U87MG Glioma Research Articles

Receptor-Targeted Carbon Nanodot Delivery through Polymer Caging and Click Chemistry-Supported LRP1 Ligand Attachment.

Carbon nanodots present resistance to photobleaching, bright photoluminescence, and superior biocompatibility, making them highly promising for bioimaging applications. Herein, nanoprobes were caged with four-armed oligomers and subsequently modified with a novel DBCO-PEG-modified retro-enantio peptide ligand reL57, enhancing cellular uptake into U87MG glioma cells highly expressing low-density lipoprotein receptor-related protein 1 (LRP1). A key point in the development of the oligomers was the incorporation of ε-amino-linked lysines instead of standard α-amino-linked lysines, which considerably extended the contour length per monomer. The four-armed oligomer 1696 was identified as the best performer, spanning a contour length of ~8.42 nm for each arm, and was based on an altering motive of two cationic ε-amidated lysine tripeptides and two tyrosine tripeptides for electrostatic and aromatic stabilization of the resulting formulations, cysteines for disulfide-based caging, and N-terminal azidolysines for click-modification. This work highlights that well-designed four-armed oligomers can be used for noncovalent coating and covalent caging of nanoprobes, and click modification using a novel LRP1-directed peptide ligand facilitates delivery into receptor-expressing target cells.

Improve BBB Penetration and Cytotoxicity of Palbociclib in U87-MG Glioblastoma Cells Delivered by Dual Peptide Functionalized Nanoparticles.

Palbociclib (PBC) is an FDA-approved CDK4/6 inhibitor used for breast cancer treatment. PBC has been demonstrated its ability to suppress the proliferation of glioma cells by inducing cell cycle arrest. However, the efflux transporters on the blood-brain barrier (BBB) restricts the delivery of PBC to the brain. The nano-delivery strategy with BBB-penetrating and glioma-targeting abilities was designed. Poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) was functionalized with the potential peptide, T7 targeting peptide and/or R9 penetrating peptide, to prepare PBC-loaded nanoparticles (PBC@NPs). The size of PBC@NPs was in the range of 168.4 ± 4.3-185.8 ± 4.4 nm (PDI < 0.2), and the zeta potential ranged from -17.8 ± 1.4 mV to -14.3 ± 1.0 mV dependent of conjugated peptide. The transport of PBC@NPs across the bEnd.3 cell model was in the order of dual-peptide modified NPs > T7-peptide modified NPs > peptide-free NPs > free PBC, indicating facilitated delivery of PBC by NPs, particularly the T7/R9 dual-peptide modified NPs. Moreover, PBC@NPs significantly enhanced U87-MG glioma cell apoptosis by 2.3-6.5 folds relative to PBC, where the dual-peptide modified NPs was the most effective one. In conclusion, the PBC loaded dual-peptide functionalized NPs improved cellular uptake in bEnd.3 cells followed by targeting to U87-MG glioma cells, leading to effective cytotoxicity and promoting cell death.

αV-Integrin-Dependent Inhibition of Glioblastoma Cell Migration, Invasion and Vasculogenic Mimicry by the uPAcyclin Decapeptide.

Among the deadliest human cancers is glioblastoma (GBM) for which new treatment approaches are urgently needed. Here, the effects of the cyclic decapeptide, uPAcyclin, are investigated using the U87-MG, U251-MG, and U138-MG human GBM and C6 rat cell models. All GBM cells express the αV-integrin subunit, the target of uPAcyclin, and bind specifically to nanomolar concentrations of the decapeptide. Although peptide exposure affects neither viability nor cell proliferation rate, nanomolar concentrations of uPAcyclin markedly inhibit the directional migration and matrix invasion of all GBM cells, in a concentration- and αV-dependent manner. Moreover, wound healing rate closure of U87-MG and C6 rat glioma cells is reduced by 50% and time-lapse videomicroscopy studies show that the formation of vascular-like structures by U87-MG in three-dimensional matrix cultures is markedly inhibited by uPAcyclin. A strong reduction in the branching point numbers of the U87-MG, C6, and U251-MG cell lines undergoing vasculogenic mimicry, in the presence of nanomolar peptide concentrations, was observed. Lysates from matrix-recovered uPAcyclin-exposed cells exhibit a reduced expression of VE-cadherin, a prominent factor in the acquisition of vascular-like structures. In conclusion, these results indicate that uPAcyclin is a promising candidate to counteract the formation of new vessels in novel targeted anti-GBM therapies.

