Patient-derived glioblastoma organoids reflect tumor heterogeneity and treatment sensitivity

Abstract

Background: Treatment resistance and tumor relapse are the primary causes of mortality in glioblastoma (GBM), with intratumoral heterogeneity playing a significant role. Patient-derived cancer organoids have emerged as a promising model capable of recapitulating tumor heterogeneity. Our objective was to develop patient-derived GBM organoids (PGO) to investigate treatment response and resistance.

Methods: GBM samples were used to generate PGOs and analyzed using whole-exome sequencing (WES) and single-cell karyotype sequencing. PGOs were subjected to temozolomide (TMZ) to assess viability. Bulk RNA sequencing was performed before and after TMZ.

Results: WES analysis on individual PGOs cultured for 3 time points (1-3 months) showed a high inter-organoid correlation and retention of genetic variants (range 92.3%-97.7%). Most variants were retained in the PGO compared to the tumor (range 58%-90%) and exhibited similar copy number variations. Single-cell karyotype sequencing demonstrated preservation of genetic heterogeneity. Single-cell multiplex immunofluorescence showed maintenance of cellular states. TMZ treatment of PGOs showed a differential response, which largely corresponded with MGMT promoter methylation. Differentially expressed genes before and after TMZ revealed an upregulation of the JNK kinase pathway. Notably, the combination treatment of a JNK kinase inhibitor and TMZ demonstrated a synergistic effect.

Conclusions: Overall, these findings demonstrate the robustness of PGOs in retaining the genetic and phenotypic heterogeneity in culture and the application of measuring clinically relevant drug responses. These data show that PGOs have the potential to be further developed into avatars for personalized adaptive treatment selection and actionable drug target discovery and as a platform to study GBM biology.

Keywords: glioblastoma; organoids; preclinical models; temozolomide resistance; tumor heterogeneity.

Figure 1.

Development of a patient-derived glioblastoma (GBM) organoid (PGO) platform and characterization of key GBM features. (A) Representative H&E stainings of PGOs. Scale bar is 500 uM. (B) Representative immunofluoresence imaging of PGOs for proliferation (EdU, top-left), hypoxia (pimonidazole, top-right), Nestin (bottom-left) and SOX2 (bottom-right). Nuclei are stained with DAPI. Scale bar is 100 uM. (C) Western blot characterization of key proteins involved in GBM tumorigenesis in PGOs (N = 6). Data is shown for epidermal growth factor receptor (EGFR), phosphatase and tensin homolog (PTEN), methylguanine methyltransferase (MGMT), platelet-derived growth factor receptor alpha (PDGFRA), phosphoinositide 3-kinase (PI3K), tumor protein 53 (p53), and cyclin-dependent kinase 4 (CDK4).

Figure 2.

Genetic characterization of patient-derived GBM organoids (PGOs). (A) Heatmap of variants identified by whole-exome sequencing (WES) of 3 individual PGOs derived from the same patient (N = 3). Pearson correlation coefficients are reported between each sample. (B) Venn diagram of variants identified by WES of PGOs derived from the same patient (N = 5) at different time points in culture (4 weeks, 8 weeks, and 12 weeks). 2012.2: 98% overlap, 2% 4 weeks only; 3128: 98% overlap, 2% 4 weeks only; 1919: 97% overlap, 3% 8 weeks only; 3565: 94% overlap, 2% 4 weeks only, 4% 8 weeks only; 1914: 93% overlap, 7% 12 weeks only. (C) Comparison between coding variants found by WES in PGOs and their corresponding parental tumors. (D) Oncoplot of top relevant mutations (top panel) and copy number variations (CNVs, bottom panel) in GBM based on WES in the tumor (T) and organoid (O).

Figure 3.

Phylogenetic trees derived from single-cell karyotype sequencing for different patient-derived glioblastoma (GBM) organoids (PGOs; N = 7). Losses and gains of (parts of) chromosomes are reported in the figure.

Figure 4.

Digital reconstructions of patient-derived glioblastoma organoid (PGO) slides after multiplex immunohistochemistry, cells were classified based on marker expression: MES (mesenchymal-like), OPC (oligodendrocyte-progenitor like), AC (astrocyte like), and NPC (neural progenitor cell like) or SOX2 (no specific classification). (A) PGO.007. (B) PGO.009. (C) PGO.027. (D) PGO.030. (E) PGO.031. (F) PGO.033. (G) Quantification of different cell populations in GBM organoids (blank bars) and their corresponding parental tumors (dashed bars). (H) Digital reconstructions of epidermal growth factor receptor (EGFR) expression heterogeneity within PGOs, representative examples are shown from a PGO with high EGFR and low EGFR expression.

Figure 5.

Treatment sensitivity of patient-derived glioblastoma organoids (PGOs): standard-of-care treatment and proof-of-principle targeted therapy. (A) Dose–response curves temozolomide (TMZ) treatment in PGOs (N = 3, mean and SEM) in methylguanine methyltransferase (MGMT)-unmethylated (left) and MGMT-methylated (right) patients. Cell viability was measured using CellTiterGlo assay and compared to untreated control (DMSO). (B) Comparison of response to human equivalent dosage (30 uM) of TMZ inPGOs (N = 3, Mean and SD). Significance was calculated using unpaired t-test. (C) Bart chart of sensitivity of PGOs toward chemoradiation treatment (N = 3, Mean and SD). PGOs were treated with 15 uM TMZ, 1 time radiotherapy (RT) 1Gy or 3 times RT 1Gy radiation or a combination treatment (RT + TMZ). (D) Dose–response curve of osimertinib treatment (N = 3, Mean and SD) as measured by cell viability. (E) Western blot analysis of PGOs for EGFR (endothelial growth factor receptor) protein, pERK (phosphorylated extracellular regulated kinase), and cleaved PARP (Poly [ADP-ribose] polymerase 1) before and after 5 uM osimertinib treatment for 24 h. Vinculin was used as a loading control.

Figure 6.

Changes in patient-derived glioblastoma (GBM) organoid (PGO) gene expression upon temozolomide (TMZ) treatment. (A) Volcano plot of differentially expressed genes (identified by RNAseq) between TMZ-treated and TMZ-untreated PGOs (N = 3). (B) Dose–response curves of TMZ and JNK inhibitor combination treatment in PGOs (N = 5). PGOs were treated with either 30 uM TMZ, 5 uM SP600125, or a combination. (C) Cell viability results of PGOs treated with different dosages of TMZ and JNK inhibitor. Data are shown for 3 PGOs combined. (D) Bliss scores showing the interaction between different concentrations of TMZ and JNK inhibitor for 3 PGOs combined. A Bliss score >10 confirms a synergistic reacting when both treatment options are combined.