Journal: Molecular & Cellular Proteomics : MCP

Article Title: Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide

doi: 10.1016/j.mcpro.2026.101610

Figure Lengend Snippet: Experimental workflow overview. This schematic illustrates the experimental design used to profile extracellular vesicles (EVs) derived from U87 glioblastoma cells cultured under 2D monolayer and eroid conditions. A , expansion of U-87 MG glioblastoma cells in T225 flasks prior to experimental setup. B , 2D experimental arm: cells cultured in T75 flasks and treated with DMSO, temozolomide (TMZ) 100 μM, or TMZ 200 μM, followed by EV isolation and cell harvesting. C , 3D-spheroid experimental arm: cells seeded in ultra-low attachment (ULA) plates and treated as in ( B ), with subsequent EV isolation and spheroid harvesting. D , EV and cell characterization by Annexin V/PI cell death assay, fluorescence microscopy, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), silver-stained SDS-PAGE, western blotting, and MACSPlex EV flow cytometry assay. E , protein extraction from EVs and cells, followed by proteomic profiling using nano-liquid chromatography tandem mass spectrometry (nLC-ESI-MS/MS). F , differential expressions and functional analysis, including heatmap clustering and identification of GBM-associated protein families (e.g., methyltransferases). G , cross-referencing of selected proteins with The Cancer Genome Atlas (TCGA) for prognostic relevance. H , EV isolation pipeline combining ultrafiltration and size-exclusion chromatography (SEC) for purification of vesicles from conditioned media.

Article Snippet: EV samples from 2D and 3D cultures were analyzed with the MACSPlex EV Kit IO (for immuno-oncology) (#130-108-813) (Miltenyi Biotec).

Techniques: Derivative Assay, Cell Culture, Isolation, Cell Harvesting, Cell Characterization, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy, Staining, SDS Page, Western Blot, Flow Cytometry, Protein Extraction, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Functional Assay, Size-exclusion Chromatography, Purification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide

doi: 10.1016/j.mcpro.2026.101610

Figure Lengend Snippet: Characterization of EV protein profiles from U87 2D and 3D cultures. A , multiplex flow cytometry using the MACSPlex EV kit was used to profile surface markers of EVs derived from U87 cells cultured under 2D and 3D conditions. CD44 was consistently detected and enriched across conditions, particularly in TMZ-treated samples. The integrins CD29 and CD49e—subunits of the α5β1 integrin heterodimer—were robustly expressed in both 2D and 3D EVs, highlighting their potential role in EV-mediated adhesion and signaling. B , silver-stained SDS-PAGE showing global protein profiles of cell lysates and EVs from 2D and 3D cultures. Distinct banding patterns reflect culture-specific proteomic signatures and successful protein isolation. C , Western blot analysis confirms the presence of established EV-associated proteins, including CD81 and CD44. GAPDH, were used as controls and were detected predominantly in cell lysates, supporting the purity of the EV preparations and absence of major cellular contamination.

Article Snippet: EV samples from 2D and 3D cultures were analyzed with the MACSPlex EV Kit IO (for immuno-oncology) (#130-108-813) (Miltenyi Biotec).

Techniques: Multiplex Assay, Flow Cytometry, Derivative Assay, Cell Culture, Staining, SDS Page, Isolation, Western Blot