Development of a Highly Specific 18F-Labeled Radioligand for Imaging of the Sigma-2 Receptor in Brain Tumors.

Novel ligands with the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline or 5,6-dimethoxyisoindoline pharmacophore were designed and synthesized for evaluation of their structure-activity relationship to the sigma-2 (σ2) receptor and developed as suitable PET radioligands. Compound 1 was found to possess nanomolar affinity (Ki(σ1) = 2.57 nM) for the σ2 receptor, high subtype selectivity (>2000-fold), and high selectivity over 40 other receptors and transporters. Radioligand [18F]1 was prepared with radiochemical yield of 37-54%, > 99% radiochemical purity, and molar activity of 107-189 GBq/μmol. Biodistribution and blocking studies in mice and micro-PET/CT imaging of [18F]1 in rats indicated excellent binding specificity to the σ2 receptors in vivo. Micro-PET/CT imaging of [18F]1 in the U87MG glioma xenograft model demonstrated clear tumor visualization with high tumor uptake and tumor-to-background ratio. Co-injection with CM398 (5 μmol/kg) led to a remarkable reduction of tumor uptake (80%, 60-70 min), indicating high specific binding of [18F]1 in U87MG glioma xenografts.

Influence of dextran-70 on the cytotoxic effect of EMAP II on glioma cells in vitro

Aim. To evaluate with the use of the MTT test how the formation of a nanocomposite complex of the cytokine EMAP II with dextran-70 affects the cytotoxic effect of the free protein in glioma cell cultures of different origins: a standart culture U251MG cells and a primary culture of cells obtained from tumor fragments. Methods. The recombinant polypeptide EMAP II was obtained using gene engineering biotechnology. The study was conducted on the standard human glioma cell line U251MG and on primary cell cultures obtained from tumor fragments after surgical removal. Cell viability was determined using the MTT test. The cells were cultivated for a day with t the different concentrations from 1.0 pM to 10 μM of EMAP II and the EMAP II + dextran-70 complex im serum-free standard DMEM growth medium. Results. In our previous works, it has been shown that EMAP II exhibited dose-dependent cytotoxic properties in the concentration range of 1.0 pM – 10 μM on U251MG glioma cells and in primary cell cultures. The dose-effect curve in all cases has a complex pattern. Dextran-70 does not fundamentally affect the cytotoxic effect of EMAP II, but depending on the dose and type of cells, it shows the ability to slightly weaken/enhance the effect of the free protein. An increased sensitivity of the primary culture of glioma cells to the cytotoxic effect of the EMAP II + dextran-70 complex was revealed. Conclusions. Dextran-70 does not fundamentally affect the cytotoxic effect of EMAP II, but depending on the dose and type of cells, it shows the ability to slightly weaken/enhance the effect of the latter.

Evaluation of (2S,4S)-4-[18F]FEBGln as a Positron Emission Tomography Tracer for Tumor Imaging.

Glutamine metabolism-related tracers have the potential to visualize numerous tumors because glutamine is the second largest source of energy for tumors. (2S,4S)-4-[18F]FEBGln was designed by introducing [18F]fluoroethoxy benzyl on carbon-4 of glutamine. The aim of this study was to investigate the pharmacokinetic properties and tumor positron emission tomography (PET) imaging characteristics of (2S,4S)-4-[18F]FEBGln in detail. The biodistribution results of nude mice bearing MCF-7 tumor showed that (2S,4S)-4-[18F]FEBGln had high initial tumor uptake, and a fast clearance rate, resulting in a high tumor-to-muscle ratio at 30 min postinjection. There was no obvious defluorination in vivo. The micro-PET-CT imaging results of (2S,4S)-4-[18F]FEBGln orthotopic MCF-7 tumor-bearing nude mice were consistent with the biological distribution results. Compared with (2S,4R)-4-[18F]FGln, (2S,4S)-4-[18F]FEBGln showed poor tumor retention, but its clearance in normal tissues was also fast, so it had better PET image contrast than the former. Unlike poor retention in MCF-7-bearing nude mice, (2S,4S)-4-[18F]FEBGln has good retention in NCI-h1975 and 22Rv1 tumor models. Since (2S,4S)-4-[18F]FEBGln has low uptake in normal lungs and high uptake in the bladder, it is expected to be used in the accurate diagnosis of lung cancer but cannot accurately determine prostate cancer. Consistent with the advantages of radiolabeled amino acids in the application of brain tumors, (2S,4S)-4-[18F]FEBGln accurately diagnoses U87MG glioma with higher contrast than [18F]FET and [18F]FDG, and there is a correlation between (2S,4S)-4-[18F]FEBGln uptake and tumor growth cycle. Further kinetic model analysis showed that (2S,4S)-4-[18F]FEBGln was similar to (2S,4R)-4-[18F]FGln, conforming to the one-compartment model and the Logan graphical model, and was expected to assess the size of the glutamine pool of the tumor. Therefore, (2S,4S)-4-[18F]FEBGln is expected to provide a strong imaging basis for the diagnosis, formulation of personalized plans, and efficacy evaluation of glioma, lung cancer, and breast cancer.

