Riassunto analitico
Background: Glioblastoma (GBM) is a highly aggressive, infiltrating, astrocytic glioma with a 5-year survival rate of just 5%. Treatment is often challenging due to the heterogeneity of the tumor microenvironment (TME), genomic variability, and the presence of more resistant stem-like cell populations. The lack of advanced in vitro models capable of accurately reproducing the TME and the complexity of GBM severely limits research progress and drug development. Current drug screening methods mostly rely on animal models and conventional in vitro systems that fail to recapitulate the tumor’s structural, molecular, and functional complexity, leading to low translational success.
To overcome his limitation, a 3D bioprinted model was developed to better mimic the structural architecture of GBM.
Materials and Methods: Bioprinting technology was used in this study. U87MG cells were encapsulated in a biocompatible hydrogel to create a bioink, which was then loaded in a printing cartridge and extruded. Dimension, geometry, and porosity of the obtained structure accurately reproduced the CAD project, assuring reproducibility and standardization of the bioprinting process. The structures, named Bioprinted-GBM (BiopGBM), were kept in culture for about one month. Viability was assessed using Live/Dead assay, while histology, immunohistochemistry (IHC), and quantitative PCR were employed to investigate the development of physiopathogenic features of GBM. To evaluate the potential of BiopGBM as a predictive platform for drug screening, two different concentrations of Temozolomide (TMZ) were administrated to both BiopGBM and U87MG spheroids, the latter being the prevalent 3D model used in oncology.
Results: U87MG viability within BiopGBM was confirmed across culture time points, while H&E staining showed diverse cell morphologies and distributions in the 3D environment. A high density of spindle-like cells was observed on the scaffold's upper part, while clusters of small, round cells were found in the necrotic core, mimicking in vivo tumor behavior. To characterize and confirm the phenotype of GBM within BiopGBM, gene expression analysis for CD133, CD44, HIF1α, and MMP9 was performed, comparing the results with 2D culture and spheroids. The three models exhibited distinct expression patterns for selected genes. CD133, a stemness marker, was significantly enriched in scaffold-cultured U87MG compared to those in 2D cultures and spheroids, as confirmed by IHC. Similarly, elevated expression levels of CD44, HIF1α, and MMP9 were observed in scaffold-extracted cells. This enrichment can be attributed to the complex 3D structure of the scaffold, that may mimic hypoxic conditions, promoting the enrichment of stem-like cell populations and migration to the upper part of the scaffold, explaining the high number of spindle-like cells. These findings enlighten the biomimicry relevance of the 3D bioprinted model, which was further tested for its response to therapy. Scaffolds and spheroids were treated with 1000 and 1600 µM TMZ for 48 hours and subsequently analyzed using viability and MTT assays. The results indicated a more evident reduction of cell viability in scaffolds, highlighting the model's capability to respond to treatment and to serve as a predictive platform.
Conclusion: This preliminary study focuses on the development of a 3D bioprinted GBM model designed to mimic the structural architecture of tumor tissue. Although the current model utilizes a single cell line and provides limited data, it represents a significant step toward establishing a standardized, high-throughput platform capable of evaluating therapeutic responses, with potential for future applications involving patient-derived cells.
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