Journal: Acta Neuropathologica Communications

Article Title: Cytotoxic activity of selinexor in BRAF V600E -driven pediatric low-grade glioma 3D cultures

doi: 10.1186/s40478-026-02461-1

Figure Lengend Snippet: High-content validation screen in proliferating and senescent pLGG 3D culture models. A-B Image-based analysis of drug responses in proliferating BT40 multicellular aggregates ( A ) and DKFZ-BT314_ON spheroids ( B ) after 6 d of treatment with single agents or in combination with binimetinib. Values represent change from baseline in TMRE-positive 3D culture area derived from the maximal intensity projection images. Grey dots show technical replicates ( n = 5) and bars show means. Horizontal lines denote response thresholds based on RECIST-like criteria. All concentrations are given in nM. C Residual metabolic activity in senescent DKFZ-BT314_OFF spheroids after 6 d of treatment. Each bar represents a technical replicate ( n = 5). All concentrations are given in nM. D Representative high-content microscopy images of BT40 multicellular aggregates illustrating cytostatic and cytotoxic treatment responses. Shown are merged brightfield images with maximal intensity projections of TMRE fluorescence (yellow). Abbreviations: MEKi, MEK inhibitor; BCL-X L i, BCL-X L inhibitor; XPO1i, exportin inhibitor; pLGG, pediatric low-grade glioma; IC, inhibitory concentration; TMRE, tetramethylrhodamine ethyl ester

Article Snippet: On 4th day after seeding cells were imaged using the ImageXpress Micro Confocal High-content microscope (Molecular Devices, San José, CA, USA) and treated with selinexor, ulixertinib and mirdametinib (DMSO and BztCl as negative and positive control, respectively).

Techniques: Biomarker Discovery, Analysis, Spheroids, Derivative Assay, Activity Assay, Microscopy, Brightfield, Fluorescence, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: Cytotoxic activity of selinexor in BRAF V600E -driven pediatric low-grade glioma 3D cultures

doi: 10.1186/s40478-026-02461-1

Figure Lengend Snippet: MEKi and selinexor induce cell death in pLGG 3D cultures, with complementary mechanistic analyses in 2D. A Representative high-content microscopy images of active caspase-3/7 (CellEvent™ Caspase-3/7 Green Detection Reagent) in BT40 multicellular aggregates after 6 d of the indicated treatments. Green fluorescence indicates cells with activated Caspase-3/7. Merged fluorescence and brightfield images are shown. Scale bars, 100 μm. B Representative DRAQ7 images of BT40 multicellular aggregates after 6 d of the indicated treatments. Red fluorescence indicates membrane-compromised and dead cells. Scale bars, 100 μm. C DRAQ7-positive cells in 3D-cultured DKFZ-BT314_OFF spheroids after the indicated treatments for 72 h. Data are presented as the percentage of DRAQ7-positive cells relative to DMSO-treated controls. Bars represent the mean, and error bars indicate standard deviation (SD) of replicates. D Percentage of DRAQ7-positive cells (late-stage cell death) relative to total cell number. BT40 cells were seeded as a monolayer and treated for 72 h with single agents or in combination with either binimetinib (upper panel) or trametinib (lower panel) at different concentrations (IC 25 , IC 50 , 2x IC 50 , IC 75 ). Data represents the mean ± SD of three independent biological replicates with small dots indicating individual wells. All concentrations indicated on the x-axis are expressed in nM. E BLISS synergy scores calculated with SynergyFinderPlus based on the percentage of dead cells measured by DRAQ7 staining in D . F Fragmented nuclei and G caspase-positive cells following 72 h treatment in BT40 monolayers. Data represents the mean ± SD of three independent biological replicates with small dots indicating individual wells. All concentrations indicated on the x-axis are expressed in nM. H Caspase-3 activity measured using a fluorescent DEVD-based substrate in cell lysates of BT40 cells, seeded as monolayers, after 48–72 h of treatment (cells were collected when they started to detach). Values are normalized to the solvent control. Bars represent the mean ± SD of three biological replicates. Treatments: binimentinib (‘b’, 58.6 nM), trametinib (‘t’, 1.74nM), selinexor (‘s’, 100 nM), navitoclax (‘n’, 100 nM), dabrafenib (‘d’, 250 nM). I Western blot analysis of cleaved PARP and BID following 72 h of treatment (same as in H ). Shown are representative blots from biological replicates. J Caspase-3/7 activity measured using a fluorescent DEVD-based substrate in cell lysates of DKFZ-BT314_OFF after 48–72 h of treatment (cells were collected once detachment was observed). Values are normalized to the solvent control. Bars represent the mean ± SD of three biological replicates. D, DMSO; b, binimetinib; t, trametinib; d, dabrafenib; n, navitoclax; s, selinexor; v, venetoclax. Abbreviations: MEKi, MEK inhibitor; IC, inhibitory concentration; CSS, combination sensitivity score