Separate and Mutual Effects of BIRB796 and Bortezomib on pHsp27 and Viability of U87MG Glioma Cells

Separate and Mutual Effects of BIRB796 and Bortezomib on pHsp27 and Viability of U87MG Glioma Cells

Recombinant SLURP-1 Inhibits Growth and Migration of U251 MG Glioma by Cell Cycle Arrest and Modulation of MAPK and AKT Signaling Pathways

A recombinant analog of the human SLURP-1 protein, rSLURP-1, effectively inhibits the growth of carcinomas by interaction with the α7-type nicotinic acetylcholine receptor. Recently, rSLURP-1 inhibition of glioma growth in vitro was shown by the authors; however, the action of rSLURP-1 was not studied. Here, we showed that rSLURP-1 selectively inhibits the growth of U251 MG glioma cells, but not of normal astrocytes, and controls glioma cell migration. In addition, rSLURP-1 induces cell cycle arrest in the G2/M phase in U251 MG glioma cells, but does not result in apoptosis. Incubation of U251 MG cells with rSLURP-1 causes inhibition of phosphorylation of ERK, p38 MAPK, and AKT kinases, whose activation contributes to the progression of gliomas. At the same time, rSLURP-1 does not affect the activity of JNK kinase. Thus, rSLURP-1 is an endogenous protein promising for the development of drugs based on it for the treatment of not only carcinomas but also gliomas.

Synthesis and in vitro anticancer activity of novel 1,4-dimethyl-9-H-carbazol-3-yl) methanamine derivatives against human glioma U87 MG cell line

Glioblastoma Multiforme (GBM) is most aggressive type of brain tumor in adults. 1,4-dimethyl carbazoles exhibits significant anticancer activities in literature. In this research article, we synthesized a series of novel 1,4-dimethyl-9-H-carbazol-3-yl)methanamine and its derivatives (13-24) and evaluated their in vitro cytotoxicity activities against human glioma U87 MG cell line using MTT assay for 24 h phase time period. All final carbazole derivatives were well confirmed by NMR and HRMS spectroscopy techniques. In series, few compound (15-17) found excellent in vitro anticancer activity (IC50) values 18.50 µM, 47 µM and 75 µM respectively against human glioma U87MG cell line compare to standard drugs used in brain cancer such as Carmustine (IC50 = 18.24 µM) and Temozolomide (IC50 = 100 µM) respectively. Among these derivatives, compound 15 was found to have the most potent cytotoxic effect on tested glioma cell line. This study supported that 1,4-dimethyl-9-H-carbazol-3-yl)methanamine derivatives found significant anticancer potential against human glioma U87MG cell line.

Intracellular Acidification in a Rat C6 Glioma Model following Cariporide Injection Investigated by CEST-MRI.

Acidification of cancerous tissue induced pharmacologically may slow tumor growth and can be detected using magnetic resonance imaging. Numerous studies have shown that pharmacologically inhibiting specific transporters, such as the Na+/H+ exchanger 1 (NHE1), can alter glycolitic metabolism and affect tumor acidosis. The sodium proton exchanger inhibitor Cariporide can acidify U87MG gliomas in mice. This study aimed to determine whether Cariporide could acidify C6 glioma tumors in rats with an intact immune system. C6 glioma cells were implanted in the right brain hemisphere of ten rats. Chemical exchange saturation transfer (CEST) MRI (9.4T) was acquired on days 7-8 and 14-15 after implantation to measure in vivo tissue intracellular pH (pHi) within the tumors and on the contralateral side. pHi was basic relative to contralateral tissue at both time points assessed using the amine and amide concentration-independent detection (AACID) value. On day 14-15, measurements were made before and up to 160 min after Cariporide injection (N = 6). Twenty minutes after drug injection, the average AACID value in the tumor significantly increased by ∼6.4% compared to pre-injection, corresponding to 0.31 ± 0.20 lower pHi, while in contralateral tissue, AACID value increased significantly by ∼4.3% compared to pre-injection, corresponding to 0.22 ± 0.19 lower pHi. Control rats without tumors showed no changes following injection of Cariporide dissolved in 10% or 1% DMSO and diluted in PBS. This study demonstrates the sensitivity of CEST-based pH-weighted imaging for monitoring the response of tumors to pharmacologically induced acidification.

Recombinant SLURP-1 Inhibits Growth and Migration of U251 MG Glioma by Cell Cycle Arrest and Modulation of MAPK and AKT Signaling Pathways

A recombinant analog of the human SLURP-1 protein (rSLURP-1) effectively inhibits the growth of carcinomas by interaction with the α7-type nicotinic acetylcholine receptor. Recently, rSLURP-1 inhibition of gliomas growth in vitro was shown by the authors, although, the mechanism of rSLURP-1 action was not studied. Here, we showed that rSLURP-1 selectively inhibits the growth of U251 MG glioma cells but not of normal astrocytes, and controls glioma cell migration. In addition, rSLURP-1 induces cell cycle arrest in the G2/M phase in U251 MG glioma cells, but does not result in apoptosis. Incubation of U251 MG cells with rSLURP-1 causes inhibition of phosphorylation of ERK, p38 MAPK, and AKT kinases, the activation of which contributes to the progression of gliomas. At the same time, rSLURP-1 does not affect the activity of JNK kinase. Thus, rSLURP-1 is an endogenous protein promising for the development of drugs based on it for the treatment of not only carcinomas, but also gliomas.

Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research

This study aimed to explore the anti-glioma effect of natural compound pterostilbene(PTE) through regulating pyroptosis and apoptosis pathways, and to analyze the possible anti-glioma pathways and targets of PTE by network pharmacology and molecular docking. In this study, the action targets of PTE and the glioma targets were obtained by network pharmacology to construct a target network and a protein-protein interaction(PPI) network to predict the possible action targets of PTE against glioma. Molecular docking was performed on the core targets by AutoDock and the action pathways of PTE against glioma were predicted by enrichment analysis. In addition, the effect of PTE on the viability of U87MG and GL261 glioma cells was detected by CCK-8 assay. Clone formation assay and cell scratching assay were used to explore the effect of different concentrations of PTE on the proliferation and migration, respectively of glioma cells. Hoechst staining was used to observe PTE-induced apoptosis in glioma cells. The changes in mitochondrial membrane potential were detected by JC-1 staining. The pyroptosis-inducing effect of PTE on glioma cells was observed by inverted microscopy and lactate dehydrogenase(LDH) assay. Hoechst 33342/PI dual staining assay was performed to detect the integrity of glioma cell membranes. The expressions of pyroptosis and apoptosis-related proteins in glioma cells after PTE induction were determined by Western blot. In this study, 37 anti-glioma targets of PTE were obtained, and enrichment analysis suggested that PTE exerted anti-glioma effects through various signaling pathways including cancer pathway, proteoglycan in cancer, PI3K/AKT pathway, and apoptosis regulatory pathway. Molecular docking revealed that PTE had good binding activity with the main targets. Compared with the control group, PTE significantly reduced the viability as well as the proliferation, migration and adhesion abilities of U87MG and GL261 cells; it induced the apoptosis of the two glioma cells and the decrease of mitochondrial membrane potential in U87MG cells, and the effects increased with the increase of drug concentration. Compared with the conditions in the control group, glioma cells in the PTE group had increased pyroptosis-specific appearance and gradually increased LDH release; the number of PI positive cells was significantly elevated with the increase of PTE concentration as revealed by Hoechst 33342/PI staining; the expression levels of apoptosis-related factors cleaved PARP1 and B-cell lymphoma-2(Bcl-2) associated X(BAX) in the PTE group were markedly up-regulated, while the expression level of Bcl-2 was markedly down-regulated; the activation levels of pyroptosis-related proteins cleaved caspase-3 and gasdermin E-N(GSDME-N) had a remarkable rise in the PTE group, while no significant changes were found in the activation levels of gasdermin D-N(GSDMD-N) and cleaved caspase-1. In summary, PTE plays an anti-glioma role by inhibiting cell viability, proliferation, and migration and activating the caspase-3/GSDME-mediated pyroptosis pathway and mitochondrial apoptosis pathway.

