Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

Abstract

Summary Glioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

Generation of glioblastoma patient-derived organoids and mouse brain orthotopic xenografts for drug screening

“… Alternatives: Alternatively, you can replace PBS with 0.9% Sodium Chloride. Before planning the surgical implantation of gliospheres or PDO cells in the mouse brain, GBM cells may optionally be labeled with luciferase reporters and/or fluorescent reporters (e.g., using lentiviral vectors) (see Chadwick et al., 2020 for details) to be able to monitor glioma formation in live mouse brains after formation of PDOXs. On the day of the mouse surgery, prepare a cell suspension from labeled gliosphere or PDO cultures and prepare the appropriate media (see Materials and equipment and Culture establishment, storage, and propagation, steps 3 and 4).…”

“…Before planning the surgical implantation of gliospheres or PDO cells in the mouse brain, GBM cells may optionally be labeled with luciferase reporters and/or fluorescent reporters (e.g., using lentiviral vectors) (see Chadwick et al., 2020 for details) to be able to monitor glioma formation in live mouse brains after formation of PDOXs.…”

“… Approximately 1 μg of RNA is used for cDNA Synthesis (explained below): cDNA synthesis is accomplished by following Thermo Fisher Superscript IV VILO Master Mix protocol ( https://assets.thermofisher.com/TFS-Assets/LSG/manuals/superscriptIV_VILO_master_mix_UG.pdf ). Resulting cDNA is used along with the desired primers for qPCR (for a list of target genes that are used to identify the expression signature of GBM subtypes, please refer to the list in Chadwick et al., 2020 ): Newly synthesized cDNA is diluted using ultra-pure PCR grade water. A SYBR green mix including forward (F), reverse (R) primers and ultra-pure PCR grade water mixture is made fresh and following the manufacturer’s specifications.…”

“…Resulting cDNA is used along with the desired primers for qPCR (for a list of target genes that are used to identify the expression signature of GBM subtypes, please refer to the list in Chadwick et al., 2020 ): Newly synthesized cDNA is diluted using ultra-pure PCR grade water. A SYBR green mix including forward (F), reverse (R) primers and ultra-pure PCR grade water mixture is made fresh and following the manufacturer’s specifications.…”

“…Promising therapies can then be validated in PDOXs for translation in precision medicine oncology trials. For complete details on the use and execution of this protocol, please refer to Chadwick et al. (2020) and Patrizii et al.…”