Abstract 228: Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients
Authors: Vooijs Marc A. , Hoosemans Linde , Piepers Jolanda , Verduin Maikel , Vanmechelen Maxime , Van Heumen Mike , De Smet Frederik , Hoeben Ann 
Published: March 2024

Glioblastoma (GBM) is the most common and incurable adult primary brain tumour. Treatment resistance and relapse is the primary cause of death in GBM. Tumour heterogeneity is one of the significant determinants of treatment failure. Patient-derived cancer organoids have been shown to maintain genetic and phenotypic tumour features and recapitulate intratumoral heterogeneity. Here, we report the development and molecular characterisation of patient-derived GBM organoid (PGO) models to study treatment response to existing and new treatment combinations. GBM biopsies were dissociated, suspended in Cultrex, and cultured in a brain stem cell medium. PGOs were characterised by whole-exome (WES) single-cell karyotype (scKaryoSeq) and RNA sequencing. The IC-50 and viability of PGO upon temozolomide (TMZ) and small molecule inhibitors were determined. Gene expression profiling was performed before and after TMZ treatment. Single-cell spatial heterogeneity was determined using Multiplex Immunostaining of PGO and matched patient samples. PGOs were established with a success rate of 80% (n=31). WES analysis between PGOs from the same patient at multiple time points (1-3 months) showed high maintenance of Single Nucleotide Variants (92.3%-97.7%). In PGOs key somatic oncogene variants and copy number variations were retained compared to the primary tumour (58%-90%). scKaryoseq (N=7) of different clinical GBM subtypes showed a high degree of genetic heterogeneity, with some PGO having up to 10 subclones. Single-cell phenotypic landscaping of patient biopsies showed mixed cellular states maintained after long-term culture in the matched PGO. PGOs showed a differential response to TMZ, corresponding with MGMT methylation and patient mutation status. RNAseq of PGO revealed TMZ-responsive genes, including upregulation of the prognostic JUN kinase pathway. Combination treatment of a JUN kinase inhibitor and TMZ showed a synergistic effect. In addition, PGOs were screened for their sensitivity to clinically approved brain-penetrable small molecule inhibitors targeting key GBM driver pathways. Combining small molecular inhibitors of MEK/PI-3/CDK4/EGFR kinase strongly reduced cell viability and activation of cell death pathways. phosphoKinome screens detected compensatory pathway activation upon kinase inhibition (MEK/CDK4 inhibitor) in several PGO. Data will show novel kinase interaction pathways that increase drug sensitivity in PGO. These data show that PGOs can be used as a stable phenotypic and genetic representative of the patient's tumour and have the potential to function as patient avatars for adaptive treatment selection and actionable drug target discovery.

Citation Format: Marc A. Vooijs, Linde Hoosemans, Jolanda Piepers, Maikel Verduin, Maxime Vanmechelen, Mike Van Heumen, Frederik De Smet, Ann Hoeben. Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 228.