S100A9, as a potential predictor of prognosis and immunotherapy response for GBM, promotes the malignant progression of GBM cells and migration of M2 macrophages.

In the past decades, the therapeutic effect of glioblastoma (GBM) has not been significantly improved. Generous evidence indicates that S100A9 has a wide range of functions in tumors, but its exploration in GBM is less. The purpose of this study is to conduct a comprehensive bioinformatics analysis and cytological experiment on S100A9 in GBM. The expression data and clinical data of GBM samples were downloaded from the public database, and comprehensive bioinformatics analysis was performed on S100A9 in GBM using R software. Wound healing assay and transwell assay were used to detect the migration activity of cells, and colony formation assay, EdU staining, and CCK-8 assay were used to detect the proliferation activity of cells. The effect of S100A9 on the migration activity of M2 macrophages was verified by the cell co-culture method. The protein expression was detected by western blotting and immunohistochemical staining. S100A9 is an independent prognostic factor in GBM patients and is related to poor prognosis. It can be used as an effective tool to predict the response of GBM patients to immune checkpoint inhibitors (ICIs). In addition, S100A9 can promote the malignant progression of GBM and the migration of M2 macrophages. On the whole, our study highlights the potential value of S100A9 in predicting prognosis and immunotherapeutic response in GBM patients. More importantly, S100A9 may promote the malignant progress of GBM by involving in some carcinogenic pathways and remodeling the tumor microenvironment (TME).

Carrier-Free Photodynamic Bioregulators Inhibiting Lactic Acid Efflux Combined with Immune Checkpoint Blockade for Triple-Negative Breast Cancer Immunotherapy.

Abnormal tumor metabolism creates a complex tumor immune microenvironment that plays a dominant role in the metastasis of triple-negative breast cancer (TNBC). TNBC is insensitive to immune checkpoint blockade (ICB) therapy because of insufficient cytotoxic T lymphocyte (CTL) infiltration and a hyper-lactic acid-suppressive immune microenvironment caused by abnormal glycolysis. Herein, we propose an amplified strategy based on lactic acid regulation to reprogram the immunosuppressive tumor microenvironment (ITM) and combine it with ICB therapy to achieve enhanced antitumor immunotherapy effects. Specifically, we constructed CASN, a carrier-free photodynamic bioregulator, through the self-assembly of the photosensitizer Chlorin e6 and monocarboxylate transporter 1 (MCT1) inhibitor AZD3965. CASN exhibited a uniform structure, good stability, and drug accumulation at the tumor site. CASN-mediated photodynamic therapy following laser irradiation inhibited primary tumor growth and induced immunogenic cell death. Furthermore, CASN reduced lactic acid-mediated regulatory T cell generation and M2 tumor-associated macrophage polarization by blocking MCT1-mediated lactic acid efflux to attenuate immune suppression, inducing the recruitment and activation of CTLs. Ultimately, CASN-mediated immunopotentiation combined with ICB therapy considerably strengthened tumor immunotherapy and effectively inhibited tumor growth and metastasis of TNBC. This synergistic amplification strategy overcomes the limitations of an acidic ITM and presents a potential clinical treatment option for metastatic tumors.

First-in-Human Evaluation of [18F]FDOPA Produced by Organo-Photoredox Reactions.

Photoredox is a powerful synthetic tool in organic chemistry and has been widely used in various fields, including nuclear medicine and molecular imaging. In particular, acridinium-based organophotoredox radiolabeling has significantly impacted the production and discovery of positron emission tomography (PET) agents. Despite their extensive use in preclinical research, no PET agents synthesized by acridinium photoredox labeling have been tested in humans. [18F]FDOPA is clinically used for tumor diagnosis and the evaluation of neuropsychiatric disorders, but its application is limited by complex synthesis methods, the need for expensive modules, and/or the high cost of consumable materials/cassettes. In this report, we integrated a photoredox labeling unit with an automated module and produced [18F]FDOPA for human study. This research not only represents the first human study of a PET agent generated by acridinium-based organophotoredox reactions but also demonstrates the safety of this novel labeling method, serving as a milestone/reference for the clinical translation of other PET agents generated by this technique in the future.

Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting In Vivo Distribution Using Multiamine Macrocycles.

