. The luminescence was emitted by cells exposed to the bile salt. A strong bioluminescent signal was emitted from mice after subcutaneous injection of bioluminescent E. coli at 107 CFU. However, no signals were emitted from mice that were administered the same cell number via intraperitoneal injection. A 2.5-fold increase in the cell number resulted in bioluminescence detection in the abdomen of mice after intraperitoneal injection and a 3.22-fold increase in signal intensity after subcutaneous injection. These results demonstrated the usefulness of P. kayaii luciferase for BLI studies.

Evaluation of a Positron Emission Tomography Tracer Targeting Colony-Stimulating Factor 1 Receptor for Detecting Pulmonary Inflammation.

Colony-stimulating factor 1 receptor (CSF1R) is a type III receptor tyrosine kinase that is crucial for immune cell activation, survival, proliferation, and differentiation. Its expression significantly increases in macrophages during inflammation, playing a crucial role in regulating inflammation resolution and termination. Consequently, CSF1R has emerged as a critical target for both therapeutic intervention and imaging of inflammatory diseases. Herein, we have developed a radiotracer, 1-[4-((7-(dimethylamino)quinazolin-4-yl)oxy)phenyl]-3-(4-[18F]fluorophenyl)urea ([18F]17), for in vivo positron emission tomography (PET) imaging of CSF1R. Compound 17 exhibits a comparable inhibitory potency against CSF1R as the well-known CSF1R inhibitor PLX647. The radiosynthesis of [18F]17 was successfully performed by radiofluorination of aryltrimethyltin precursor with a yield of approximately 12% at the end of synthesis, maintaining a purity exceeding 98%. In vivo stability and biodistribution studies demonstrate that [18F]17 remains >90% intact at 30 min postinjection, with no defluorination observed even at 60 min postinjection. The PET/CT imaging study in lipopolysaccharide-induced pulmonary inflammation mice indicates that [18F]17 offers a more sensitive characterization of pulmonary inflammation compared to traditional [18F]FDG. Notably, [18F]17 shows a higher discrepancy in uptake ratio between mice with pulmonary inflammation and the sham group. Furthermore, the variations in [18F]17 uptake ratio observed on day 7 and day 14 correspond to lung density changes observed in CT imaging. Moreover, the expression levels of CSF1R on day 7 and day 14 follow a trend similar to the uptake pattern of [18F]17, indicating its potential for accurately characterizing CSF1R expression levels and effectively monitoring the pulmonary inflammation progression. These results strongly suggest that [18F]17 has promising prospects as a CSF1R PET tracer, providing diagnostic opportunities for pulmonary inflammatory diseases.

Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Reliable differentiation of necrosis and active metabolically contours of glioblastoma multiforme using susceptibility-based imaging

PurposeGadolinium-enhancing necrosis in glioblastoma multiforme (GBM), as an occasionally occurring false positive in contrast enhancement (CE) imaging, leads to trouble for segmentation of GBM and treatment. Therefore, the investigation of complementary detection way to identify the metabolically active volume of the tumor with high reliability is very worth to be addressed. Here, we reported on a case of GBM with gadolinium-enhancing necrosis in an experimental CE imaging study in mice and evaluated the discrimination of the necrosis and metabolically active parts of the GBM using conventional and state-of-the-art susceptibility-based MRI. MethodsIn this study, following 5-aminolevulinic acid (ALA) and iron supplements (FAC, 6 h after ALA, intra-tumoral injection) to animal, T2*-W imaging and quantitative susceptibility mapping (QSM) were performed, and compared with CE imaging. ResultsThe signal intensity (SI) of the active and necrosis areas of the case in the CE image demonstrated no significant difference while the SI on the T2*-W images and susceptibility value in QSM changed 24 and 150%, respectively. ConclusionThe preclinical case report provides valuable insights into the potential of susceptibility-based MRI using ALA + FAC to apply as a robust discriminator between necrotic and viable tumors.

Contrast-Free Super-Resolution Power Doppler (CS-PD) Based on Deep Neural Networks.

