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Abstract
Glioblastoma multiforme (GBM) is a highly aggressive central nervous system tumor with poor prognosis and resistance to therapy. This study aimed to investigate Semaphorin 6D (SEMA6D) expression and apoptosis related responses in U87 glioblastoma cells cultured under two dimensional (2D) and three dimensional (3D) conditions, while also optimizing an agarose based spheroid culture model as an alternative 3D system. U87 cells were cultured in conventional 2D conditions and in 3D systems using Matrigel and 1.5% agarose coated plates. SEMA6D protein expression was analyzed by flow cytometry, apoptosis was assessed using Annexin V/propidium iodide staining, and the expression levels of SEMA6D, VEGF, BCL2, BAK1, and CASP3 were evaluated by RT qPCR. SEMA6D protein expression increased modestly in 3D cultures compared with 2D cultures and reached statistical significance in agarose based spheroids at later time points. Although SEMA6D mRNA levels showed an increasing trend, the differences were not statistically significant. In contrast, VEGF, BCL2, BAK1, and CASP3 transcripts were significantly upregulated in 3D cultures relative to 2D controls (***p < 0.001; ****p < 0.0001). Apoptosis rates were also significantly elevated in both 3D culture systems.These findings indicate that the 3D microenvironment strongly modulates survival and apoptosis related pathways in glioblastoma cells. Both Matrigel and agarose based spheroid models provide physiologically relevant in vitro platforms for studying GBM biology and may support the identification of novel biomarkers and therapeutic targets.
