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Cancer Technologies

Cancer Technologies - Poster Session E

S14 - Three-Dimensional Spheroids as In Vitro Models of the Tumor Microenvironment for Glioblastoma Treatment

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Glioblastoma (GBM) is a grade IV glioma and the most aggressive primary brain tumor in adults, characterized by poor prognosis and limited treatment options, including surgical resection, radiotherapy, and chemotherapy. Despite aggressive multimodal therapy, median survival remains approximately 15 months, and recurrence is nearly universal [1]. To improve the prognosis of GBM, new treatments are being investigated. However, the current two-dimensional (2D) cell culture model for testing new therapeutics does not provide an accurate model reflective of the physiological tumor environment [2]. Three-dimensional (3D) models, such as spheroids, can provide an in vitro model that better mimics the in vivo physiological environment [3]. Spheroids are 3D cell aggregates composed of a necrotic core, quiescent middle layer, and proliferative outer layer [4]. These layers mimic that of the in vivo tumor microenvironment, providing a physiologically relevant model for in vitro testing. This study aims to improve in vitro models for GBM therapeutics by analyzing the growth and formation of U87MG and U118MG GBM spheroids, characterizing their cellular layers, and comparing three-dimensional to two-dimensional models through gene expression analyses.

Materials and

Methods: : U87MG and U118MG GBM spheroids were formed by centrifuging cells in ultra-low attachment plates to form cell aggregates. To examine the growth rates of various densities, spheroids were seeded at densities ranging from 5,000 to 20,000 cells/mL and were imaged using 4x magnification on days 3, 5, and 7. Flow cytometry was conducted on spheroids stained with Annexin V and Propidium Iodide to characterize the cellular layers of the spheroids from days 4 through 7 of growth. Live immunofluorescent staining was performed on U87MG spheroids to further analyze spheroid composition. U87MG spheroids were stained with Propidium Iodide and Hoechst or Wheat Germ Agglutinin and Hoechst and imaged using confocal microscopy. The half-maximal inhibitory concentration (IC50) of temozolomide (TMZ) was also measured to compare the concentration of the chemotherapeutic drug needed to kill half of the cellular population in both 2D and 3D culture models. U87MG and U118MG spheroids were seeded at 5,000, 12,000, and 20,000 cells/well and treated with TMZ, and a viability assay was conducted on both 2D and 3D cultures. Western blots and RT-qPCR were conducted using U87MG 2D monolayers and 3D spheroids to compare the basal expression of various genes, including genes overexpressed in glioblastoma.

Results, Conclusions, and Discussions:: The U87MG and U118MG cell lines successfully formed spheroids at various seeding densities (Figure 1). On average, the growth rates of the spheroids were directly related to seeding density; however, high-density U87MG spheroids exhibited a slower growth rate. At 20,000 cells/well, there was no growth in U87MG spheroid size after seeding, likely because of cell contact-induced apoptosis. As suggested by Annexin V/PI staining, both U87MG and U118MG spheroids contain viable, apoptotic, and necrotic cellular layers (Figure 1). The U118MG spheroids contain a smaller percentage of viable cells compared to the U87MG spheroids, which could contribute to their overall smaller size. As a higher percentage of cells become necrotic, the dense, nutrient-deficient core becomes more abundant, reducing the overall size of the U118MG spheroids. Live immunofluorescent staining revealed that the core of U87MG spheroids contained no live cells compared to strong fluorescence on the surface, further confirming the presence of the proliferative, quiescent, and necrotic layers of the spheroid reflective of the physiological tumor microenvironment. TMZ IC50 analysis revealed that the 3D spheroids have a higher resistance to TMZ treatment compared to the 2D monolayers, further confirming the relevance of 3D models in accurately mimicking the tumor microenvironment. 2D U87MG cells had an IC50 of 0.96 mg/mL, while the 3D U87MG spheroids had IC50 values of 1.986, 1.635, and 1.343 mg/mL for seeding densities of 5K, 12K, and 20K cells/well, respectively. Likewise, the 2D U118MG cells had an IC50 of 0.9311 mg/mL, while the 3D U118MG spheroids had IC50 values of 1.547, 1.987, and 1.967 mg/mL for seeding densities of 5K, 12K, and 20K cells/well. Western blots and RT-qPCR confirmed the expression of oncogenes in both 2D cell cultures and 3D spheroids and provided a comparison of expression between the two models.
In conclusion, the 3D spheroids offer a promising model for accurate in vitro studies for glioblastoma treatments. The U87MG and U118MG glioblastoma cell lines can effectively aggregate into spheroids and grow over several days with three layers that mimic the tumor environment. Future studies include continued spheroid gene expression analysis for accurate comparison of 2D and 3D cell models.

Acknowledgements and/or References (Optional): : Acknowledgements: This research was supported in part by the National Science Foundation (NSF) Faculty Early Career Development Program under NSF Award #204669, the MADE in SC Program through the NSF EPSCoR Program under NSF Award # OIA-1655740, SCBioCRAFT through the National Institute of Health (NIH) COBRE Initiative under NIH Award # P30GM131959, and Clemson Creative Inquiry.

References: [1] Pitz, M. et al. Biomedicines 2022;10(9):1-2. [2] Kapałczyńska, M. et al. Archives of Medical Science 2016;14 (4):1. [3] Henrique, R. B. L. et al. Life Sciences 2022;302:1-2. [4] Pinto, B. F. et al. Pharmaceutics 2020;12(12):1-3.