Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: Hypoxia impacts gene expression and signaling pathways in GBM cells (A) Hypoxia-responsive pathways were identified using Partek Flow software under double filter MI50 and fold change >+1.5 criteria. (B) Expression of candidate genes in PBT030 cells following 48-h hypoxia exposure compared to cells under normoxic conditions. (C) Expression of candidate genes in U-87 MG cells after 48 h of hypoxia compared to cells maintained under normoxic conditions. Representative bar graphs are derived from at least two independent experiments, with 28S used as the reference gene for all experiments.

Article Snippet: U-251 MG and U-87 MG cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Gene Expression, Protein-Protein interactions, Software, Expressing, Derivative Assay

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Overview of Research Workflow. (A) Representative bright‐field micrographs showing the morphology of U87MG and U251MG cells cultured under 2D and 3D spheroid conditions. Representative images were obtained from three independent cultures ( n = 3), Scale bar: 100 µm, 4× magnification. (B) Graphical summary of research workflow.

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques: Cell Culture

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Identification of total and differentially expressed proteins. Total proteins (A) and differentially expressed proteins (B) were identified in U87MG and U251MG cells cultured under two‐dimensional (2D) and three‐dimensional (3D) conditions using LC‐MS/MS. Volcano plots illustrate the upregulated and downregulated proteins in U87MG (C) and U251MG (D) based on comparisons between 2D and 3D cultures. The significant difference in protein expression was determined using the Mann‐Whitney U test at p < 0.05.

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Expressing, MANN-WHITNEY

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Gene ontology classification based on the basis of DEPs. A bar chart representing the top ten gene ontologies. The annotated functions were classified into three major categories: biological process (A, B), molecular function (C, D), and cellular component (E, F), based on 220 differentially expressed proteins (DEPs) of U87MG cells (A, C, E) and 254 DEPs of U251MG cells (B, D, F), respectively. The size of the circle represents the protein count, and the color represents the enrichment score.

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques:

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Principal component analysis. The PCA illustrated the data distribution using total proteins (A) and differentially expressed proteins (B) as features between U87MG and U251MG in 2D and 3D culture. PCA considering only 220 DEPs of U87MG (C) and 254 DEPs of U251MG (D). A comparative analysis of differentially expressed proteins between U87MG and U251MG is presented in panel (E).

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques:

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Gene ortholog classification based on DEPs. A bar graph representing gene orthologs identified using the KOG database is shown, based on 220 differentially expressed proteins (DEPs) from U87MG cells and 254 DEPs from U251MG cells. Functional categories include cellular processes and signaling (A), information storage and processing (B), and metabolism (C). Gene orthologs were also identified using the KEGG Orthology (KO) database, with annotated functions classified into signaling and cellular processes (D), genetic information processing (E), and metabolism (F).

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques: Functional Assay

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Correlation Between Gene Expression and Proteomic Analysis of Selected Proteins. The relative mRNA levels of selected candidate proteins in 3D spheroid models compared to 2D monolayer cultures (3D relative fold change to 2D, where 2D is normalized to 1) for U87MG (A) and U251MG (B) cell lines. A corresponding heatmap further illustrates the gene expression patterns of U87MG (C) and U251MG cells (D).

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques: Gene Expression

Journal: Biology of the Cell

Article Title: Integrated 2D–3D Proteomic Profiling Identifies MLK4 as a Microenvironment‐Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells

doi: 10.1111/boc.70068

Figure Lengend Snippet: Targeting MLK4 sensitizes glioblastoma cells to Temozolomide treatment. Western blot analysis of MLK4 expression in U87MG and U251MG after treatment with siRNA against MLK4 (siMLK4) and nontarget siRNA control (siNT) compared to WT at 0, 48, and 72 h. β‐actin was used as a loading control (A). Band intensities were normalized to β‐actin and converted to a relative fold change in siRNA treatment compared to wild type (B). Cell viability of GBM cells after 24 h of treatment with 50 and 100 µM TMZ, with siMLK4 or siNT compared to wild‐type cells (C). * p < 0.05, ** p < 0.01, and *** p < 0.001. Original blots from Western blot analysis associated with Figure are shown in Figure .

Article Snippet: GBM cell lines, U251MG and U87MG, were obtained from the American Type Culture Collection (ATCC, Rockville, USA).