Super-resolution ultrasound microvessel imaging based on ultrasound localization microscopy (ULM) is an emerging imaging modality that is capable of resolving micrometer-scaled vessels deep into tissue. In practice, ULM is limited by the need for contrast injection, long data acquisition, and computationally expensive postprocessing times. In this study, we present a contrast-free super-resolution power Doppler (CS-PD) technique that uses deep networks to achieve super-resolution with short data acquisition. The training dataset is comprised of spatiotemporal ultrafast ultrasound signals acquired from in vivo mouse brains, while the testing dataset includes in vivo mouse brain, chicken embryo chorioallantoic membrane (CAM), and healthy human subjects. The in vivo mouse imaging studies demonstrate that CS-PD could achieve an approximate twofold improvement in spatial resolution when compared with conventional power Doppler. In addition, the microvascular images generated by CS-PD showed good agreement with the corresponding ULM images as indicated by a structural similarity index of 0.7837 and a peak signal-to-noise ratio (PSNR) of 25.52. Moreover, CS-PD was able to preserve the temporal profile of the blood flow (e.g., pulsatility) that is similar to conventional power Doppler. Finally, the generalizability of CS-PD was demonstrated on testing data of different tissues using different imaging settings. The fast inference time of the proposed deep neural network also allows CS-PD to be implemented for real-time imaging. These features of CS-PD offer a practical, fast, and robust microvascular imaging solution for many preclinical and clinical applications of Doppler ultrasound.

Shape Anisotropy-Governed High-Performance Nanomagnetosol for In Vivo Magnetic Particle Imaging of Lungs.

Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease 2019 (COVID-19) has shown extensive lung manifestations in vulnerable individuals, putting lung imaging and monitoring at the forefront of early detection and treatment. Magnetic particle imaging (MPI) is an imaging modality, which can bring excellent contrast, sensitivity, and signal-to-noise ratios to lung imaging for the development of new theranostic approaches for respiratory diseases. Advances in MPI tracers would offer additional improvements and increase the potential for clinical translation of MPI. Here, a high-performance nanotracer based on shape anisotropy of magnetic nanoparticles is developed and its use in MPI imaging of the lung is demonstrated. Shape anisotropy proves to be a critical parameter for increasing signal intensity and resolution and exceeding those properties of conventional spherical nanoparticles. The 0D nanoparticles exhibit a 2-fold increase, while the 1D nanorods have a > 5-fold increase in signal intensity when compared to VivoTrax. Newly designed 1D nanorods displayed high signal intensities and excellent resolution in lung images. A spatiotemporal lung imaging study in mice revealed that this tracer offers new opportunities for monitoring disease and guiding intervention.

Automated Synthesis and Preclinical Evaluation of Optimized Integrin α6-Targeted Positron Emission Tomography Imaging of Pancreatic Cancer.

Integrin α6 has been considered a promising biomarker, is overexpressed in many tumors, and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we identified a novel high-affinity integrin α6-targeted peptide named RD2 (Arg-Trp-Tyr-Asp-PEG4)2-Lys-Lys and developed a 18F-radiolabeled peptide tracer ([18F]-AlF-NOTA-RD2) and evaluated its potential application in positron emission tomography (PET) imaging of pancreatic cancer. [18F]-AlF-NOTA-RD2 was produced using GMP (Good Manufacturing Practice of Medical Products)-compliant automatic radiosynthesis on a single GE FASTLab2 cassette-type synthesis module. The stability of [18F]-AlF-NOTA-RD2 was analyzed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). The cell uptake assay of the tracer was assessed using PANC-1 cells. In addition, small-animal PET imaging and biodistribution studies of [18F]-AlF-NOTA-RD2 were performed in pancreatic cancer subcutaneous tumor-bearing mice. The PET tracer [18F]-AlF-NOTA-RD2 was obtained with a radiochemical yield of 23.7 ± 4.7%, radiochemical purity of >99%, and molar activity of 165.7 ± 59.1 GBq/μmol. [18F]-AlF-NOTA-RD2 exhibited good in vitro stability in PBS and FBS. LogP octanol water value for the tracer was -2.28 ± 0.05 (n = 3). The binding affinity of RD2 to the integrin α6 protein (Kd = 0.13 ± 3.65 μM, n = 3) was significantly higher than that of the RWY (CRWYDENAC) (Kd = 6.97 ± 1.44 μM, n = 3). Small-animal PET imaging and biodistribution also revealed that [18F]-AlF-NOTA-RD2 displayed rapid and good tumor uptake and lower liver background uptake in PANC-1 tumor-bearing mice. [18F]-AlF-NOTA-RD2 showed significant radioactivity accumulation in tumors and was successfully blocked by NOTA-RD2. Compared with [18F]-FDG, [18F]-AlF-NOTA-RD2 PET imaging and biodistribution studies in PANC-1 xenograft tumor-bearing mice confirmed a good tumor-to-muscle ratio (8.69 ± 2.03 vs 1.41 ± 0.23, respectively) at 0.5 h and (2.99 ± 3.02 vs 1.43 ± 0.17, respectively) at 1 h post injection. Autoradiography of human pancreatic cancer tumor tissues further confirmed high accumulation of [18F]-AlF-NOTA-RD2. In summary, we developed an optimized integrin α6-targeted imaging tracer and obtained high radioactivity products with a cassette-type synthesis module; moreover, the tracer exhibited good binding affinity with integrin α6 and good target specificity for PANC-1 cells in xenograft pancreatic tumor-bearing mice, demonstrating its promising application as a noninvasive PET radiotracer of integrin α6 expression in pancreatic cancer.

Synthesis of novel DOTA-/AAZTA-based bifunctional chelators: Solution thermodynamics, peptidomimetic conjugation, and radiopharmaceutical evaluation

Bifunctional chelators (BFCs), which link metallic radionuclide and a targeting vector, are some of the most crucial components of metallic radionuclide-based radiopharmaceuticals for positron-emission computed tomography (PET) imaging. In this study, we designed and synthesized two versatile BFCs, p-NCS-Ph-DE4TA and p-NCS-Ph-AAZ4TA, and we conjugated them with a prostate-specific membrane antigen (PSMA) inhibitor. These two chelators showed high affinity for Ga (III) according to a study of the thermodynamics and kinetics and DFT calculations. The labeled PSMA targeted probes, [68Ga]Ga-p-NCS-Ph-DE4TA-PSMA and [68Ga]Ga-p-NCS-Ph-AAZ4TA-PSMA, maintained excellent stability in vitro, and they exhibited high specific activity when binding to PSMA. A PET/CT imaging study in mice bearing SMMC-7721 hepatocellular carcinoma xenografts demonstrated clear visualization of tumors with a high tumor uptake and low background level, indicating the excellent performance in vivo and specific activity when targeting hepatocellular carcinomas. In summary, p-NCS-Ph-DE4TA and p-NCS-Ph-AAZ4TA are leading developmental candidates for PET imaging for tumor diagnosis.

Development of [177Lu]Lu-LNC1003 for radioligand therapy of prostate cancer with a moderate level of PSMA expression.

Evans blue as an albumin binder has been widely used to improve pharmacokinetics and enhance tumor uptake of radioligands, including prostate-specific membrane antigen (PSMA) targeting agents. The goal of this study is to develop an optimal Evans blue-modified radiotherapeutic agent that could maximize the absolute tumor uptake and tumor absorbed dose thus the therapeutic efficacy to allow treatment of tumors even with moderate level of PSMA expression. [177Lu]Lu-LNC1003 was synthesized based on PSMA-targeting agent and Evans blue. Binding affinity and PSMA targeting specificity were verified through cell uptake and competition binding assay in 22Rv1tumor model that has moderate level of PSMA expression. SPECT/CT imaging and biodistribution studies in 22Rv1 tumor-bearing mice were performed to evaluate the preclinical pharmacokinetics. Radioligand therapy studies were conducted to systematically assess the therapeutic effect of [177Lu]Lu-LNC1003. LNC1003 showed high binding affinity (IC50 = 10.77nM) to PSMA in vitro, which was comparable with that of PSMA-617 (IC50 = 27.49nM) and EB-PSMA-617 (IC50 = 7.91nM). SPECT imaging of [177Lu]Lu-LNC1003 demonstrated significantly improved tumor uptake and retention as compared with [177Lu]Lu-EB-PSMA and [177Lu]Lu-PSMA-617, making it suitable for prostate cancer therapy. Biodistribution studies further confirmed the remarkably higher tumor uptake of [177Lu]Lu-LNC1003 (138.87 ± 26.53%ID/g) over [177Lu]Lu-EB-PSMA-617 (29.89 ± 8.86%ID/g) and [177Lu]Lu-PSMA-617 (4.28 ± 0.25%ID/g) at 24h post-injection. Targeted radioligand therapy results showed noteworthy inhibition of 22Rv1 tumor growth after administration of a single dose of 18.5MBq [177Lu]Lu-LNC1003. There was no obvious antitumor effect after [177Lu]Lu-PSMA-617 treatment under the same condition. In this study, [177Lu]Lu-LNC1003 was successfully synthesized with high radiochemical purity and stability. High binding affinity and PSMA targeting specificity were identified in vitro and in vivo. With greatly enhanced tumor uptake and retention, [177Lu]Lu-LNC1003 has the potential to improve therapeutic efficacy using significantly lower dosages and less cycles of 177Lu that promises clinical translation to treat prostate cancer with various levels of PSMA expression.

