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Cancer Technologies

Cancer Technologies - Poster Session A

Poster H4 - Characterization of Three-Dimensional Spheroids for Glioblastoma Treatment

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Glioblastoma (GBM) is a grade IV glioma tumor with limited treatment options, including surgical resection, radiotherapy, and chemotherapy. To improve the prognosis of GBM, new treatments are being investigated. However, the current two-dimensional cell culture model for testing new therapeutics does not provide an accurate model reflective of the physiological tumor environment. Three-dimensional models, such as spheroids, can provide an in vitro model that better mimics the in vivo physiological environment. Three-dimensional spheroids are cell aggregates composed of a necrotic core, quiescent middle layer, and proliferative outer layer. This study aims to improve in vitro models for GBM therapeutics by analyzing the growth and formation of U87MG GBM spheroids, characterizing their cellular layers, and comparing three-dimensional to two-dimensional models through gene expression analyses.

Materials and

Methods:  U87MG spheroids were formed by spinning U87MG cells in ultra-low attachment plates until cell aggregates formed. 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 at 4x magnification microscopy on days three, five, and seven. Live immunofluorescent staining was performed on U87MG spheroids to analyze spheroid composition. U87MG spheroids were stained with Propidium Iodide and Hoechst or Wheat Germ Agglutinin and Hoechst and imaged using confocal microscopy. Basal expression western blots and RT-qPCR were conducted with both U87MG two-dimensional cells and three-dimensional spheroids to compare the expression of various genes, including genes overly expressed in glioblastoma.

Results, Conclusions, and Discussions: The U87MG cell line successfully formed spheroids at various seeding densities. On average, the growth rates of low-density spheroids were directly related to seeding density; however, high-density seedings revealed a slowed growth rate (Figure 1). At 20,000 cells/well, there was no growth in spheroid size after seeding, likely because of cell contact-induced apoptosis. Other high-density spheroids stopped proliferating after day 3, revealing 5,000 cells/well as the optimal seeding density for spheroid formation. Live immunofluorescent staining revealed that the necrotic core of the spheroid contained no live cells (Figure 2A). Hoechst binds to live DNA, and Propidium Iodide binds to dead DNA. As the image was taken closer to the surface, there was a strong blue signal of Hoechst. Wheat Germ Agglutinin binds the cell membrane and fluoresces green. At all image positions, a green ring was seen around the spheroid; this signal was intense toward the surface, indicating that the cell membrane of the surface proliferating cells was identified (Figure 2B). These images characterized the layers of the spheroids as having proliferative cells on the outer surface and a necrotic core in the center. Western blots and RT-qPCR confirmed the expression of oncogenes, such as EGFR, in both two-dimensional cell cultures and three-dimensional spheroids and provided a comparison of expression between the two models. In conclusion, the three-dimensional spheroids offer a promising model for more accurate in vitro studies for glioblastoma treatments. The glioblastoma U87MG cell line 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 two-dimensional and three-dimensional cell models and Annexin V Staining to better characterize the U87MG spheroid cellular layers.

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