Deciphering pericyte-induced temozolomide resistance in glioblastoma with a 3D microphysiological system mimicking the biomechanical properties of brain tissue

Glioblastoma (GBM) is a highly aggressive malignancy with a poor prognosis and frequent resistance to temozolomide (TMZ), the standard-of-care chemotherapy. The complex mechanisms underlying GBM chemoresistance, particularly the role of pericytes, remain poorly understood due to the lack of physiologically relevant in vitro models replicating the complex tumor microenvironment (TME). Here, we present a biomimetic 3D GBM microphysiological system that replicates the biomechanical properties of brain tissue (G'∼800Pa, G"∼100Pa) and enables the study of pericyte-mediated TMZ resistance. GBM spheroids (U87, LN229, PDM140) were cultured alone or co-cultured with pericytes in a composite hydrogel for 14 days and remained viable and proliferative. In response to TMZ, PDM140 was the most sensitive (IC₅₀=73μM), followed by LN229 (IC₅₀=278μM) and U87 (IC₅₀=446μM). Co-culture with pericytes significantly increased GBM spheroid viability by 22.7% (PDM140), 32.5% (LN229), and 22.1% (U87), confirming pericyte-induced TMZ resistance. Notably, pericytes exhibited a 160-fold upregulation of C-C motif chemokine ligand 5 (CCL5) upon TMZ treatment, implicating the CCL5-mediated pathway in chemoresistance. This innovative brain-mimicking 3D GBM model provides a physiologically relevant platform for studying tumor-pericyte interactions and testing therapeutic strategies targeting CCL5-mediated resistance mechanisms in GBM. STATEMENT OF SIGNIFICANCE: We developed a multicellular 3D glioblastoma microphysiological system mimicking the physicochemical properties of brain tissues and pericyte-mediated TMZ resistance that can be used to screen for standard-of-care chemotherapy. This advanced hydrogel-based platform demonstrated the critical role of the glioblastoma tumor microenvironment in modulating chemotherapy sensitivity, particularly the pericyte-induced CCL5-CCR5 paracrine axis that can lead to the identification of therapeutic targets within the CCL5-CCR5 pathway toward more effective treatments disrupting these resistance mechanisms. Overall, the proposed 3D glioblastoma microphysiological system can transform drug screening and personalized treatment for GBM by offering ethical and cost-effective alternatives to animal testing and more effective drug screening and discovery efforts, ultimately improving GBM patient outcomes.