Atraumatic access to human glioblastoma in a xenograft animal model by cerebral open flow microperfusion

BackgroundOrthotopic xenograft studies promote the development of targeted/personalized therapies to improve the still poor life expectancy of glioblastoma patients. New methodWe implemented an atraumatic access to glioblastoma with cerebral Open Flow Microperfusion (cOFM) by implantation of xenograft cells in rat brain with intact blood brain barrier (BBB) and subsequent development of a xenograft glioblastoma at the interface between the cOFM probe and surrounding brain tissue. Human glioma U87MG cells were implanted at a well-defined position into immunodeficient Rowett nude rat´s brain via cOFM (cOFM group) and syringe (control group). Characteristics of the mature tumors from both groups were assessed. ResultsFor the first time xenograft cells were successfully introduced into rat brain with intact BBB using cOFM, and the tumor tissue developing around the cOFM probe was unaffected by the presence of the probe. Thereby an atraumatic access to the tumor was created. The success rate of glioblastoma development in the cOFM group was high (>70%). The mature cOFM-induced tumors (20–23 days after cell-implantation) resembled the syringe-induced ones and showed typical features of human glioblastoma. Comparison with existing methodExamining xenograft tumor microenvironment with currently available methods inevitably causes trauma that could affect the reliability of obtained data. ConclusionThis novel atraumatic access to human glioblastoma in rat brain provides the possibility to collect interstitial fluid from functional tumor tissue in vivo without trauma generation. Thereby, reliable data can be generated promoting drug research, biomarker identification, and enabling investigation of the BBB of an intact tumor.

Holographic Focused Ultrasound Hyperthermia System for Uniform Simultaneous Thermal Exposure of Multiple Tumor Spheroids.

Hyperthermia is currently used to treat cancer due to its ability to radio- and chemo-sensitize and to stimulate the immune response. While ultrasound is non-ionizing and can induce hyperthermia deep within the body non-invasively, achieving uniform and volumetric hyperthermia is challenging. This work presents a novel focused ultrasound hyperthermia system based on 3D-printed acoustic holograms combined with a high-intensity focused ultrasound (HIFU) transducer to produce a uniform iso-thermal dose in multiple targets. The system is designed with the aim of treating several 3D cell aggregates contained in an International Electrotechnical Commission (IEC) tissue-mimicking phantom with multiple wells, each holding a single tumor spheroid, with real-time temperature and thermal dose monitoring. System performance was validated using acoustic and thermal methods, ultimately yielding thermal doses in three wells that differed by less than 4%. The system was tested in vitro for delivery of thermal doses of 0-120 cumulative equivalent minutes at 43 °C (CEM43) to spheroids of U87-MG glioma cells. The effects of ultrasound-induced heating on the growth of these spheroids were compared with heating using a polymerase chain reaction (PCR) thermocycler. Results showed that exposing U87-MG spheroids to an ultrasound-induced thermal dose of 120 CEM43 shrank them by 15% and decreased their growth and metabolic activity more than seen in those exposed to a thermocycler-induced heating. This low-cost approach of modifying a HIFU transducer to deliver ultrasound hyperthermia opens new avenues for accurately controlling thermal dose delivery to complex therapeutic targets using tailored acoustic holograms. Spheroid data show that thermal and non-thermal mechanisms are implicated in the response of cancer cells to non-ablative ultrasound heating.

A novel prognostic related lncRNA signature associated with amino acid metabolism in glioma.

