Genetically engineered cerebral organoids model brain tumor formation

Abstract

Brain tumors are among the most lethal and devastating cancers. Their study is limited by genetic heterogeneity and the incompleteness of available laboratory models. Three-dimensional organoid culture models offer innovative possibilities for the modeling of human disease. Here we establish a 3D in vitro model called a neoplastic cerebral organoid (neoCOR), in which we recapitulate brain tumorigenesis by introducing oncogenic mutations in cerebral organoids via transposon- and CRISPR-Cas9-mediated mutagenesis. By screening clinically relevant mutations identified in cancer genome projects, we defined mutation combinations that result in glioblastoma-like and central nervous system primitive neuroectodermal tumor (CNS-PNET)-like neoplasms. We demonstrate that neoCORs are suitable for use in investigations of aspects of tumor biology such as invasiveness, and for evaluation of drug effects in the context of specific DNA aberrations. NeoCORs will provide a valuable complement to the current basic and preclinical models used to study brain tumor biology.

Figure 1. Introducing genome-editing constructs into neural stem/precursor cells (NS/PCs) of cerebral organoids.

(a) Schematic of cerebral organoid culture and nucleofection strategy. (b) The images show immunofluorescence staining for the indicated markers in EBs 1 day after nucleofection. Lower panel shows high-magnification images of nucleofected cells. Arrowheads point to nucleofected cells (GFP, green) that express NS/PC markers; arrows point to cells expressing mesodermal (BRA or FOXF1) or endodermal (SOX17 or CD31) markers. This experiment was performed twice independently with same results. EB, embryoid body; bFGF, basic fibroblast growth factor; hESCs, human embryonic stem cells; hiPSCs, human induced pluripotent stem cells; RA, retinoic acid; N-CAD: N-CADHERIN; NES: NESTIN; BRA: BRACHYURY. Scale bar: b, upper panel: 200 μm; lower panel: 100 μm.

Figure 2. Clonal mutagenesis in organoids induces tumor overgrowth.

(a-c) Immunofluorescence images (a) and quantification of the GFP fluorescence intensity (b,c) of organoids mutagenized with the indicated mutation combinations 1 day (b) and 1 month (c) after nucleofection. Organoids from four groups exhibited significant overgrowth at one month: MYC^OE (n=7; adjusted p<0.0001 v.s. CTRL(SB)), CDKN2A^-/CDKN2B^-/EGFR^OE/EGFRvIII^OE (n=5; adjusted p<0.0001 v.s. CTRL(dT+SB)), NF1^-/PTEN^-/p53^- (n=9; adjusted p<0.0001 v.s. CTRL(dT+SB)), EGFRvIII/PTEN^-/CDK2A^- (n=6; adjusted p<0.0001 v.s. CTRL(dT+SB)). This experiment was performed once. Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data are presented as mean±SD; full details including all sample sizes are provided Source Data. ***, p<0.001. Scale bar: a, 1 day: 200 μm; 1 month: 500 μm.

Figure 3. MYC^OE and GBM-like neoCORs have distinct transcriptional profiles and cellular identities.

(a) Principle component analysis (PCA) of the top 500 variable genes between normal cells from CTRL organoids and tumour cells from different neoCOR groups. (b) The heatmap shows normalized expression levels for differentially expressed genes (adjusted absolute log2fc value >1 or <-1 and adjusted p value <0.05) between Cluster 2 and Cluster 3 (n=3 for Cluster 2 and n=7 for Cluster 3 from one experiment) selected from differentially expressed genes between human primary CNS-PNET and GBM tumours. The heatmap was created from log2(TPM) transformed data that was row (gene) normalized using the “Median Center Genes/Rows” and “Normalize Genes/Rows” functions to report data as relative expression between samples. (c) Representative immunofluorescence images of four-month-old organoids from CTRL, MYC^OE, and GBM-1. The staining was performed from six independent experiments with the similar results. (d-i) Quantification of the indicated markers from panel c (magenta) and Supplemental Fig. 5 in CTRL and all neoCOR groups. Quantification was performed on organoids from three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s test. Data are presented as mean±SD, with details of sample sizes and values, as well as adjusted p value in Source Data. *, p<0.05; **, p<0.01; ***, p<0.001. Scale bar: c: 100 μm.

Figure 4. NeoCORs expand upon renal subcapsular xenografts.

(a) Brightfield and immunofluorescence images of the indicated renal subcapsular implants 1.5 months after implantation. (b) H&E staining of neoCORs under the renal capsule. Arrows indicate glial cells, arrowhead indicates a neuron. (c-e) Immunohistochemical staining of the indicated markers in implanted organoids. Scale bar: a, 500 mm; b-f, 200 μm and 50 μm (inset).

Figure 5. GBM neoCORs exhibit features of GBM invasion.

(a-c) Representative images of the tumour-normal interface in GBM-1 neoCORs. