Optimization of In-Situ Exosome Enrichment Methodology On-a-Chip to Mimic Tumor Microenvironment Induces Cancer Stemness in Glioblastoma Tumor Model

Understanding cancer etiology requires replicating the tumor microenvironment (TME), which significantly differs from standard in vitro cultures due to nutrient limitations, acidic pH, and oxidative stress. To address this, a microfluidic bioreactor (µBR) with an expanded culture surface was designed to optimize exosome enrichment and glioblastoma cell behavior. Using response surface methodology (RSM), key parameters-including medium exchange volume and interval time-were optimized, leading to about a six-fold increase in exosome concentration without artificial inducers. Characterization techniques (SEM, AFM, DLS, RT-qPCR, and ELISA) confirmed significant alterations in exosome profiles, cancer stemness, and epithelial-mesenchymal transition (EMT)-related markers. Notably, EMT was induced in the µBR system, with a six-fold increase in HIF-1α protein despite normoxic conditions, suggesting activation of compensatory signaling pathways. Molecular analysis showed upregulation of <i>SOX2</i>, <i>OCT4</i>, and <i>Notch1</i>, with SOX2 protein reaching 28 ng/mL, while it was undetectable in traditional culture. Notch1 concentration tripled in the µBR system, correlating with enhanced stemness and phenotypic heterogeneity. Immunofluorescent microscopy confirmed nuclear SOX2 accumulation and co-expression of SOX2 and HIF-1α in dedifferentiated CSC-like cells, demonstrating tumor heterogeneity. These findings highlight the µBR's ability to enhance stemness and mimic glioblastoma's aggressive phenotype, establishing it as a valuable platform for tumor modeling and therapeutic development.