Engineering a modular 44Ti/44Sc generator: eluate evaluation in preclinical models and estimation of human radiation dosimetry

Background44Sc/47Sc is an attractive theranostic pair for targeted in vivo positron emission tomographic (PET) imaging and beta-particle treatment of cancer. The 44Ti/44Sc generator allows daily onsite production of this diagnostic isotope, which may provide an attractive alternative for PET facilities that lack in-house irradiation capabilities. Early animal and patient studies have demonstrated the utility of 44Sc. In our current study, we built and evaluated a novel clinical-scale 44Ti/44Sc generator, explored the pharmacokinetic profiles of 44ScCl3, [44Sc]-citrate and [44Sc]-NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid) in naïve mice, and estimated the radiation burden of 44ScCl3 in humans.Methods44Ti/44Sc (101.2 MBq) in 6 M HCl solution was utilized to assemble a modular ZR resin containing generator. After assembly, 44Sc was eluted with 0.05 M HCl for further PET imaging and biodistribution studies in female Swiss Webster mice. Based on the biodistribution data, absorbed doses of 44/47ScCl3 in human adults were calculated for 18 organs and tissues using the IDAC-Dose software.Results44Ti in 6 M HCl was loaded onto the organic resin generator with a yield of 99.97%. After loading and initial stabilization, 44ScCl3 was eluted with 0.05 M HCl in typical yields of 82.9 ± 5.3% (N = 16), which was normalized to the estimated generator capacity. Estimated generator capacity was computed based on elution time interval and the total amount of 44Ti loaded on the generator. Run in forward and reverse directions, the 44Sc/44Ti ratio from a primary column was significantly improved from 1038 ± 440 to 3557 ± 680 (Bq/Bq) when a secondary, replaceable, ZR resin cartridge was employed at the flow outlet. In vivo imaging and ex vivo distribution studies of the reversible modular generator for 44ScCl3, [44Sc]-citrate and [44Sc]-NODAGA show that free 44Sc remained in the circulation significantly longer than the chelated 44Sc. The dose estimation of 44ScCl3 reveals that the radiation burden is 0.146 mSv/MBq for a 70 kg adult male and 0.179 mSv/MBq for a 57 kg adult female. Liver, spleen and heart wall will receive the highest absorbed dose: 0.524, 0.502, and 0.303 mGy/MBq, respectively, for the adult male.ConclusionsA clinical-scale 44Ti/44Sc generator system with a modular design was developed to supply 44ScCl3 in 0.05 M HCl, which is suitable for further radiolabeling and in vivo use. Our data demonstrated that free 44ScCl3 remained in the circulation for extended periods, which resulted in approximately 10 times greater radiation burden than stably chelated 44Sc. Stable 44Sc/47Sc-complexation will be more favorable for in vivo use and for clinical utility.

Oxytocin mediated excitation of hypoglossal motoneurons: implications for treating obstructive sleep apnea.

Clinical studies have shown that oxytocin administered intranasally (IN) decreased the incidence and duration of obstructive events in patients with obstructive sleep apnea (OSA). Although the mechanisms by which oxytocin promotes these beneficial effects are unknown, one possible target of oxytocin could be the excitation of tongue-projecting hypoglossal motoneurons in the medulla, that exert central control of upper airway patency. This study tested the hypothesis that IN oxytocin enhances tongue muscle activity via the excitation of hypoglossal motoneurons projecting to tongue protrudor muscles (PMNs). To test this hypothesis we performed in vivo and in vitro electrophysiological studies in C57BL6/J mice as well as fluorescent imaging studies in transgenic mice in which neurons that express oxytocin receptors co-express fluorescent protein. IN oxytocin significantly increased the amplitude of inspiratory-related tongue muscle activity. This effect was abolished by severing the medial branch of hypoglossal nerve that innervates PMNs of the tongue. Oxytocin receptor-positive neurons were more prevalent in the population of PMNs than in retractor-projecting hypoglossal motoneurons (RMNs). Oxytocin administration increased action potential firing in PMNs, but had no significant effect on firing activity in RMNs. In conclusion, IN oxytocin stimulates respiratory-relating tongue muscle activity likely acting on central hypoglossal motoneurons that provide tongue protrusion and upper airway opening. This mechanism may play a role in oxytocin-induced reductions in upper airway obstructions in patients with OSA.

