Digestion-Free Middle-Down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from Antibody-Drug Conjugates.

Antibody-drug conjugate (ADC) is a therapeutic modality that aims to improve payload delivery specificity and reduce systemic toxicity. Considering the complex structure of ADCs, various bioanalytical methods by liquid chromatography coupled with mass spectrometry (LC-MS), ligand binding assay (LBA) and hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable drug-antibody ratio (DAR) sensitive quantification of the conjugated payload. Typically, for quantitative, DAR-sensitive, assessment by LC-MS/MS,the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of noncleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using a collision-induced dissociation approach for absolute quantification of conjugated payload from four different ADCs in a biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a noncleavable linker ADC, trastuzumab emtansine. It also shows that the assay sensitivity is comparable to the conventional ADC quantification method by linker-payload cleavage using enzyme, while the assay dynamic range depends on factors including payload ionization and dissociation efficiency, DAR and its distribution, and species abundance. By demonstrating absolute quantification of both cleavable and noncleavable linker ADCs, this novel middle-down ADC approach demonstrates its potential application in bioanalysis and analytical characterization, especially for early discovery where high-throughput screening is required as the new approach saves time and resources by not requiring enzymatic digestion for cleavable ADCs or development of anti-payload antibodies for noncleavable linker ADCs.

Reference-free thio-succinimide isomerization characterization by electron-activated dissociation.

RATIONALE: Isomerism can be an important aspect in pharmaceutical drug development. Identification of isomers can provide insights into drug pharmacology and contribute to better design of drug molecules. The general approaches to differentiate isomers include Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and circular dichroism. Additionally, a commonly used method to differentiate isomers is liquid chromatography coupled with mass spectrometry (LC-MS). Notably, LC-MS is routinely applied to leucine and isoleucine differentiation to facilitate protein sequencing. This work focuses on isomer differentiation of widely employed thio-succinimide structure bridging the antibody backbone and linker-payload of antibody-drug conjugates (ADCs). Thio-succinimide hydrolysis stabilizes the payload-protein structure while generating a pair of constitutional isomers: thio-aspartyl and thio-isoaspartyl. METHODS: This paper introduces a hybrid method using ligand binding assay (LBA) and liquid chromatography coupled with tandem MS (LC-MS/MS) to reveal isomerization details of thio-succinimide hydrolysis over time in plasma samples incubated with ADC. Application of two orthogonal dissociation methods, collision-induced dissociation (CID) and electron-activated dissociation (EAD) revealed different MS/MS spectra for this pair of isomers. This observation enables a unique approach in distinguishing thio-succinimide hydrolysis isomers. RESULTS: We observed signature [R1 + Thio + 57 + H]+, [R2 + Succ + H2O - 57 + H]+, and [R2 + Succ + H2O - 44 + 2H]2+ product ions (Succ = succinimide) that differentiated thio-aspartyl and thio-isoaspartyl isomers using EAD. A newly discovered [R2 + ThioSucc + H2O - 44 + 2H]2+ ion also served as additional evidence that further supported our findings. CONCLUSIONS: This study is a first-to-date identification of thio-succinimide hydrolysis isomers without using synthesized reference materials. This approach should be applicable to all thio-succinimide-linked molecules. Correct identification of thio-succinimide hydrolysis isomers may eventually benefit the development of ADCs in the future.

Drug-Loaded Bioscaffolds for Osteochondral Regeneration.

Osteochondral defect is a complex tissue loss disease caused by arthritis, high-energy trauma, and many other reasons. Due to the unique structural characteristics of osteochondral tissue, the repair process is sophisticated and involves the regeneration of both hyaline cartilage and subchondral bone. However, the current clinical treatments often fall short of achieving the desired outcomes. Tissue engineering bioscaffolds, especially those created via three-dimensional (3D) printing, offer promising solutions for osteochondral defects due to their precisely controllable 3D structures. The microstructure of 3D-printed bioscaffolds provides an excellent physical environment for cell adhesion and proliferation, as well as nutrient transport. Traditional 3D-printed bioscaffolds offer mere physical stimulation, while drug-loaded 3D bioscaffolds accelerate the tissue repair process by synergistically combining drug therapy with physical stimulation. In this review, the physiological characteristics of osteochondral tissue and current treatments of osteochondral defect were reviewed. Subsequently, the latest progress in drug-loaded bioscaffolds was discussed and highlighted in terms of classification, characteristics, and applications. The perspectives of scaffold design, drug control release, and biosafety were also discussed. We hope this article will serve as a valuable reference for the design and development of osteochondral regenerative bioscaffolds and pave the way for the use of drug-loaded bioscaffolds in clinical therapy.

Drug-target prediction based on Dynamic Heterogeneous Graph Convolutional Network.

Novel drug-target interaction (DTI) prediction is crucial in drug discovery and repositioning. Recently, graph neural network (GNN) has shown promising results in identifying DTI by using thresholds to construct heterogeneous graphs. However, an empirically selected threshold can lead to loss of valuable information, especially in sparse networks, a common scenario in DTI prediction. To make full use of insufficient information, we propose a DTI prediction model based on Dynamic Heterogeneous Graph (DT-DHG). And progressive learning is introduced to adjust the receptive fields of node. The experimental results show that our method significantly improves the performance of the original GNNs and is robust against the choices of backbones. Meanwhile, DT-DHG outperforms the state-of-the-art methods and effectively predicts novel DTIs. The source code is available at https://github.com/kissablemt/DT-DHG.

Diatomite-incorporated hierarchical scaffolds for osteochondral regeneration.

Osteochondral regeneration involves the highly challenging and complex reconstruction of cartilage and subchondral bone. Silicon (Si) ions play a crucial role in bone development. Current research on Si ions mainly focuses on bone repair, by using silicate bioceramics with complex ion compositions. However, it is unclear whether the Si ions have important effect on cartilage regeneration. Developing a scaffold that solely releases Si ions to simultaneously promote subchondral bone repair and stimulate cartilage regeneration is critically important. Diatomite (DE) is a natural diatomaceous sediment that can stably release Si ions, known for its abundant availability, low cost, and environmental friendliness. Herein, a hierarchical osteochondral repair scaffold is uniquely designed by incorporating gradient DE into GelMA hydrogel. The adding DE microparticles provides a specific Si source for controlled Si ions release, which not only promotes osteogenic differentiation of rBMSCs (rabbit bone marrow mesenchymal stem cells) but also enhances proliferation and maturation of chondrocytes. Moreover, DE-incorporated hierarchical scaffolds significantly promoted the regeneration of cartilage and subchondral bone. The study suggests the significant role of Si ions in promoting cartilage regeneration and solidifies their foundational role in enhancing bone repair. Furthermore, it offers an economic and eco-friendly strategy for developing high value-added osteochondral regenerative bioscaffolds from low-value ocean natural materials.

Predict lncRNA-drug associations based on graph neural network.

LncRNAs are an essential type of non-coding RNAs, which have been reported to be involved in various human pathological conditions. Increasing evidence suggests that drugs can regulate lncRNAs expression, which makes it possible to develop lncRNAs as therapeutic targets. Thus, developing in-silico methods to predict lncRNA-drug associations (LDAs) is a critical step for developing lncRNA-based therapies. In this study, we predict LDAs by using graph convolutional networks (GCN) and graph attention networks (GAT) based on lncRNA and drug similarity networks. Results show that our proposed method achieves good performance (average AUCs > 0.92) on five datasets. In addition, case studies and KEGG functional enrichment analysis further prove that the model can effectively identify novel LDAs. On the whole, this study provides a deep learning-based framework for predicting novel LDAs, which will accelerate the lncRNA-targeted drug development process.

Comparison of consistency in acute stroke: Automated ASPECT score software tools and manual consensus scores.

PURPOSE: The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) is a standardized semi-quantitative scoring system. It is widely used to assess the extent of acute ischemic stroke (AIS). We evaluated the consistency of three automatic software packages with the overall and region-specific manual ASPECTS scores of AIS patients. METHODS: Retrospectively, we gathered patients who presented with stroke symptoms between February 2019 and June 2022, and 174 cases were eventually included in the trial. Two radiologists reviewed the NCCT images independently; After four weeks, the same two radiologists began randomly reviewing the DWI images, discussed different scores and give consistent results as ground truth. RESULTS: Median ASPECTS of the expert consensus reading was 7 (5-9). Good to excellent correlation of ASPECTS total scores among the three software tools (0.70, 0.74 and 0.83). Correlation among ground truth and Rapid-ASPECTS, RealNow-ASPECTS, ShuKun-ASPECTS (ICC = 0.51, Cronbach's α = 0.53), (ICC = 0.60, Cronbach's α = 0.70) and (ICC = 0.52, Cronbach's α = 0.64) respectively. The AUCs for Rapid, RealNow and ShuKun were 0.61, 0.67, and 0.62 respectively. The region-specific results showed a poor to good correlation. The correlations between the non-dominant and dominant cerebral hemispheres and the ground truth were statistically different (P < 0.05). CONCLUSIONS: Overall, the scoring consistency between the three automated scoring software and the ground truth is comparable, with RealNow-ASPECTS being no less consistent and effective than Rapid-ASPECTS and ShuKun-ASPECTS, and even better than both. But the consistency grade that still is developable.

Quantitative evaluation of trastuzumab deruxtecan pharmacokinetics and pharmacodynamics in mouse models of varying degrees of HER2 expression.

Trastuzumab deruxtecan (T-DXd; DS-8201; ENHERTU®) is a human epithelial growth factor receptor 2 (HER2)-directed antibody drug conjugate (ADC) with demonstrated antitumor activity against a range of tumor types. Aiming to understand the relationship between antigen expression and downstream efficacy outcomes, T-DXd was administered in tumor-bearing mice carrying NCI-N87, Capan-1, JIMT-1, and MDA-MB-468 xenografts, characterized by varying HER2 levels. Plasma pharmacokinetics (PK) of total antibody, T-DXd, and released DXd and tumor concentrations of released DXd were evaluated, in addition to monitoring γΗ2AX and pRAD50 pharmacodynamic (PD) response. A positive relationship was observed between released DXd concentrations in tumor and HER2 expression, with NCI-N87 xenografts characterized by the highest exposures compared to the remaining cell lines. γΗ2AX and pRAD50 demonstrated a sustained increase over several days occurring with a time delay relative to tumoral-released DXd concentrations. In vitro investigations of cell-based DXd disposition facilitated the characterization of DXd kinetics across tumor cells. These outputs were incorporated into a mechanistic mathematical model, utilized to describe PK/PD trends. The model captured plasma PK across dosing arms as well as tumor PK in NCI-N87, Capan-1, and MDA-MB-468 models; tumor concentrations in JIMT-1 xenografts required additional parameter adjustments reflective of complex receptor dynamics. γΗ2AX longitudinal trends were well characterized via a unified PD model implemented across xenografts demonstrating the robustness of measured PD trends. This work supports the application of a mechanistic model as a quantitative tool, reliably projecting tumor payload concentrations upon T-DXd administration, as the first step towards preclinical-to-clinical translation.

Identification and Fungicide Sensitivity of Fusarium spp. Associated with Root Rot of Scutellaria baicalensis in Shanxi Province, China.

Fusarium root rot is usually classified as an extremely destructive soilborne disease. From 2020 to 2021, Fusarium root rot was observed in production areas and seriously affected the yield and quality of Scutellaria baicalensis in Shanxi Province, China. Based on morphological characteristics and combined analysis of the internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-alpha sequences, 68 Fusarium isolates obtained in this work were identified as F. oxysporum (52.94%), F. acuminatum (20.59%), F. solani (16.17%), F. proliferatum (5.88%), F. incarnatum (2.94%), and F. brachygibbosum (1.47%). In the pathogenicity tests, all Fusarium isolates could infect S. baicalensis roots, presenting different pathogenic ability. Among these isolates, F. oxysporum was found to have the highest virulence on S. baicalensis roots, followed by F. acuminatum, F. solani, F. proliferatum, F. brachygibbosum, and F. incarnatum. According to fungicide sensitivity tests, Fusarium isolates were more sensitive to fludioxonil and difenoconazole, followed by carbendazim, thiophanate-methyl, and hymexazol. In brief, this is the first report of Fusarium species (F. oxysporum, F. acuminatum, F. solani, F. proliferatum, F. incarnatum, and F. brachygibbosum) as causal agents of root rot of S. baicalensis in Shanxi Province, China. The fungicide sensitivity results will be helpful for formulating management strategies of S. baicalensis root rot.

Hsa_circ_0021205 enhances lipolysis via regulating miR-195-5p/HSL axis and drives malignant progression of glioblastoma.

Abnormal lipid metabolism is an essential hallmark of glioblastoma. Hormone sensitive lipase (HSL), an important rate-limiting enzyme contributed to lipolysis, which was involved in aberrant lipolysis of glioblastoma, however, its definite roles and the relevant regulatory pathway have not been fully elucidated. Our investigations disclosed high expression of HSL in glioblastoma. Knock-down of HSL restrained proliferation, migration, and invasion of glioblastoma cells while adding to FAs could significantly rescue the inhibitory effect of si-HSL on tumor cells. Overexpression of HSL further promoted tumor cell proliferation and invasion. Bioinformatics analysis and dual-luciferase reporter assay were performed to predict and verify the regulatory role of ncRNAs on HSL. Mechanistically, hsa_circ_0021205 regulated HSL expression by sponging miR-195-5p, which further promoted lipolysis and drove the malignant progression of glioblastoma. Besides, hsa_circ_0021205/miR-195-5p/HSL axis activated the epithelial-mesenchymal transition (EMT) signaling pathway. These findings suggested that hsa_circ_0021205 promoted tumorigenesis of glioblastoma through regulation of HSL, and targeting hsa_circ_0021205/miR-195-5p/HSL axis can serve as a promising new strategy against glioblastoma.

Adipose-derived stem cell exosomes promote tumor characterization and immunosuppressive microenvironment in breast cancer.

Adipose-derived stem cells (ASC) or autologous fat transplantation could be used to ameliorate breast cancer postoperative deformities. This study aims to explore the action of ASC and ASC-exosomes (ASC-exos) in breast cancer characterization and tumor microenvironment immunity, which provided a new method into the application of ASC-exos. ASC were extracted from human adipose tissue for the isolation and verification of ASC-exos. ASC-exos were co-cultured with CD4+T cells, CD14+ monocytes and MCF-7 cells, respectively. The tumor formation of nude mice was also constructed. Cell characterization was determined by CCK8, scratch assay, and Transwell. Hematoxylin-eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to observe the histopathology and protein expression. CD4+T cell and CD14+ monocytes differentiation was detected by flow cytometry. Western blot, qRT-PCR and RNAseq were used to detect the action of ASC-exos on gene and protein expression. CD4+T cells could take up ASC-exos. ASC-exos inhibited Th1 and Th17 differentiation and promoted Treg differentiation of CD4+T cells. ASC-exos inhibited M1 differentiation and promoted M2 differentiation of CD14+ monocytes. ASC-exos promoted the migration, proliferation, and invasion, while inhibited apoptosis of MCF-7 cells. ASC-exos promoted the tumor formation of breast cancer. The effect of ASC-exos on tumor microenvironment immunity was in accordance with the above in vitro results. TOX, CD4 and LYZ1 genes were upregulated, while Mettl7b and Serpinb2 genes were downregulated in ASC-exos group. Human T-cell leukemia virus 1 infection pathway was significantly enriched in ASC-exos. Thus, ASC-exos promoted breast cancer characterization and tumor microenvironment immunosuppression by regulating macrophage and T cell differentiation.

Discrete dynamics of light in an anti-parity-time symmetric photonic lattice in atomic vapors.

We demonstrate the realization of an anti-parity-time (PT)-symmetric photonic lattice in a coherent three-level Λ-type 85Rb atomic system both experimentally and theoretically. Such an instantaneously reconfigurable anti-PT-symmetric photonic lattice is "written" by two one-dimensional coupling fields, which are arranged alternately along the x direction and can modulate the refractive index of the atomic vapor in a spatially periodical manner via controllable atomic coherence. By properly adjusting the relevant atomic parameters, the phase shift between two adjacent lattice channels occurs in the constructed non-Hermitian photonic system. Such a readily reconfigurable anti-PT-symmetric photonic lattice may open the door for demonstrating the discrete characteristics of the optical waves in periodic anti-PT-symmetric photonic systems.

Dielectric optical waveguide fabricated on a transparent substrate.

Transparent glass substrates are routinely used in the fabrication of metasurfaces, augmented reality (AR), virtual reality (VR), and holographic devices. While readily compatible with photolithographic patterning methods, when electron beam (E-Beam) techniques are used, field distortion and stitching errors can result due to the buildup of charge. A common approach to overcome this issue is to deposit a thin conductive polymer layer (E-Spacer). However, if high-voltage E-Beam is used to achieve nano-features, the polymer conductivity is not sufficient. We have shown that by using chromium (Cr) as an overcoating conductive layer on the resist, we can achieve accurate and seamless patterning in multiple writing fields and used the method to fabricate on-chip Si3N4 waveguides on SiO2. This technique has the potential to enable the fabrication of large-scale integrated photonic systems on transparent or dielectric substrates.

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

3,6'-Disinapoyl sucrose alleviates cognitive deficits in APP/PS1 transgenic mice.

Alzheimer's disease (AD) is a neurodegenerative disorder with insidious onset and progressive development. There is an urgent need to find drugs that prevent and slow AD progression. We focus our attention on 3,6'-disinapoyl sucrose (DISS), an oligosaccharide with antidepressant and antioxidant activities. In this work, APP/PS1 transgenic mice were used to explore the neuroprotective impact of DISS to provide new applications for prevention and therapy of AD. This study aims to assess DISS's neuroprotective impact on learning and memory deficits in APP/PS1 transgenic mice using behavioral tests (Morris water maze, novel object recognition test, and passive avoidance test). Morphological alterations of hippocampus neurons were observed by Nissl staining and neuronal apoptosis was assessed by TUNEL assay. By using ELISA, the expressions of inflammatory factors were evaluated, and Western blotting was used to measure the protein expressions of neuron-related regulators in the hippocampus. DISS significantly ameliorated the cognitive disorder in APP/PS1 transgenic mice, reduced apoptosis by decreasing the ratio of Bax/B-cell lymphoma/leukemia-2 (Bcl-2) in hippocampal neurons, and restored the abnormal secretion of inflammatory factors (IL-2, TNF-α, IL-1ß, and IL-6). Moreover, the gavage of high-dose DISS can boost the expressions of CREB/brain-derived neurotrophic factor (BDNF). Overall, our results indicate that DISS improves cognitive function in APP/PS1 transgenic mice by inhibiting neural apoptosis and activating the CREB/BDNF signal pathway.NEW & NOTEWORTHY In this study, for the first time, DISS was used in APP/PS1 transgenic mice to explore its neuroprotective effect. After gavage DISS for 1 mo, the impairment of learning and spatial memory ability and the loss of neurons in APP/PS1 mice were alleviated. DISS reduced a neuroprotective effect in AD mice via decreasing neuronal apoptosis, enhancing the expressions of CREB phosphorylation and BDNF, pointing to DISS as a new therapeutic target for AD.

YCH1899, a Highly Effective Phthalazin-1(2H)-one Derivative That Overcomes Resistance to Prior PARP Inhibitors.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have significant efficacy in treating BRCA-deficient cancers, although resistance development remains an unsolved challenge. Herein, a series of phthalazin-1(2H)-one derivatives with excellent enzymatic inhibitory activity were designed and synthesized, and the structure-activity relationship was explored. Compared with olaparib and talazoparib, compound YCH1899 exhibited distinct antiproliferation activity against olaparib- and talazoparib-resistant cells, with IC50 values of 0.89 and 1.13 nM, respectively. Studies of the cellular mechanism revealed that YCH1899 retained sensitivity in drug-resistant cells with BRCA1/2 restoration or 53BP1 loss. Furthermore, YCH1899 had acceptable pharmacokinetic properties in rats and showed prominent dose-dependent antitumor activity in olaparib- and talazoparib-resistant cell-derived xenograft models. Overall, this study suggests that YCH1899 is a new-generation antiresistant PARPi that could provide a valuable direction for addressing drug resistance to existing PARPi drugs.

Exosomal miR-6733-5p mediates cross-talk between glioblastoma stem cells and macrophages and promotes glioblastoma multiform progression synergistically.

AIM: Exosomal miRNAs derived from glioblastoma stem cells (GSCs) are important mediators of immunosuppressive microenvironment formation in glioblastoma multiform (GBM), especially in M2-like polarization of tumor-associated macrophages (TAMs). However, the exact mechanisms by which GSCs-derived exosomes (GSCs-exo) facilitate the remodeling of the immunosuppressive microenvironment of GBM have not been elucidated. METHODS: Transmission electron microscopy (TME) and nanoparticle tracking analysis (NTA) were applied to verify the existence of GSCs-derived exosomes. Sphere formation assays, flow cytometry, and tumor xenograft transplantation assays were performed to identify the exact roles of exosomal miR-6733-5p. Then, the mechanisms of miR-6733-5p and its downstream target gene regulating crosstalk between GSCs cells and M2 macrophages were further investigated. RESULTS: GSCs-derived exosomal miR-6733-5p induce macrophage M2 polarization of TAMs by positively targeting IGF2BP3 to activate the AKT signaling pathway, which further facilitates the self-renewal and stemness of GSCs. CONCLUSION: GSCs secrete miR-6733-5p-rich exosomes to induce M2-like polarization of macrophages, as well as enhance GSCs stemness and promote malignant behaviors of GBM through IGF2BP3 activated AKT pathway. Targeting GSCs exosomal miR-6733-5p may provide a potential new strategy against GBM.

HDAC1 mediates epithelial-mesenchymal transition and promotes cancer cell invasion in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most malignant tumors of the central nervous system, and its treatment has always been a difficult clinical problem. Here, we evaluated HDAC1 expression patterns and their effect on prognosis based on GBM cases from TCGA and CGGA databases. Expression was compared between GBM samples and normal controls. High HDAC1 expression was found to be an indicator of poor prognosis in glioblastoma. We also established a protein-protein interaction network to explore HDAC1-related interacting proteins, including the epithelial-mesenchymal transition (EMT)-related protein VIM, which is closely associated with HDAC1. Consistently, functional enrichment analysis showed that several GBM tissues with high HDAC1 were enriched in the expression of cancer markers, such as those involved in glycolysis, hypoxia, inflammation, and some signaling pathways. Next, this study analyzed the effect of HDAC1 on invasive ability and the EMT signaling pathway in GBM cells in vitro. The results showed that an HDAC1 inhibitor (RGFP109) could inhibit the EMT process in glioma cells in vitro, thereby affecting the invasion and migration of cells. Similar results were obtained based on in vivo studies. Our data suggest that HDAC1 has the potential to be a powerful prognostic biomarker, which might provide a basis for developing therapeutic targets for GBM.

Upregulation of the ZNF148/PTX3 axis promotes malignant transformation of dendritic cells in glioma stem-like cells microenvironment.

BACKGROUND: The recent development of dendritic cell (DC)-based immunotherapy has resulted in advances in glioblastoma multiforme (GBM) treatment. However, the cell fate of DCs in the GBM microenvironment, especially in microenvironments in which glioma stem cell (GSCs)-mediated remodeling has resulted in highly immunosuppressive conditions, has not yet been fully investigated. METHODS: Observed the interaction between GSCs and primary cultured DCs in a dual-color tracing model, monoclonal and continuously passaged highly proliferative DCs, and named transformed DCs (t-DCs). The expression of DC-specific surface markers was analyzed using RT-PCR, chromosome karyotype, and flow cytometry. The expression of long pentraxin 3 (PTX3) and its transcription factor zinc finger protein 148 (ZNF148) in t-DCs was detected using qRT-PCR and western blot. CCK8 and transwell assays were conducted to assess the effect of ZNF148 and PTX3 on the proliferation, migration, and invasion of t-DCs. Bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP)-qPCR assay were used to explore the relation between ZNF148 and PTX3. RESULTS: Transformed DCs (t-DCs) still expressed DC-specific surface markers, namely, CD80 and CD11c, and immune-related costimulatory molecules, namely, CD80, CD86, CD40, and ICAM-1. However, the expression levels of these molecules in t-DCs decreased moderately compared to those in naive DCs. Stable overexpression of PTX3 further promoted the proliferation and migration of t-DCs in vitro, decreased the expression of costimulatory molecules, and increased the tumorigenicity of t-DCs in vivo. The transcription factor zinc finger protein 148 (ZNF148) was directly bound to the PTX3 promoter region and enhanced PTX3 expression. Downregulation of ZNF148 significantly decreased PTX3 expression and reduced the proliferation and migration of t-DCs. Overexpression of ZNF148 significantly increased PTX3 expression and promoted the proliferation and migration of t-DCs, achieving the same biological effects as PTX3 overexpression in t-DCs. Simultaneously, the downregulation of ZNF148 partially reversed the effect of PTX3 overexpression in t-DCs. CONCLUSION: The ZNF148/PTX3 axis played an important role in regulating the malignant transformation of DCs after cross-talk with GSCs, and this axis may serve as a new target for sensitizing GBM to DC-based immunotherapy.

Prognostic value and immune-infiltration pattern of FOXD3-AS1 in patients with glioma.

Gliomas are difficult-to-treat brain tumors due to their aggressive nature, rapid proliferation, and high invasiveness (Zhang et al., J Cell Biochem, 2019, 120 (9), 15106-15118; Ge et al., Int J Biochem Cell Biol, 2021, 139, 106054). FOXD3-AS1 has been identified as an emerging potential target for tumor prediction and treatment in many studies (Qin et al., Front Oncol, 2021, 11, 688027). However, the utility of FOXD3-AS1 has not been reported in glioma patients (Li et al., Cancer Manag Res, 2021, 13, 9037-9048). The differential profiles of FOXD3-AS1 in TCGA-GBMLGG database were analyzed across clinical subgroups. The analysis of overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) revealed that a high level of FOXD3-AS1 was associated with a poor prognosis and survival outcome. Based on the Cox regression analysis, FOXD3-AS1 was found to be a high-risk factor for glioma that affects prognosis outcomes independently. More importantly, because oxidative stress is closely linked to glioma prognosis, we focused on the potential mechanisms of six oxidative stress co-expressed genes with FOXD3-AS1. In addition, the predictive value of FOXD3-AS1 was determined for each clinical subgroup status. The ROC curve results showed that FOXD3-AS1 had a good predictive performance. A stratified clinicopathological subgroup analysis revealed that high expression of FOXD3-AS1 is associated with a poor prognosis. This also indicates a link between FOXD3-AS1 and tumorigenesis and prognosis, which has potential application value. Furthermore, the immune cell infiltration of FOXD3-AS1 and the signal marker correlation suggested that immune cell infiltration differed significantly between immune cell subsets. To the best of our knowledge, this is the first report to investigate FOXD3-AS1 in glioma and how it may modulate GBM and LGG immune microenvironments. Furthermore, FOXD3-AS1 was detected in tumor and paraneoplastic tissues using RT-qPCR. Transwell analysis verified the migration and invasion of the FOXD3-AS1 knockout group in vitro to a certain extent. In conclusion, FOXD3-AS1 can be used as a prognostic indicator for GBM and LGG, and it is closely related to immune infiltration and response to oxidative stress, which may contribute to the advancement of glioma immunotherapy research.