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BackgroundGlioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults. Myeloid-derived suppressor cells (MDSCs), particularly the granulocytic subset (gMDSCs), are highly abundant in GBM and suppress anti-tumor immunity, making them a high-priority therapeutic target. In mice, gMDSCs are identified by Ly6G expression. Due to tissue limitations, tumor heterogeneity, and the need to assess gMDSC levels in survival studies without euthanasia, we aimed to develop a noninvasive tool to quantify gMDSCs in preclinical GBM using positron emission tomography (PET).MethodsFlow cytometry confirmed Ly6G+ gMDSC infiltration in orthotopic syngeneic GBM tumors. An anti-Ly6G antibody (clone 1A8) was conjugated to a DFO chelator and radiolabeled with Zirconium-89 (89Zr). Longitudinal PET/CT imaging and biodistribution studies were performed at 48- and 120-hours post-injection in tumor-bearing mice injected with resulting radio-conjugate, 89Zr-DFO-anti-Ly6G, at multiple specific activities as compared to blocking and vector controls.ResultsOur data confirms high MDSC levels in murine GBM. The radiolabeled tracer (89Zr-DFO-anti-Ly6G) was produced with high radiolabeling yield (>95%) and in vivo stability over 120 hours. Initial PET imaging demonstrated that tumor uptake increased at 100 µg (SUVmean = 3.08 ± 0.101) vs. 40 µg (SUVmean = 1.98 ± 1.062). Additionally, pre-injection of cold antibody (25x) enhanced tumor uptake by blocking peripheral Ly6G, reducing tracer accumulation in blood and immune organs while increasing tumor signal (SUVmean = 3.25 ± 0.724).Isotype controls showed a strong correlation between tumor and non-tumor uptake (SUVmean = 1.59 ± 0.703), indicating peripheral receptor saturation as a mechanism of enhanced tumor targeting. Tumor tracer uptake increased over time in all groups. Ex vivo biodistribution studies confirmed PET imaging findings. PET imaging studies are currently underway to further optimize specific activity identify an optimal balance between peripheral blocking and tumor targeting, and further validate specificity of 89Zr-DFO-anti-Ly6G through blocking (100x) studies.ConclusionsgMDSCs are a critical therapeutic target in GBM. We developed Ly6G-PET, a novel, non-invasive imaging strategy using 89Zr-DFO-anti-Ly6G to detect and monitor gMDSCs in vivo. This approach enables longitudinal tracking of gMDSCs in preclinical models and supports the clinical potential of MDSC-targeted PET imaging in GBM.

Nanomedical research necessarily involves the study of the interactions between nanoparticulates and the biological environment. Transmission electron microscopy has proven to be a powerful tool in providing information about nanoparticle uptake, biodistribution and relationships with cell and tissue components, thanks to its high resolution. This article aims to overview the transmission electron microscopy techniques used to explore the impact of nanoconstructs on biological systems, highlighting the functional value of ultrastructural morphology, histochemistry and microanalysis as well as their fundamental contribution to the advancement of nanomedicine.