Article Snippet: On 4th day after seeding cells were imaged using the ImageXpress Micro Confocal High-content microscope (Molecular Devices, San José, CA, USA) and treated with selinexor, ulixertinib and mirdametinib (DMSO and BztCl as negative and positive control, respectively).

Techniques: 3D Cultures, Microscopy, Fluorescence, Brightfield, Membrane, Cell Culture, Spheroids, Standard Deviation, Staining, Activity Assay, Solvent, Control, Western Blot, Analysis, Concentration Assay, Sensitivity

Journal: Acta Neuropathologica Communications

Article Title: Cytotoxic activity of selinexor in BRAF V600E -driven pediatric low-grade glioma 3D cultures

doi: 10.1186/s40478-026-02461-1

Figure Lengend Snippet: High-content imaging drug screen identifies MEKi combination partners. A Representative high-content microscope images of a three-dimensional BT40 multicellular aggregate cultured for 6 d with DMSO and TMRE. From left to right: brightfield image, maximum-intensity projection of TMRE fluorescence, and merged image. Complete TMRE staining (yellow) indicates full cell viability within the whole aggregate after 6 d. B-C BT40 multicellular aggregates treated for 6 d with increasing concentrations of binimetinib ( B ) or trametinib ( C ). Bars show change from baseline of TMRE-positive aggregate area; each bar represents an independent technical replicate ( n = 7). Horizontal lines indicate response thresholds according to RECIST-like criteria, modified for aggregate growth. Dose-response curves show metabolic activity measured using CellTiter-Glo ® 2.0. Data are shown as percent inhibition relative to DMSO control. Each grey dot represents a technical replicate ( n = 7); yellow dots the mean value. Calculated concentrations resulting in 25%, 50%, and 75% inhibition of metabolic activity (IC 25 , IC 50 , IC 75 ) are indicated on the graph. D Drug sensitivity profiling of BT40 multicellular aggregates with 87 drugs combined with 6.35 nM binimetinib (left) or 0.18 nM trametinib (right) for 6 d (IC 25 ). Drugs were tested at five concentrations and classified using the indicated RECIST-like thresholds applied descriptively to change in 3D culture area. A reduction of at least 30% in comparison to the baseline was defined as a partial response (PR; green color in heatmap). An increase in size of at least 20% was classified as progression (PD; red color), while the range between a 20% increase and a 30% decrease was designated as stable disease (SD; orange). Corresponding brightfield-based area measurements and percent inhibition of metabolic activity of the binimetinib screen are shown for comparison. E Overlap of top hits from the two independent screens. Abbreviations: MEKi, MEK inhibitor; TMRE, tetramethylrhodamine ethyl ester; IC, inhibitory concentration; PD, progressive disease; PR, partial response; SD, stable disease; RECIST, Response Evaluation Criteria in Solid Tumors

Article Snippet: On 4th day after seeding cells were imaged using the ImageXpress Micro Confocal High-content microscope (Molecular Devices, San José, CA, USA) and treated with selinexor, ulixertinib and mirdametinib (DMSO and BztCl as negative and positive control, respectively).

Techniques: Imaging, Microscopy, Cell Culture, Brightfield, Fluorescence, Staining, Modification, Activity Assay, Inhibition, Control, Sensitivity, Profiling, Comparison, Concentration Assay