Techniques: Western Blot, Expressing, Control

Journal: Redox Biology

Article Title: Alexander disease mutations differentially sensitize glial fibrillary acidic protein (GFAP) to posttranslational modifications and network disruption by oxidants

doi: 10.1016/j.redox.2026.104103

Figure Lengend Snippet: Analysis of disulfide-mediated oligomerization of GFAP AxD mutants under basal and oxidizing conditions in U-87 MG cells . U-87 MG astrocytoma cells expressing GFP-GFAP wt or AxD mutants were lysed, and cell lysates were prepared in the absence (non-reducing conditions) or presence (reducing conditions) of β-mercaptoethanol for western blot analysis. ( A ) Basal oligomerization was examined in cells expressing GFP-GFAP wt or the R239C, R79C, or E373K mutants. The amount of sample loaded was adjusted according to the levels of GFP-GFAP in each lysate as follows: 10 μg total protein for wt and E373K, 2 μg for R79C, and 5 μg for R239C. ( B ) Glutathione levels in U87-MG cells expressing GFP-GFAP wt or AxD mutants. Total (GSH + GSSG) and oxidized glutathione (GSSG) were measured in U87-MG cells expressing GFP-GFAP wt or the AxD mutants, R79C, R239C or E373K. The figure shows normalized data of the three parameters, where values for GFP-GFAP E373K-expressing cells were taken as 100% due to their lower interexperimental variability between independent experiments. Top panel depicts total glutathione results (100% = 3082.4 ± 849.7 pmol/mg protein) and bottom panel shows GSSG concentrations (100% = 115.9 ± 73.3 pmol/mg protein). Statistical analysis was performed with GraphPad Prism v9 using Kruskal-Wallis test and Dunn's post-hoc analysis. ( C ) Oxidant-induced oligomerization was assessed in cells expressing GFP-GFAP wt or R239C treated with 1 mM H 2 O 2 for 30 min, 0.5 mM ONOOH for 15 min, or 50 μM HOCl for 15 min at 37 o C. Sample loading was adjusted according to expression levels: 30 μg total protein for wt and 4 μg for R239C. Asterisks indicate GFP-GFAP wt monomers, and arrowheads indicate oligomers in panels A and C. Molecular mass markers (kDa) are shown on the left of the gels. Dashed lines separate two sections of the same blot. Lower panels in (A) and (C) show long time exposures of the same blot to ensure detection of low-abundance oligomeric bands.

Article Snippet: The U-87 MG glioblastoma astrocytoma cell line was obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling via microsatellite amplification at Secugen, SL (Madrid, Spain).

Techniques: Expressing, Western Blot

Journal: Redox Biology

Article Title: Alexander disease mutations differentially sensitize glial fibrillary acidic protein (GFAP) to posttranslational modifications and network disruption by oxidants

doi: 10.1016/j.redox.2026.104103

Figure Lengend Snippet: Organization of the GFP-GFAP wt and R239C networks in astrocytoma cells. (A) U-87 MG cells were transfected with GFP-GFAP wt and R239C constructs and treated with the indicated oxidants, as described in the Materials and Methods. After treatment, cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with Triton X-100, blocked with 1% BSA (w/v) and stained with Phalloidin-Alexa Fluor 568 and DAPI. The GFAP networks were directly visualized by the GFP fluorescence. Overall projections of GFP-GFAP (green), Phalloidin (red, actin) and DAPI (blue, nuclei) are shown. Cell contours are outlined in white. Scale bars, 20 μm. (B) Quantification of GFAP-covered area in cells expressing GFP-GFAP wt under control or oxidative treatment conditions. ROIs were defined using phalloidin staining for the cell body and GFP for GFAP-positive filamentous structures. Data represent the mean ± SEM of 20 cells per condition from three independent experiments. (C) Quantitation of effects of the oxidative agents in cells expressing GFP-GFAP R239C. Left graph, percentage of GFP-GFAP R239C-expressing cells containing aggregates under control or treated conditions. At least 150 cells per experiment were analyzed across three independent experiments, each symbol represents an individual experiment. Right graph, average number of aggregates per GFP-GFAP R239C-expressing cell determined in 25 cells per condition across three independent experiments. Aggregates were identified using manual thresholding in ImageJ to include discrete aggregates while excluding bundles. Data are shown as mean ± SEM.