Barium sulfate and pigment admixture for photoacoustic and x-ray contrast imaging of the gut.

X-ray imaging is frequently used for gastrointestinal imaging. Photoacoustic imaging (PAI) of the gastrointestinal tract is an emerging approach that has been demonstrated for preclinical imaging of small animals. A contrast agent active in both modalities could be useful for imaging applications. We aimed to develop a dual-modality contrast agent comprising an admixture of barium sulfate with pigments that absorb light in the second near-infrared region (NIR-II), for preclinical imaging with both x-ray and PAI modalities. Eleven different NIR-II dyes were evaluated after admixture with a 40% w/v barium sulfate mixture. The resulting NIR-II absorption in the soluble fraction and in the total mixture was characterized. Proof-of-principle imaging studies in mice were carried out. Pigments that produced more uniform suspensions were assessed further for photoacoustic contrast signal at a wavelength of 1064nm that corresponds to the output of the Nd:YAG laser used. Phantom imaging studies demonstrated that the pigment-barium sulfate mixture generated imaging contrast in both x-ray and PAI modalities. The optimal pigment selected for further study was a cyanine tetrafluoroborate salt. Ex-vivo and whole-body mouse imaging demonstrated that photoacoustic and x-ray contrast signals co-localized in the intestines for both imaging modalities. These data demonstrate that commercially-available NIR-II pigments can simply be admixed with barium sulfate to generate a dual-modality contrast agent appropriate for small animal gastrointestinal imaging.

Enhanced apoptosis and mitochondrial cell death by paclitaxel-loaded TPP-TPGS1000-functionalized nanoemulsion.

Background: The present research was designed to develop a nanoemulsion (NE) of triphenylphosphine-D-α-tocopheryl-polyethylene glycol succinate (TPP-TPGS1000) and paclitaxel (PTX) to effectively deliver PTX to improve breast cancer therapy. Materials & methods: A quality-by-design approach was applied for optimization and in vitro and in vivo characterization were performed. Results: The TPP-TPGS1000-PTX-NE enhanced cellular uptake, mitochondrial membrane depolarization and G2M cell cycle arrest compared with free-PTX treatment. In addition, pharmacokinetics, biodistributionandin vivo live imaging studies in tumor-bearing mice showed that TPP-TPGS1000-PTX-NE had superior performance compared with free-PTX treatment. Histological and survival investigations ascertained the nontoxicity of thenanoformulation, suggesting new opportunities and potential to treat breast cancer. Conclusion: TPP-TPGS1000-PTX-NE improved the efficacy of breast cancer treatmentby enhancing its effectiveness and decreasing drugtoxicity.

Development and evaluation of 89Zr-trastuzumab for clinical applications.

