Abstract 3568: Developing pediatric glioblastoma microtumors with a three-dimensional bioprinter
Authors: Beierle Andee M. , Schanel Taylor L. , Alrefai Hasan , Anderson Joshua C. , Hicks Patricia H. , Nassour Lauren C. , Willey Christopher D. 
Published: April 2023

Abstract
Background:Glioblastoma multiforme is one of the most devastating forms of brain cancer seen in children. Children diagnosed with high-grade gliomas have poor prognosis, and there remains a need for improvement in the standard of care. In order to improve the standard of care for this condition, it is necessary to develop in vitro models which better recapitulate the nature of the tumors. Three-dimensional bioprinting has proven to be an effective technique in creating microtumors using adult glioblastoma cell lines but remains unexplored in pediatric glioblastoma. As such, we sought to develop a protocol which would allow for the creation of three-dimensionally bioprinted microtumors using patient-derived pediatric glioblastoma cells.
Methods:A Bio X three-dimensional bioprinter (Cellink) was utilized to create the microtumor constructs. The composition of the bioink was modified through varying concentrations of sodium alginate and gelatin, or gelatin-methacrylate (Gel-MA). The microtumor size and volume was optimized through the variation of extrusion time, nozzle pressure, and both nozzle and print bed temperature. The crosslinking technique was also optimized through the utilization of 2% calcium chloride for varying exposure times, and also a 405-nm light (for Gel-MA bioink) at varied light intensity, length of time of application to, and distance from, the printed structure. Bioprinted pediatric XD456 brain tumor initiating cell constructs were treated with both chemotherapeutics and radiation to determine the model’s capacity to respond to treatment. PamStation 12 (PamGene Inc.) Kinomic data and RNA-seq (Illumina) data were generated for the constructs. Imaging was performed using Cytation 5 imager (Agilent).
Results:Fluorescent microscopy with Calcein AM and Sytox™ Orange (ThermoFisher), along with viability assessments via CellTiter-Glo®, demonstrated expected cytotoxic effects after exposure to 10 μM Cisplatin. Additionally, PamStation 12 Kinomic and RNA-seq data indicated a difference in kinomic signaling between the bioprinted constructs as compared to the XD456 cells cultured as neurospheres.
Conclusions:The utilization of a three-dimensional bioprinter allows for the creation of microtumors using patient-derived pediatric glioblastoma cells. Future studies will investigate the microtumors’ capacity as a drug screening model. The model will also be developed to further recapitulate the native tumor microenvironment of pediatric glioblastoma.
Citation Format: Andee M. Beierle, Taylor L. Schanel, Hasan Alrefai, Joshua C. Anderson, Patricia H. Hicks, Lauren C. Nassour, Christopher D. Willey. Developing pediatric glioblastoma microtumors with a three-dimensional bioprinter. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3568.