Atherosclerosis is a cardiovascular disease initiated by the deposition of Low Density Proteins (LDL) within the intima and their subsequent oxidation to (oxLDL). Currently, there are no available Positron Emission Tomography (PET) tracers for clinical imaging of atherosclerosis. LO1 and LO9 are novel antibodies that target modified low-density lipoprotein, a component of atherosclerotic plaques. Despite their high specificity for their molecular target, the use of full antibodies as PET tracers is limited due to their long circulation time. Pretargeted labelling of antibodies overcomes this limitation by decoupling the antibodies’ slow pharmacokinetics from the half-life of the radioisotope, injecting the two components separately and facilitating their use as tracers.The methodology requires a trans-cyclooctene (TCO) conjugation to the antibody, followed by the selective in vivo reaction with a radiolabelled tetrazine (Tz) injected after the conjugated antibody has accumulated on target and partially cleared form the blood.The components necessary for the pretargeted PET imaging of atherosclerosis were prepared and characterised. The radiolabelle tetrazine [18F]FpyTz was prepared and purified with a radiochemical yield of 10.5% and a radiochemical purity <99%. In addition, a near-infrared (NIRF) emitting tetrazine Tz-VT was also synthesized to mimic the behavior of the radiolabeled analogues in some in vitro and ex vivo tests.Conjugation of LO9 and LO1 yielded LO9-TCO and LO1-TCO, respectively. Aortic root sections from LDL-R-/- mice fed a high-fat diet for 14 weeks were exposed to LO9, LO1, LO9-TCO and LO1-TCO respectively, followed by Tz -VT, and examined using fluorescent microscopy. The stained sections exposed to LO9-TCO or LO1-TCO and Tz showed areas of positive signal along the plaque edges a representative example obtained using LO9-TCO is depicted in figure, while control slides exposed to LO9 or LO1 showed no signal. Ldlr-/- mice fed a high-fat diet for 22 weeks were injected with the modified antibodies first and with the radiolabelled tetrazine after 72h. PET/CT studies and Ex vivo biodistribution studies were via γ-counter were performed. Additionally, PET/CT and ex vivo biodistribution studies were performed with directly labelled antibodies LO1-89Zr and LO9-89Zr to enable comparison.Abstract 163 Figure 1NIRF tetrazine selectively reacts with LO9-TCO on tissueThe Figure shows a microscopy study on sequential series of atherosclerotic mouse tissue. Panel A shows H&E stain of atherosclerotic mouse tissue (magnified in D). In panel B the tissue was coated with unmodified LO9 and then exposed to the NIRF tetrazine. In panel C the tissue was coated with LO9-TCO and then exposed to the NIRF tetrazine (in red). NIRF tetrazine signal can only be seen on slide coated with LO9-TCO.Abstract 163 Figure 2PET images of mice injected with pretargeted antibodiesPET images of three IdIr-/- mice fed a high-fat diet for 22 weeks; animals were injected (from left to right) with LO1-TCO, control-TCO, LO9-TCO then after 72 h, animals received a second injection with [18F]FpyTz Animals were imaged 5minutes post-injection. Red arrows indicate areas of positive accumulation corresponding to atherosclerotic plaquesAnimals injected with LO1 and LO9 showed a higher aorta uptake when compared with the control antibody. Analysis of PET images of pretargeted antibodies showed co-localisation of LO9 and LO1 at bifurcations of the aorta. The first studies of in vivo pretargeted PET detection of atherosclerosis were performed, our studies successfully demonstrated pretargeting of native low-density lipoprotein within atherosclerotic plaques in mice, in-vivo and ex-vivo using plaque specific antibodies.Conflict of Interestnone

Engineered nanomaterials (ENMs) have gained huge importance in technological advancements over the past few years. Among the various ENMs, silver nanoparticles (AgNPs) have become one of the most explored nanotechnology-derived nanostructures and have been intensively investigated for their unique physicochemical properties. The widespread commercial and biomedical application of nanosilver include its use as a catalyst and an optical receptor in cosmetics, electronics and textile engineering, as a bactericidal agent, and in wound dressings, surgical instruments, and disinfectants. This, in turn, has increased the potential for interactions of AgNPs with terrestrial and aquatic environments, as well as potential exposure and toxicity to human health. In the present review, after giving an overview of ENMs, we discuss the current advances on the physiochemical properties of AgNPs with specific emphasis on biodistribution and both in vitro and in vivo toxicity following various routes of exposure. Most in vitro studies have demonstrated the size-, dose- and coating-dependent cellular uptake of AgNPs. Following NPs exposure, in vivo biodistribution studies have reported Ag accumulation and toxicity to local as well as distant organs. Though there has been an increase in the number of studies in this area, more investigations are required to understand the mechanisms of toxicity following various modes of exposure to AgNPs.