Article Snippet: The U-87 MG glioblastoma astrocytoma cell line was obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling via microsatellite amplification at Secugen, SL (Madrid, Spain).

Techniques: Transfection, Construct, Staining, Fluorescence, Expressing, Control, Quantitation Assay

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Generation of CAR T cell workflow, assessment of CAR transduction, and quantification of on-target antigens on U87. A) Pictographic representation of timeline for CAR T cell culturing and functional assessment. B) Flow cytometric gating strategy of representative donor to quantify CAR transduction applicable to both IL-13 and TV-13 CAR transduced cells. C) Comparative CAR expression distinguished between CD4 and CD8 from a representative donor of Control T Cells (UTD), TV-13, and IL-13 CARs. D) Flow cytometric verification of IL13Rα1 and IL13Rα2 expression on U87 cells.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Transduction, Cell Culture, Functional Assay, Expressing, Control

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: 2D in vitro cytotoxic assessment of CARs polyfunctionality. A) Workflow for intracellular cytokine assay. Created with BioRender.com . B) Flow cytometric gating strategy of the representative donor to identify CAR + T cells from viable singlets. C) Comparative release of IL-2 and TNF-α by CAR + T cells from the representative donor between UTD, TV-13, and IL-13 CAR transduced cells. D ) Comparative release of IFN-γ from the representative between UTD, TV-13, and IL-13 CAR T cells. E ) Graphical display of perforin and granzyme B release ( n = 3 ). ∗ p < 0.05. F) Quantification of the amount of INF-γ released into bulk media across UTD, TV-13, and IL-13 ( n = 3 ). One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. G) Lactate Dehydrogenase (LDH) based quantification rate of tumor lysis across different T cell treatment conditions ( n = 3 ). One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. H) Simplified Presentation of Incredibly Complex Evaluations (SPICE) analysis showing the number of intracellular cytokines (TNF-α, IFN-γ, and IL-2) produced per T cell by TV-13 and IL-13 CAR T cells, in response to U87 target stimulation indicating their polyfunctionality. The purple quadrant denotes the percentage of T cells producing all three cytokines, green represents cells producing two cytokines, blue denotes cells producing one, and grey represents cells producing none. Comparable levels of polyfunctionality were observed between the TV-13 and IL-13 groups. Data collected from three biological replicates ( n = 3 ).

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: In Vitro, Cytokine Assay, Lysis, Produced

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Formation of 3D self-assembled microvascular network (μVN) and its influence on U87 cells. A) Establishment of the μVN. (i) Schematic representation detailing the formation of the self-assembled μVN, and (ii) Representative phase contrast tile image of the device showing the progression of μVN formation on day 0 (left) and day 7 (right). B) Characterization of the μVN. (i) 10X tile image of vascular region stained for endothelial marker CD31 (green), junctional protein CD144 (red), and counterstained for nuclei with DAPI (blue) (scale bar: 200 μm), (ii) Phase contrast region of interest (ROI) image highlighting the vascular bundle formed within the vascular region (left), alongside 20X immunofluorescent image showing the expression of CD31(middle), and wrapping of pericytes (α-SMA) around the vascular bundle (right). Scale bars: 100 μm. C) orthogonal sectioning of established μVN confirming the open lumen formation (white arrowhead indicates the open lumen in the orthogonal view). Scale bar: 50 μm. D) Representative immunofluorescent and phase contrast overlap image after injection of 70 kDa fluorescent dextran dye captured at 30s, 1,2, and 4min. Scale bars: 100 μm. E) Line graph image of co-localization of pericytes with endothelial cells based on the scan line (white line) from figure Bii (right). F) Representative immunofluorescent image captured after perfusion of 2 μm fluorescent bead (red) through the CD31 (green) stained vascular bundle. Scale bar: 100 μm. G) Characterization of the μVN in the presence of tumor cells. (i) 10X tile image showing the intact μVN in the vascular (V) region and the migration of the tumor cells (U87-green) from the tumor (T) to the stroma (S) region. Yellow dashed trapezoids and hexagons mark the microposts of the 3D GOC. Scale bar: 200 μm, and (ii) Orthogonal sectioning of the vascular region confirming the maintenance of lumens post U87 injection (white arrowhead indicates the open lumen with white dashed box showing a zoomed-in lumen). Scale bar: 50 μm. Actin acquired with Alexa 647 and CD31 stained with Alexa 555 were pseudo colored in gray and magenta, respectively, for visualization. T, S, V represent the tumor, stroma, and vascular regions of the GOC system.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Staining, Marker, Expressing, Injection, Migration