Due to the suitable physical characteristics of 89Zr as a PET radionuclide and affinity of Trastuzumab monoclonal antibody against HER2, [89Zr]Zr-Trastuzumab was prepared and went through preclinical evaluations for ultimate human applications. 89Zr was produced by using 89Y(p,n)89Zr reaction at a 30 MeV cyclotron (radionuclide purity>99.9%, specific activity of 17 GBq/µg). p-SCN-Bn-Deferoxamine (DFO); was conjugated to trastuzumab, followed by labeling with 89Zr in oxalate form at optimized condition. Cell binding, internalization and, radioimmuno-activity assays were studied using HER2+ BT474 and HER2- CHO cell lines. Finally, the biodistribution of the radioimmunoconjugate was assessed in normal and HER2+ BT474 tumor-bearing mice using tissue counting and imaging at different intervals after injection. Also, a woman with HER2-positive metastatic breast cancer under treatment with Herceptin underwent both [89Zr]Zr-Trastuzumab and, [18F]FDG PET/CTs. 89Zr was produced with high radionuclidic and radiochemical purities (>99%) and [89Zr]Zr-DFO-Trastuzumab was prepared with radiochemical purity of >98% and specific activity of 9.85 GBq/µmol. The radioimmunoconjugate was stable both in PBS buffer and in human serum for at least 48 h. The radioimmunoactivity assay demonstrated about 70% of [89Zr]Zr-DFO-Trastuzumab is bound to the BT474 cells at the number of 250×106 cells. Cell binding studies showed that about 28% of radioimmunoconjugate is attached to BT474 cells after 90 min. Internalization studies showed that 50% of [89Zr]Zr-Trastuzumab is internalized to BT474 cells only in 6 h. The biodistribution study of the labeled compound in normal mice demonstrated the same pattern of the monoclonal antibodies which is entirely different from the biodistribution of free 89Zr. Biodistribution and imaging studies in tumor-bearing mice showed the significant uptake values of [89Zr]Zr-Trastuzumab in tumor sites. [89Zr]Zr-Trastuzumab PET/CT revealed metastatic lesions documented previously with [18F]FDG PET/CT scan in a woman with breast cancer who was under treatment with Herceptin. Although the [18F]FDG PET/CT scan had better quality images, the valuable and unique advantage of [89Zr]Zr-Trastuzumab PET/CT is delineating HER2+ metastasis, which is essential in diagnosis and HER2-based treatments. The prepared [89Zr]Zr-Trastuzumab has a high potential radio-pharmaceutical for immune-PET imaging of the patients with HER2+ tumors.

Synthesis and characterization of a macromolecular magnetic resonance imaging and delivery system with hyaluronic acid as a carrier

Using hyaluronic acid (HA) as macromolecular drug carriers, a glutathione-responsive imaging drug delivery system HA-SS-a-Gd-DOTA was formed by conjugating gadolinium chelates and cytarabine. This system exhibited T1-reflexivity (21.9 mmol−1 L s−1, 0.5 T) that was higher than that of gadoterate meglumine. In an acidic environment, in vitro drug release reached 63.4% in 24 h. Low cytotoxicity indicated that this system has good biocompatibility. In vivo mouse imaging studies showed that tumor signaling was significantly enhanced. About 58% of the signal enhancement was obtained 50 min after injection of the drug. The degradation of the hyaluronic acid macromolecular chains in vivo makes it an ideal tumor imaging diagnostic agent because it did not cause damage to important organs of the mice.

A hemicyanidin-based NIR fluorescent probe for detection of H2S and imaging study in cells and mice.

The selective detection of hydrogen sulfide in physiological and pathological processes has gained substantial attention in recent years. However, the real-time detection of hydrogen sulfide remains an elusive goal. In this work, a new type of hemicyanidin-based fluorescent "turn-on" probe NTR-HS (Ex = 680nm, Em = 760nm) was developed to detected H2S in a very short time (3min).Thefluorescence quantum yield is 0.15 and accompanied with a noticeable color change from violet to bluethatcan be used to detect H2S in the range 1.04 × 10-7-4 × 10-5Mwith a limit of detection of 1.04 x 10-7 M. TheNTR-HS probewas also used for imaging of endogenous hydrogen sulfide and mitochondrial localization in HCT116 and HeLa cells. The detection mechanism was studied through fluorescence, UV-Vis, NMR, and mass analysis. Notably, the probe was successfully used to imaging H2S in mice and locating hydrogen sulfide in the large intestine of mice.

Unravelling the Potential of Lutetium-177 Labeled Pertuzumab: Targeting HER 2 Receptors for Theranostic Applications