Glioma is one of the deadliest malignant brain tumors in adults, which is highly invasive and has a poor prognosis, and long non-coding RNAs (lncRNAs) have key roles in the progression of glioma. Amino acid metabolism reprogramming is an emerging hallmark in cancer. However, the diverse amino acid metabolism programs and prognostic value remain unclear during glioma progression. Thus, we aim to find potential amino-related prognostic glioma hub genes, elaborate and verify their functions, and explore further their impact on glioma. Glioblastoma (GBM) and low-grade glioma (LGG) patients' data were downloaded from TCGA and CCGA datasets. LncRNAs associated with amino acid metabolism were discriminated against via correlation analysis. LASSO analysis and Cox regression analysis were conducted to identify lncRNAs related to prognosis. GSVA and GSEA were performed to predict the potential biological functions of lncRNA. Somatic mutation data and CNV data were further built to demonstrate genomic alterations and the correlation between risk scores. Human glioma cell lines U251 and U87-MG were used for further validation in vitro experiments. There were eight amino-related lncRNAs in total with a high prognostic value that were identified via Cox regression and LASSO regression analyses. The high risk-score group presented a significantly poorer prognosis compared with the low risk-score group, with more clinicopathological features and characteristic genomic aberrations. Our results provided new insights into biological functions in the above signature lncRNAs, which participate in the amino acid metabolism of glioma. LINC01561 is one of the eight identified lncRNAs, which was adopted for further verification. In in vitro experiments, siRNA-mediated LINC01561 silencing suppresses glioma cells' viability, migration, and proliferation. Novel amino-related lncRNAs associated with the survival of glioma patients were identified, and a lncRNA signature can predict glioma prognosis and therapy response, which possibly has vital roles in glioma. Meanwhile, it emphasized the importance of amino acid metabolism in glioma, particularly in providing deeper research at the molecular level.

Abstract 2605: LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells

Abstract Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target. Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.

Drug-Loading Content Influences Cellular Uptake of Polymer-Coated Nanocellulose.

While the effects of nanoparticle properties such as shape and size on cellular uptake are widely studied, influences exerted by drug loading have so far been ignored. In this work, nanocellulose (NC) coated by Passerini reaction with poly(2-hydroxy ethyl acrylate) (PHEA-g-NC) was loaded with various amounts of ellipticine (EPT) by electrostatic interactions. The drug-loading content was determined by UV-vis spectroscopy to range between 1.68 and 8.07 wt %. Dynamic light scattering and small-angle neutron scattering revealed an increased dehydration of the polymer shell with increasing drug-loading content, which led to higher protein adsorption and more aggregation. The nanoparticle with the highest drug-loading content, NC-EPT8.0, displayed reduced cellular uptake in U87MG glioma cells and MRC-5 fibroblasts. This also translated into reduced toxicity in these cell lines as well as the breast cancer MCF-7 and the macrophage RAW264.7 cell lines. Additionally, the toxicity in U87MG cancer spheroids was unfavorable. The nanoparticle with the best performance was found to have intermediate drug-loading content where the cellular uptake was adequately high while each nanoparticle was able to deliver a sufficiently toxic amount into the cells. Medium drug loading did not hinder uptake into cells while maintaining sufficiently toxic drug concentrations. It was concluded that while striving for a high drug-loading content is appropriate when designing clinically relevant nanoparticles, it needs to be considered that the drug can cause changes in the physicochemical properties of the nanoparticles that might cause unfavorable effects.

Identification of cancer stem cells derived from U118MG and the involvement of LncRNA-DC and STAT3 in promoting their malignant transformation

Abstract Objectives Cancer stem cells (CSCs) are a subpopulation of cancer cells that share similarities with somatic stem cells. CSCs are believed to play a key role in carcinogenesis, metastasis, cancer relapse, and drug resistance. Despite their significant impacts, the specific biological markers for the identification of CSCs and their differentiation/transformation mechanisms have not yet been fully characterized. Methods Utilizing stem cell markers, the ability to differentiate in multiple directions, and resistance to radiotherapy and chemotherapy, CSCs were identified. To assess the variations in gene expression, gene alterations, protein expression, and cell proliferation between CSCs and U118MG glioma cells, second generation sequencing, Real-Time PCR, Western Blotting, and CCK-8 were employed. Results In this study, we identified a subset of CSCs in human U118MG glioma cells that expressed the stem cell biomarkers CD133+, OCT4+, and CD44+. These cells exhibited stem cell-like characteristics such as multilineage differentiation and resistance to chemical and radiation stresses. Notably, they can form neurons with electrical signals and sodium currents. Further study also revealed that the malignant growth of this CSC subset was controlled by long noncoding RNA (Lnc-DC) through the STAT3 pathway. Conclusions As a potential therapeutic approach, inhibiting Lnc-DC may be beneficial in hindering carcinogenesis and drug resistance, as it selectively targets the growth of CSCs.

