Abstract 7523: A vascularized glioblastoma tumor spheroid model for studying tumor invasion and therapeutic response

Abstract Introduction: GBM is characterized by its extensive invasiveness; its highly infiltrative cells penetrate surrounding brain tissue and often cannot be completely removed through surgery, leading to tumor recurrence and poor survival. The extent of vascularization directly correlates with GBM malignancy. Previous studies have demonstrated that sequentially adding fibroblasts to a pre-formed spheroid resulted in their peripheral concentration and enhanced vascularization in a SN12C kidney cancer model. We are interested in investigating whether sequentially adding fibroblasts promote vasculature formation in vitro and induces angiogenesis in the presence of endothelial cells. The objective of this experiment is to develop a representative model of the GBM tumor spheroid, with a focus on how vascularization and stromal cell populations could affect invasion and therapeutic response. Materials & Methods: Sequential spheroids are synthesized by adding 2.5x103 U87-MG cells to an ultra-low attachment plate and incubated at 37°C for 24 hours to allow spheroid aggregation. After 24 hours, an equal amount of NHLFs are added to the preformed spheroids at a 1:1 ratio. Cells were incubated at 37°C for an additional 24 hours to allow complete spheroid formation. Co-mixed spheroids are synthesized using a similar method but adding U87-MG and NHLFs concurrently. NHLF and HUVECs were resuspended in 5% GelMA solution at a ratio of 2: 1 (2x106 NHLF:1x106 HUVEC). Individual spheroids were pipetted into each well of a 20 µL hydrogel mold and polymerized using UV radiation. Results: We observed distinct patterns in fibroblast distribution and spheroid behavior. In sequential spheroids, fibroblasts localized primarily at the periphery, whereas the co-mixed method produced a more uniform distribution throughout the spheroid. When sequential and co-mixed spheroids are encapsulated with endothelial cells (HUVECs) and NHLFs, sequential spheroids demonstrated more vessel formation around the spheroids, while limited network development occurred around co-mixed spheroids. Additionally, AlamarBlue data confirmed significant higher metabolic activity in hydrogels containing sequential spheroid on Day 5. We also observed that sequential spheroid outgrowth correlated to fibroblast density, while co-mixed spheroid outgrowth was more evenly distributed around the spheroid. Temozolomide treated sequential spheroids showed no significant difference compared to untreated controls, suggesting a reduced response to TMZ. Conclusion: Future work will focus on understanding how spheroid organization influences population dynamics. We plan to track changes in cellular populations and investigate whether spheroid organization affects cytokine release in a 3D model. Finally, we aim to validate the effects of fibroblasts on tumor invasion using different cancer cell lines. Citation Format: Anagha Shreesha, Sheridan Ke-Wing Fok, Brendan Harley. A vascularized glioblastoma tumor spheroid model for studying tumor invasion and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7523.