<h2>ABSTRACT</h2><h3>Purpose</h3> Pertuzumab, a humanized antibody against HER2 receptors has shown promising results in combination with trastuzumab and docetaxel for the neoadjuvant treatment of patients with HER2-positive breast cancer. The aim of the present study was to demonstrate the potential of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab for targeting HER2 positive tumors. Comparative studies with <b>[</b>¹⁷⁷Lu]-Lu-DTPA-trastuzumab and synergism in presence of cold trastuzumab was also envisaged. <h3>Methods & Materials</h3> Pertuzumab and trastuzumab were conjugated with p-NCS-CHX-A''-DTPA and radiolabeled with Lutetium-177. The stability of the formulations was assessed and <i>in-vitro</i> binding parameters (immunoreactivity, saturation and competitive binding) were examined in HER2 overexpressing and HER2 negative cell lines. SPECT imaging studies were performed in SCID mice bearing HER2 positive tumors. <h3>Results</h3> <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab and trastuzumab could be prepared with >95% radiochemical purity (% RCP). <i>In-vitro</i> binding studies revealed high affinity and specificity of the formulations towards HER2 receptors. Specific tumor uptake of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab in HER2 positive tumors was observed by SPECT imaging studies in tumor bearing mice, comparable to <b>[</b>¹⁷⁷Lu]-Lu-DTPA-trastuzumab. Significant retention of the formulations was observed in the tumor for up to 120 h. The studies also showed enhanced uptake of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab in the presence of cold trastuzumab in HER2 positive tumors. <h3>Conclusions</h3> The present study revealed significant uptake of both ¹⁷⁷Lu labeled pertuzumab and trastuzumab in HER2 positive tumors. Further studies are warranted to determine efficacy of these formulations against tumor growth for confirming their potential as effective radioimmunotheranostic agents. Increase in uptake of radiolabeled pertuzumab in presence cold trastuzumab provides an avenue for more effective radionuclide imaging and therapy of HER2 positive tumors.

PET Imaging of Active Invasive Fungal Infections with d-[5-11C]-Glutamine.

The increasing prevalence and severity of invasive fungal infections (IFIs), especially in immunocompromised populations, has amplified the need for rapid diagnosis of fungal pathogens. Radiotracers derived from d-amino acids (DAAs) show promise as bacterial-specific positron emission tomography (PET) imaging agents due to their preferential consumption by bacteria and largely nonutilization by hosts. Unlike mammals, fungi can utilize external DAAs including d-glutamine for their growth by rapidly upregulating DAA oxidases. Additionally, glutamine is essential for fungal nitrogen assimilation, survival, and virulence. We previously validated d-[5-11C]-glutamine (d-[5-11C]-Gln) as an efficient radiotracer targeting live bacterial soft-tissue infections. Here, we further expanded this investigation to evaluate its translational potential for PET imaging of IFIs in immunocompetent mouse models subcutaneously (SubQ) and intramuscularly (IM) infected with Candida albicans (C. albicans), using its l-isomer counterpart (l-[5-11C]-Gln) as a control. Comparative studies between pathogens showed significantly (p < 0.05) higher uptake in fungi (C. albicans and C. tropicalis) versus tested bacterial species for d-[5-11C]-Gln, suggesting that it could potentially serve as a more sensitive radiotracer for detection of fungal infections. Additionally, comparative PET imaging studies in immunocompetent infected mice demonstrated significantly higher infection-to-background ratios for d- versus l-[5-11C]-Gln in both SubQ (ratio = 1.97, p = 0.043) and IM (ratio = 1.97, p = 0.028) infections. Fungal infection imaging specificity was confirmed with no significant difference observed between localized inflammation sites versus untreated muscle background (heat-killed injection site/untreated muscle: ∼1.1). Taken together, this work demonstrates the translational potential of d-[5-11C]-Gln for noninvasive PET imaging of IFIs.

Abstract 3303: Pre-clinical characterization of the novel FAP targeting ligand PNT6555 for imaging and therapy of cancer