Currently, metformin is the first-line medication to treat type 2 diabetes mellitus (T2DM) in most guidelines and is used daily by >200 million patients. Surprisingly, the mechanisms underlying its therapeutic action are complex and are still not fully understood. Early evidence highlighted the liver as the major organ involved in the effect of metformin on reducing blood levels of glucose. However, increasing evidence points towards other sites of action that might also have an important role, including the gastrointestinal tract, the gut microbial communities and the tissue-resident immune cells. At the molecular level, it seems that the mechanisms of action vary depending on the dose of metformin used and duration of treatment. Initial studies have shown that metformin targets hepatic mitochondria; however, the identification of a novel target at low concentrations of metformin at the lysosome surface might reveal a new mechanism of action. Based on the efficacy and safety records in T2DM, attention has been given to the repurposing of metformin as part of adjunct therapy for the treatment of cancer, age-related diseases, inflammatory diseases and COVID-19. In this Review, we highlight the latest advances in our understanding of the mechanisms of action of metformin and discuss potential emerging novel therapeutic uses.This Review highlights the latest advances in our understanding of the mechanisms of action of metformin. Potential repurposing of metformin for other indications is also discussed.

The unique properties of chitosan make it a useful choice for various nanoparticulate drug delivery applications. Although chitosan is biocompatible and enables cellular uptake, its interactions at cellular and systemic levels need to be studied in more depth. This review focuses on the various physical and chemical properties of chitosan that affect its performance in biological systems. We aim to analyze recent research studying interactions of chitosan nanoparticles (NPs) upon their cellular uptake and their journey through the various compartments of the cell. The positive charge of chitosan enables it to efficiently attach to cells, increasing the probability of cellular uptake. Chitosan NPs are taken up by cells via different pathways and escape endosomal degradation due to the proton sponge effect. Furthermore, we have reviewed the interaction of chitosan NPs upon in vivo administration. Chitosan NPs are immediately surrounded by a serum protein corona in systemic circulation upon intravenous administration, and their biodistribution is mainly to the liver and spleen indicating RES uptake. However, the evasion of RES system as well as the targeting ability and bioavailability of chitosan NPs can be improved by utilizing specific routes of administration and covalent modifications of surface properties. Ongoing clinical trials of chitosan formulations for therapeutic applications are paving the way for the introduction of chitosan into the pharmaceutical market and for their toxicological evaluation. Chitosan provides specific biophysical properties for effective and tunable cellular uptake and systemic delivery for a wide range of applications.

Structural imaging remains an essential component of diagnosis, staging and response assessment in patients with cancer; however, as clinicians increasingly seek to noninvasively investigate tumour phenotypes and evaluate functional and molecular responses to therapy, theranostics — the combination of diagnostic imaging with targeted therapy — is becoming more widely implemented. The field of radiotheranostics, which is the focus of this Review, combines molecular imaging (primarily PET and SPECT) with targeted radionuclide therapy, which involves the use of small molecules, peptides and/or antibodies as carriers for therapeutic radionuclides, typically those emitting α-, β- or auger-radiation. The exponential, global expansion of radiotheranostics in oncology stems from its potential to target and eliminate tumour cells with minimal adverse effects, owing to a mechanism of action that differs distinctly from that of most other systemic therapies. Currently, an enormous opportunity exists to expand the number of patients who can benefit from this technology, to address the urgent needs of many thousands of patients across the world. In this Review, we describe the clinical experience with established radiotheranostics as well as novel areas of research and various barriers to progress.Radiotheranostics enables the clinician to image and then target lesions using the same probe. Despite this appealing potential, interest in the field of radiotheranostics has long been constrained by a lack of expertise, high infrastructure costs and the availability of non-radioactive alternative approaches. Nonetheless, several recent successes have led to renewed research interest. In this Review, the authors summarize the current challenges and opportunities in this rapidly emerging area.