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Evaluation of cytotoxic abilities of T cells against GBM cells within the GOC model. A) Microfluidic 3D invasion assay. (i) Schematic representation depicting the culture of tumor cells with T cells on day 0 (top) and day 3 (bottom), (ii) Representative phase contrast tile image overlapped with GFP (tumor cells) channel captured on day 0 to show the distribution of tumor and T cells across the experimental conditions (Scale bars: 200 μm), and (iii) Representative phase contrast tile image overlapped with GFP channel showing the migration of the U87 cells (green) from the tumor region to the stroma region across three different T cell populations. The densities of U87 are kept consistent across all conditions, and the density of T cells varies from 4 × 10 6 to 15 × 10 6 cells/mL. Images were captured 72 h after the interaction of cells within the GOC model (Scale bars: 200 μm). T-tumor, S-stroma, and V-vascular regions of GOC. B) Assessment of tumor cell migration in the presence of different T cells. (i) Quantification of migration distance from the 3D microfluidic model showing dose-dependent inhibition of U87 migration by the CAR T cells. Data were measured on Day 3 from three biological replicates ( n = 3 ) and represented as mean ± SD, T cell donors: DN26, DN28, and DN31, ∗ p < 0.05, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (ii) Comparison of migration distance of the U87 cells in the presence of different concentrations of the T cell population. Analysis performed on samples captured on Day 3 of migration ( n = 3 ) and represented as mean ± SD, T cell donors: DN26, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. C) xCELLigence-based real-time evaluation of T cell cytolytic capacity. (i) Time-course of the average cell index ( n = 3 donors ) for UTD, TV-13, and IL-13 CAR T cell groups under a 10:1 E: T condition over a 7-day co-culture, measured using the xCELLigence platform, (ii) Bar plot of xCELLigence data comparing averaged cell index values of tumor cells at Day 0 and Day 7 across UTD, TV-13, and IL-13 CAR T cell groups. Data represent mean ± SEM ( n = 3 donors ), ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001, Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, (iii) xCELLigence data from a representative donor (Donor 31) showing dose-dependent killing of U87 cells achieved by five doses of TV-13 CAR T cells, and (iv) IL-13 CAR T cells during a 7-day co-culture period.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Invasion Assay, Migration, Inhibition, Comparison, Co-Culture Assay

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Assessment of migratory behavior and proliferative potential of GBM tumor cells in the presence of engineered T cells. A) Evaluation of changes in migratory behavior of tumor cells across UTD, TV-13, and IL-13 T cells based on cytoskeletal organization. (i) Representative tile image of the 3D GOC model stained for actin cytoskeleton (red) showing the tumor-stroma-vascular interface (left), zoomed-in view highlighting the chain migration of the tumor cells from the tumor to the stroma region (middle), 20X region of interest (ROI) showing the disruption in the migratory pattern of the tumor cells and the formation of immune synapse (IS) (right). The white dashed box represents the ROIs alongside an inset image (ROI1) that highlights the formation of multiple IS between the tumor (green) and T cell within the stroma interface. The white arrow shows the IS formation, and the white dashed arrow represents the line scan utilized for intensity profiling to confirm the reorganization of actin cytoskeleton at the tumor-T cell interface . Red- Actin, Green- U87 cells, and DAPI – Blue . Scale bars: 200 μm (left and middle), 50 μm (right). (ii) Quantification of the number of cells migrating in a chain from near and far regions across three different T cell conditions . Data are represented as mean ± SD measured from three biological replicates ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗∗ p < 0.001 , ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, (iii) Quantification of the number of cells within a field of view (FOV) from two distinct areas, namely near and far regions, Data are represented as mean ± SD measured from three biological replicated ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. B) Immunofluorescent images of the devices stained for proliferation marker Ki-67. (i) Representative 20X ROI image showing the Ki-67 (red) expression on U87 cells (green) and (ii) Quantification of the number of Ki-67 positive cells across each condition through the proliferative index (Ki-67/Nuclei Ratio), Data are represented as mean ± SD measured from three biological replicates ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗ p < 0.01. One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Staining, Migration, Disruption, Marker, Expressing