RTA dh404 Induces Cell Cycle Arrest, Apoptosis, and Autophagy in Glioblastoma Cells.

RTA dh404 is a novel synthetic oleanolic acid derivative that has been reported to possess anti-allergic, neuroprotective, antioxidative, and anti-inflammatory properties, and exerts therapeutic effects on various cancers. Although CDDO and its derivatives have anticancer effects, the actual anticancer mechanism has not been fully explored. Therefore, in this study, glioblastoma cell lines were exposed to different concentrations of RTA dh404 (0, 2, 4, and 8 µM). Cell viability was evaluated using the PrestoBlue™ reagent assay. The role of RTA dh404 in cell cycle progression, apoptosis, and autophagy was analyzed using flow cytometry and Western blotting. The expression of cell cycle-, apoptosis-, and autophagy-related genes was detected by next-generation sequencing. RTA dh404 reduces GBM8401 and U87MG glioma cell viability. RTA dh404 treated cells had a significant increase in the percentage of apoptotic cells and caspase-3 activity. In addition, the results of the cell cycle analysis showed that RTA dh404 arrested GBM8401 and U87MG glioma cells at the G2/M phase. Autophagy was observed in RTA dh404-treated cells. Subsequently, we found that RTA dh404-induced cell cycle arrest, apoptosis, and autophagy were related to the regulation of associated genes using next-generation sequencing. Our data indicated that RTA dh404 causes G2/M cell cycle arrest and induces apoptosis and autophagy by regulating the expression of cell cycle-, apoptosis-, and autophagy-related genes in human glioblastoma cells, suggesting that RTA dh404 is a potential drug candidate for the treatment of glioblastoma.

PROS1 shapes the immune-suppressive tumor microenvironment and predicts poor prognosis in glioma.

Glioma is the most malignant cancer in the brain. As a major vitamin-K-dependent protein in the central nervous system, PROS1 not only plays a vital role in blood coagulation, and some studies have found that it was associated with tumor immune infiltration. However, the prognostic significance of PROS1 in glioma and the underlying mechanism of PROS1 in shaping the tumor immune microenvironment (TIME) remains unclear. The raw data (including RNA-seq, sgRNA-seq, clinicopathological variables and prognosis, and survival data) were acquired from public databases, including TCGA, GEPIA, CGGA, TIMER, GEO, UALCAN, and CancerSEA. GO enrichment and KEGG pathway analyses were performed using "cluster profiler" package and visualized by the "ggplot2" package. GSEA was conducted using R package "cluster profiler". Tumor immune estimation resource (TIMER) and spearman correlation analysis were applied to evaluate the associations between infiltration levels of immune cells and the expression of PROS1. qRT-PCR and WB were used to assay the expression of PROS1. Wound-healing assay, transwell chambers assays, and CCK-8 assays, were performed to assess migration and proliferation. ROC and KM curves were constructed to determine prognostic significance of PROS1 in glioma. The level of PROS1 expression was significantly increased in glioma in comparison to normal tissue, which was further certificated by qRT-PCR and WB in LN-229 and U-87MG glioma cells. High expression of PROS1 positively correlated with inflammation, EMT, and invasion identified by CancerSEA, which was also proved by downregulation of PROS1 could suppress cells migration, and proliferation in LN-229 and U-87MG glioma cells. GO and KEGG analysis suggested that PROS1 was involved in disease of immune system and T cell antigen receptor pathway. Immune cell infiltration analysis showed that expression of PROS1 was negatively associated with pDC and NK CD56 bright cells while positively correlated with Macrophages, Neutrophils in glioma. Immune and stromal scores analysis indicated that PROS1 was positively associated with immune score. The high level of PROS1 resulted in an immune suppressive TIME via the recruitment of immunosuppressive molecules. In addition, Increased expression of PROS1 was correlated with T-cell exhaustion, M2 polarization, poor Overall-Survival (OS) in glioma. And it was significantly related to tumor histological level, age, primary therapy outcome. The results of our experiment and various bioinformatics approaches validated that PROS1 was a valuable poor prognostic marker. Increased expression of PROS1 was correlated with malignant phenotype and associated with poor prognosis in glioma. Besides, PROS1 could be a possible biomarker and potential immunotherapeutic target through promoting the glioma immunosuppressive microenvironment and inducing tumor-associated macrophages M2 polarization.