Abstract 238: Investigating size-dependent invasion behaviors and therapeutic responses of the 42MGBA glioblastoma spheroids
Authors: Fok Sheridan , Ivanova Yoanna , Kriuchkovskaia Victoria , Harley Brendan 
Published: March 2024

Abstract
Introduction: Glioblastoma (GBM) is one of the most aggressive malignant brain tumors. Many studies have reported that the tumor microenvironment (TME) contributes greatly to invasion and drug resistance of GBM. Our lab has previously described a tri-culture model using methacrylamide-functionalized gelatin (GelMA) with brain microvascular endothelial cells (BMVEC), human normal astrocytes (NHA), and human brain vascular pericyte (HBVP) recapitulated the in vivo brain microenvironment. However, phenotypic drug response was only observed under supraphysiological dosage. To evaluate therapeutic response more precisely, we decided to combine the Tri-culture system with the IdenTx3 microfluidic device (AIM Biotech). By separating the immature perivascular network from the GBM spheroids, we are hoping to generate a model that can more realistically mimic the TME. While many studies have used cancer spheroid in GBM studies, little is known about the impact of spheroids size. Since spheroid size correlates with nutrient and oxygen gradients, we are also curious to find out whether an increase in spheroid size increases the necrotic population and affects drug response and invasion.
Materials: Wild-type (WT) and TMZ resistant 42MGBA cell lines were seeded at different cell numbers to generate spheroids of different sizes. Spheroids were dissociated for flow analysis to quantify the necrotic population. In the invasion assay, spheroids were encapsulated in GelMA hydrogel with or without the addition of perivascular cells including BMVECs, HBVP, and NHA (3:1:1). Vasculature formation inside the chip was initiated 5 days prior to the introduction of GBM spheroids.
Results: 42MGBA-WT and 42MGBA-TMZres spheroids demonstrated phenotypical and proliferative differences. Spheroids sizes showed a direct relationship with necrotic cell numbers. Small spheroids (1000 cells) and large spheroids (10,000 cells) were encapsulated inside the hydrogel and subjected to different treatments of temozolomide. A difference in invasion area and proliferation was found when subjected to bulk or metronomic dosages. The two 42MGBA cell lines displayed an increase in invading area when co-cultured with perivascular cells. This result coincided with our previous finding that perivascular network increases GBM proliferation and invasion in GBM6 spheroid (Ngo et al, 2022). In addition, a different invasion behavior was observed between the 42MGBA-WT and 42MGBA-TMZres spheroids when introduced inside the microfluidic device. Surprisingly, the wildtype showed a larger invading area compared to the TMZ resistant.
Conclusion: We demonstrated a direct relationship between size and necrotic population in the 42MGBA spheroids. We also showed that bulk or metronomic treatment exert different effects on the cancer spheroids. Finally, we have verified that TME is critical to invasion and drug resistance.
Citation Format: Sheridan Fok, Yoanna Ivanova, Victoria Kriuchkovskaia, Brendan Harley. Investigating size-dependent invasion behaviors and therapeutic responses of the 42MGBA glioblastoma spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 238.