Abstract Background: Fibroblast Activation Protein-α (FAP) is a transmembrane glycoprotein highly expressed on activated fibroblasts. It is a constitutively active 170 kDa serine protease and a member of the dipeptide peptidase (DPP) family, sharing ~50% homology with DPPIV. FAP expression is only rarely expressed in normal adult tissues and is overexpressed in many epithelial cancers through upregulation on cancer-associated fibroblasts present in the stroma of various types of tumor. POINT BioPharma is developing PNT6555, which comprises a DOTA chelator linked to a FAP-targeting moiety, for imaging and therapeutic applications. Methods: PNT6555 and its radiometal chelates were evaluated for potency, selectivity, biodistribution and efficacy using biochemical and cellular assays as well as imaging, biodistribution and efficacy studies in tumor bearing mice. Results: PNT6555 and its gallium (natGa-PNT6555) and lutetium (natLu-PNT6555) chelates showed potent activity in FAP inhibition assays using human, mouse, and rat sources of FAP. PNT6555, natLu-PNT6555 and natGa-PNT6555 also showed significantly reduced potency when tested against PREP and DPPIV, two closely related homologous proteins. In vivo time-course biodistribution studies (by PET-imaging) with 68Ga-PNT6555 showed rapid clearance of 68Ga-PNT6555 from blood through the kidneys and urinary tract, with rising 68Ga-PNT6555 activity observed in the tumor through 60 minutes. At 60 minutes, the tumor was the only site of significant retained activity (&amp;gt;10 %ID/g). In vivo biodistribution studies (by SPECT imaging and direct organ assay) with 177Lu-PNT6555 showed rapid renal clearance into the bladder. After 24 hours, the tumor was the only tissue with significant activity retention. Direct organ assay showed little 177Lu-PNT6555 accumulation and retention in normal tissues with a high level of tumor retention observed out to 168h (&amp;gt;10 %ID/g). Therapeutic studies, using a single dose of 177Lu-PNT6555 or 225Ac-PNT6555, were completed in pre-clinical mouse models of cancer. In the HEK-mFAP model, significant dose responsive efficacy was observed in mice treated with either 177Lu-PNT6555 or 225Ac-PNT6555, with no apparent weight loss observed at all tested dose levels. Several mice experienced long-term survival &amp;gt;100 days at multiple of the tested dose levels. Conclusions: PNT6555, and its radiometal chelates, are potent and specific inhibitors of FAP. 68Ga/177Lu-PNT6555 showed rapid and prolonged uptake into FAP expressing tumors with limited uptake or retention observed in normal tissues. 177Lu/225Ac-PNT6555 showed compelling efficacy in pre-clinical tumor models that expressed FAP. Clinical studies with imaging and therapeutic chelates of PNT6555 are warranted. Citation Format: Robin M. Hallett, Sarah E. Poplawski, Mark H. Dornan, Shin Hye Ahn, Shuang Pan, Wu Wengen, Liu Yuxin, David G. Sanford, Valerie S. Hergott, Quang-De Nguyen, Anthony P. Belanger, Jack H. Lai, William Bachovchin, Joe A. McCann. Pre-clinical characterization of the novel FAP targeting ligand PNT6555 for imaging and therapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3303.

Cocaine's effects on the reactivity of the medial prefrontal cortex to ventral tegmental area stimulation: optical imaging study in mice.

The prefrontal cortex (PFC) is modulated by dopaminergic and glutamatergic neurons that project from the ventral tegmental area (VTA) and disruption of this modulation might facilitate impulsive behaviors during cocaine intoxication. Here, we assessed the effects of acute cocaine (30 mg/kg, i.p.) on the reactivity of the PFC to VTA stimulation. Using a genetically encoded calcium indicator (GCaMP6f), we optically imaged the neuronal Ca2+ reactance in medial PFC (mPFC) in response to 'tonic-like' (5Hz) and 'phasic-like' (50 Hz) electrical VTA stimulation. The high temporal and spatial resolutions of our optical system allowed us to capture single Ca2+ neuronal transients from individual stimuli with 'tonic-like' stimulation and to visualize single neuronal activation evoked by 'phasic-like' VTA stimulation. 'Tonic-like' VTA stimulation induced a rapid increase in mean neuronal Ca2+ in mPFC followed by a plateau and recovery upon termination of stimulation. After cocaine, the mPFC sensitivity to 'tonic-like' VTA stimulation was attenuated, with a 50.4% reduction (P = 0.03) in the number of Ca2+ transients corresponding to single electrical stimuli but the recovery time was lengthened (4.30 ± 0.25 sec to 5.41 ± 0.24 sec, P = 0.03). 'Phasic-like' stimulation evoked a rapid Ca2+ fluorescence increase in mPFC with an immediate decay process, and while cocaine did not affect the peak response (7.17 ± 1.07% versus 7.13 ± 0.96%, P = 0.98) it shortened the recovery time to baseline (3.27 ± 0.11 sec versus 2.38 ± 0.23 sec, P = 0.005). Acute cocaine impairs reactivity of medial prefrontal cortex (mPFC) to ventral tegmental area stimulation, decreasing its sensitivity to 'tonic-like' stimulation and lengthening the recovery time to return to baseline while shortening it for phasic stimulation. These changes in mPFC might contribute to cocaine binging during intoxication.