BackgroundA critical need in adoptive T cell therapy is the ability to noninvasively and longitudinally track engineered T cells in vivo, as biodistribution and persistence directly impact efficacy and safety. We previously developed a novel theranostic platform by engineering CD19 CAR-T cells to express a membrane-bound scFv, huC825, which binds DOTA-haptens with picomolar affinity, rendering it suitable for labeling with diagnostic or therapeutic radionuclides. Here, we applied this platform to newly developed anti-IL13Rα2-scFv-derived CAR-T cells (KLG3BBz-huC825) and evaluated their biodistribution and persistence in a human melanoma xenograft model using immunoPET.MethodsSecond-generation IL13Rα2-targeted CAR-T cells co-expressing huC825 were engineered and validated for antigen-specific cytotoxicity and cytokine production in vitro. We evaluated in vivo detection sensitivity using a T cell titration assay. Furthermore, NSG mice were engrafted with A375 melanoma tumors and seven days later intravenously (IV) injected with KLG3BBz-huC825 or non-transduced (NT) T cells. We performed weekly [8 6Y]Y-ABD-based whole-body PET/CT and monitored the outcome.ResultsKLG3BBz-huC825 CAR-T cells exhibited potent antigen-specific cytotoxicity and cytokine production in vitro. ImmunoPET visualized uptake at the tumor site already on day 7 post T cell infusion (4.67 ± 1.76 %ID/g; n=4), peaking at day 14 (16 ± 7.67 %ID/g, n=3), with persistence up to day 49 (3.51%ID/g, n=1) in mice injected with KLG3BBz-huC825 CAR-T cells. Uptake in normal organs and tumor site correlated with T cell infiltration on histologic assessment. In contrast, minimal uptake at the tumor site and normal tissues was seen in control mice receiving NT T cells. Mice treated with KLG3BBz-huC825 showed significantly prolonged survival compared to mice treated with NT (median 44.5 vs. 20 days).ConclusionsThis immunoPET-enabled tracking platform offers exquisite sensitivity and spatiotemporal resolution to monitor CAR-T cell dynamics in vivo. It provides a clinically translatable strategy to guide next-generation CAR-T cell therapy through improved understanding of trafficking, persistence, and tumor engagement. Such approaches could support early therapeutic monitoring, adaptive dosing, and stratification in clinical immunotherapy trials.

BackgroundAdvances in precision medicine utilizing antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) as therapeutic platforms have brought promising solutions for various neurodegenerative/neuromuscular disorders and rare diseases. Currently, 20 oligonucleotide drug products have been commercially approved by the FDA and EMA, and many more are in clinical phase I-III trials. The FDA has issued recommendations to generate nonclinical biodistribution (BD) data for gene therapy products to evaluate and interpret nonclinical pharmacology and toxicology findings before initiating human clinical trials. In situ hybridization (ISH) and immunohistochemistry (IHC) are increasingly used to spatially visualize the delivered therapeutic, target gene, transgene, and/or cell markers and complement information gathered from molecular technologies such as quantitative polymerase chain reaction (qPCR) and digital PCR. Direct visualization of oligonucleotides can also monitor the risk of off-target events by studying BD of potential therapies in various organs.MethodsA new RNAscope™ ISH assay enabling the detection of ASOs, siRNAs, microRNAs (miRNAs), and other small RNAs was developed on the Roche DISCOVERY™ ULTRA System. This fully automated assay allows the co-detection of small RNAs and mRNAs and can be combined with protein detection on the same formalin-fixed paraffin-embedded sections of tissues. This workflow includes harmonized cell conditioning for an efficient combined RNA and antigen retrieval and the new protease-free pretreatment reagent VS PretreatPro, allowing for RNA detection without disrupting protease-sensitive epitopes. Visualization of multiple RNA species and protein targets at the single-cell level is achieved by combining different HRP and AP-based detection, leveraging Roche’s translucent chromogens.ResultsWe investigated the expression profile of miR-21, a microRNA implicated in cancer proliferation and progression, in the human cancer TMA tissue. MicroRNA detection was combined with PTEN RNA to assess the impact of miR21 on the expression of PTEN, a tumor suppressor that regulates cell growth, proliferation, and survival. Ki67 protein was co-detected in the same sample to correlate with tumor cell proliferation.ConclusionsThis new automated ISH assay showed high sensitivity and specificity for the detection of small RNAs with different expression profiles, and subcellular resolution in intact tissue context. This novel assay will be particularly valuable for the study of ASO/siRNAs delivery, biodistribution, stability, and expression profile of their associated RNA targets for the development of new oligonucleotide therapeutics.