Enhanced Glioblastoma-Targeted Aptamer Discovery by 3D Cell-SELEX with Hyaluronic Acid Hydrogel

Glioblastoma (GBM), a highly aggressive brain tumor, presents significant treatment challenges due to the lack of targeted therapies. Aptamers offer a promising solution, with their small size and high affinity for GBM targets, enabling enhanced tumor selectivity. Notably, they can be newly identified through the systematic evolution of ligands by exponential enrichment (SELEX). However, traditional SELEX using two-dimensional (2D) cell cultures does not accurately replicate the inherently three-dimensional (3D) native tumor microenvironment. To overcome this limitation, we present here the first attempt to conduct SELEX in a 3D cellular microenvironment for targeting GBM, rather than using the traditional 2D format, introducing a novel 3D cell-SELEX approach. Compared to 2D culture, the 3D model cultured in a hyaluronic acid (HA) hydrogel that can better mimic the natural GBM microenvironment, providing a more physiologically relevant platform for aptamer selection, preserved unpolarized native cell morphology, and exhibited different GBM surface marker gene expression. By comparing 2D and 3D SELEX using U87MG GBM cells as the selection target and T98G cells as the negative control, we observed distinct differences in aptamer binding profiles. Unlike 2D SELEX, our 3D SELEX approach exhibited a steady increase in the ssDNA yield, indicating enhanced stability and efficiency in aptamer enrichment. Confocal imaging confirmed that aptamers obtained from the final round of 3D SELEX exhibited a strong and selective affinity for GBM cells. Principal component analysis (PCA) of sequence diversity further revealed distinct clustering patterns derived from 2D and 3D SELEX, with the latter pool exhibiting greater sequence complexity. This approach will improve the clinical relevance of aptamer selection, paving the way for more effective GBM diagnostics and targeted therapeutic strategies.