The development of theranostic radiotracers relies on their binding to specific molecular markers of a particular disease and the use of corresponding radiopharmaceutical pairs thereafter. This study reports the use of multiamine macrocyclic moieties (MAs), as linkers or chelators, in tracers targeting the neurotensin receptor-1 (NTSR-1). The goal is to achieve elevated tumor uptake, minimal background interference, and prolonged tumor retention in NTSR-1-positive tumors. Methods: We synthesized a series of neurotensin antagonists bearing MA linkers and metal chelators. The MA unit is hypothesized to establish a strong interaction with the cell membrane, and the addition of a second chelator may enhance water solubility, consequently reducing liver uptake. Small-animal PET/CT imaging of [64Cu]Cu-DOTA-SR-3MA, [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, [64Cu]Cu-NT-CB-DOTA, and [64Cu]Cu-NT-Sarcage was acquired at 1, 4, 24, and 48 h after injection using H1299 tumor models. [55Co]Co-NT-CB-NOTA was also tested in HT29 (high NTSR-1 expression) and Caco2 (low NTSR-1 expression) colorectal adenocarcinoma tumor models. Saturation binding assay and internalization of [55Co]Co-NT-CB-NOTA were used to test tracer specificity and internalization in HT29 cells. Results: In vivo PET imaging with [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, and [55Co]Co-NT-CB-NOTA revealed high tumor uptake, high tumor-to-background contrast, and sustained tumor retention (≤48 h after injection) in NTSR-1-positive tumors. Tumor uptake of [64Cu]Cu-NT-CB-NOTA remained at 76.9% at 48 h after injection compared with uptake 1 h after injection in H1299 tumor models, and [55Co]Co-NT-CB-NOTA was retained at 60.2% at 24 h compared with uptake 1 h after injection in HT29 tumor models. [64Cu]Cu-NT-Sarcage also showed high tumor uptake with low background and high tumor retention 48 h after injection Conclusion: Tumor uptake and pharmacokinetic properties of NTSR-1-targeting radiopharmaceuticals were greatly improved when attached with different nitrogen-containing macrocyclic moieties. The study results suggest that NT-CB-NOTA labeled with either 64Cu/67Cu, 55Co/58mCo, or 68Ga (effect of 177Lu in tumor to be determined in future studies) and NT-Sarcage labeled with 64Cu/67Cu or 55Co/58mCo may be excellent diagnostic and therapeutic radiopharmaceuticals targeting NTSR-1-positive cancers. Also, the introduction of MA units to other ligands is warranted in future studies to test the generality of this approach.

Mitochondria-targeted polyprodrug nanoparticles induce mitochondrial stress for immunogenic chemo-photodynamic therapy of ovarian cancer.

Hypoimmunogenicity and the immunosuppressive microenvironment of ovarian cancer severely restrict the capability of immune-mediated tumor killing. Immunogenic cell death (ICD) introduces a theoretical principle for antitumor immunity by increasing antigen exposure and presentation. Despite recent research progress, the currently available ICD inducers are still very limited, and many of them can hardly induce sufficient ICD based on traditional endoplasmic reticulum (ER) stress. Accumulating evidence indicates that inducing mitochondrial stress usually shows a higher efficiency in evoking large-scale ICD than that via ER stress. Inspired by this, herein, a mitochondria-targeted polyprodrug nanoparticle (named Mito-CMPN) serves as a much superior ICD inducer, effectively inducing chemo-photodynamic therapy-caused mitochondrial stress in tumor cells. The rationally designed stimuli-responsive polyprodrugs, which can self-assemble into nanoparticles, were functionalized with rhodamine B for mitochondrial targeting, cisplatin and mitoxantrone (MTO) for synergistic chemo-immunotherapy, and MTO also serves as a photosensitizer for photodynamic immunotherapy. The effectiveness and robustness of Mito-CMPNs in reversing the immunosuppressive microenvironment is verified in both an ovarian cancer subcutaneous model and a high-grade serous ovarian cancer model. Our results support that the induction of abundant ICD by focused mitochondrial stress is a highly effective strategy to improve the therapeutic efficacy of immunosuppressive ovarian cancer.

Chelator boosted tumor-retention and pharmacokinetic properties: development of 64Cu labeled radiopharmaceuticals targeting neurotensin receptor.

PURPOSE: Accumulating evidence suggests that neurotensin (NTS) and neurotensin receptors (NTSRs) play key roles in lung cancer progression by triggering multiple oncogenic signaling pathways. This study aims to develop Cu-labeled neurotensin receptor 1 (NTSR1)-targeting agents with the potential for both imaging and therapeutic applications. METHOD: A series of neurotensin receptor antagonists (NRAs) with variable propylamine (PA) linker length and different chelators were synthesized, including [64Cu]Cu-CB-TE2A-iPA-NRA ([64Cu]Cu-4a-c, i = 1, 2, 3), [64Cu]Cu-NOTA-2PA-NRA ([64Cu]Cu-4d), [64Cu]Cu-DOTA-2PA-NRA ([64Cu]Cu-4e, also known as [64Cu]Cu-3BP-227), and [64Cu]Cu-DOTA-VS-2PA-NRA ([64Cu]Cu-4f). The series of small animal PET/CT were conducted in H1299 lung cancer model. The expression profile of NTSR1 was also confirmed by IHC using patient tissue samples. RESULTS: For most of the compounds studied, PET/CT showed prominent tumor uptake and high tumor-to-background contrast, but the tumor retention was strongly influenced by the chelators used. For previously reported 4e, [64Cu]Cu-labeled derivative showed initial high tumor uptake accompanied by rapid tumor washout at 24 h. The newly developed [64Cu]Cu-4d and [64Cu]Cu-4f demonstrated good tumor uptake and tumor-to-background contrast at early time points, but were less promising in tumor retention. In contrast, our lead compound [64Cu]Cu-4b demonstrated 9.57 ± 1.35, 9.44 ± 2.38 and 9.72 ± 4.89%ID/g tumor uptake at 4, 24, and 48 h p.i., respectively. Moderate liver uptake (11.97 ± 3.85, 9.80 ± 3.63, and 7.72 ± 4.68%ID/g at 4, 24, and 48 h p.i.) was observed with low uptake in most other organs. The PA linker was found to have a significant effect on drug distribution. Compared to [64Cu]Cu-4b, [64Cu]Cu-4a had a lower background, including a greatly reduced liver uptake, while the tumor uptake was only moderately reduced. Meanwhile, [64Cu]Cu-4c showed increased uptake in both the tumor and the liver. The clinical relevance of NTSR1 was also demonstrated by the elevated tumor expression in patient tissue samples. CONCLUSIONS: Through the side-by-side comparison, [64Cu]Cu-4b was identified as the lead agent for further evaluation based on its high and sustained tumor uptake and moderate liver uptake. It can not only be used to efficiently detect NTSR1 expression in lung cancer (for diagnosis, patient screening, and treatment monitoring), but also has the great potential to treat NTSR-positive lesions once chelating to the beta emitter 67Cu.

WTAP regulates the production of reactive oxygen species, promotes malignant progression, and is closely related to the tumor microenvironment in glioblastoma.

RNA modifications have been substantiated to regulate the majority of physiological activities in the organism, including the metabolism of reactive oxygen species (ROS), which plays an important role in cells. As for the effect of RNA modification genes on ROS metabolism in glioblastoma (GBM), it has not been studied yet. Therefore, this study aims to screen the RNA modification genes that are most related to ROS metabolism and explore their effects on the biological behavior of GBM in vitro. Here, an association between WTAP and ROS metabolism was identified by bioinformatics analysis, and WTAP was highly expressed in GBM tissue compared with normal brain tissue, which was confirmed by western blotting and immunohistochemical staining. When using a ROS inducer to stimulate GBM cells in the WTAP overexpression group, the ROS level increased more significantly and the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) also increased. Next, colony formation assay, wound healing assay, and transwell assay were performed to investigate the proliferation, migration, and invasion of GBM cells. The results showed that WTAP, as an oncogene, promoted the malignant progression of GBM cells. Functional enrichment analysis predicted that WTAP was involved in the regulation of tumor/immune-related functional pathways. Western blotting was used to identify that WTAP had a regulatory effect on the phosphorylation of PI3K/Akt signaling. Finally, based on functional enrichment analysis, we further performed immune-related analysis on WTAP. In conclusion, this study analyzed WTAP from three aspects, which provided new ideas for the treatment of GBM.

Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

Hexafluoroisopropanol as a Bioconjugation Medium of Ultrafast, Tryptophan-Selective Catalysis.

The past decade has seen a remarkable growth in the number of bioconjugation techniques in chemistry, biology, material science, and biomedical fields. A core design element in bioconjugation technology is a chemical reaction that can form a covalent bond between the protein of interest and the labeling reagent. Achieving chemoselective protein bioconjugation in aqueous media is challenging, especially for generally less reactive amino acid residues, such as tryptophan. We present here the development of tryptophan-selective bioconjugation methods through ultrafast Lewis acid-catalyzed reactions in hexafluoroisopropanol (HFIP). Structure-reactivity relationship studies have revealed a combination of thiophene and ethanol moieties to give a suitable labeling reagent for this bioconjugation process, which enables modification of peptides and proteins in an extremely rapid reaction unencumbered by noticeable side reactions. The capability of the labeling method also facilitated radiofluorination application as well as antibody functionalization. Enhancement of an α-helix by HFIP leads to its compatibility with a certain protein, and this report also demonstrates a further stabilization strategy achieved by the addition of an ionic liquid to the HFIP medium. The nonaqueous bioconjugation approaches allow access to numerous chemical reactions that are unavailable in traditional aqueous processes and will further advance the chemistry of proteins.

Three Stages of Dynamic Assembly Process of Dipeptide-Based Supramolecular Gel Revealed by In Situ Infrared Spectroscopy.

The exploration of short peptide-based assembly is vital for understanding protein-misfolding-associated diseases and seeking strategies to attenuate aggregate formation. While, the molecular mechanism of their structural evolution remains poorly studied in view of the dynamic and unpredictable assembly process. Herein, infrared (IR) spectroscopy, which serves as an in situ and real-time analytical technique, was intelligently employed to investigate the mechanism of phase transition and aggregate formation during the dynamic assembly process of diphenylalanine. Combined with other spectroscopy and electron microscopy technologies, three stages of gel formation and the main driving forces in different stages were revealed. A variety of stoichiometric methods such as continuous wavelet transform, principal component analysis, and two-dimensional correlation spectroscopy techniques were conducted to analyze the original time-dependent IR spectra to obtain detailed information on the changes in the amide bands and hydration layer. The microenvironment of hydrogen bonding among amide bands was significantly changed with the addition of pyridine derivatives, resulting in great differences in the properties of co-assembled gels. This work not only provides a universal analytical way to reveal the dynamic assembly process of dipeptide-based supramolecular gel but also expands their applications in supramolecular regulation and high-throughput screens in situ.

Expression of the Calcium-Sensing Receptor on Normal and Abnormal Parathyroid and Thyroid Tissue.

INTRODUCTION: Current imaging techniques have several limitations in detecting parathyroid glands. We have investigated the calcium-sensing receptor (CaSR) as a potential target for specifically labeling parathyroid glands for radiologic detection. For accurate imaging it is vital that a large differential expression exists between the target tissue and adjacent structures. We sought to investigate the relative abundance of the CaSR in normal and abnormal parathyroid tissue, as well as normal and abnormal thyroid. METHODS: Existing clinical specimens were selected that represented a wide variety of pathologically and clinically confirmed malignant and benign thyroid and parathyroid specimens. Sections were stained for the CaSR using immunohistochemistry and scored for intensity and abundance of expression. (H score = intensity scored from 0 to 3 multiplied by the % of cells at each intensity. Range 0-300). RESULTS: All parathyroid specimens expressed the CaSR to a high degree. Normal parathyroid had the highest H score (271, s.d. 25.4). Abnormal parathyroid specimens were slightly lower but still much higher than normal thyroid (H score 38.3, s.d. 23.3). Medullary thyroid cancer also expressed the CaSR significantly higher than normal thyroid (H score 182, s.d. 69.1, P < 0.001) but below parathyroid levels. Hürthle cell carcinoma expressed the CaSR to a lesser degree but higher than normal thyroid (H score 101, s.d. 46.4, P = 0.0037). CONCLUSIONS: The CaSR is differentially expressed on parathyroid tissue making it a feasible target for parathyroid imaging. False positives might be anticipated with medullary and Hürthle cell cancers.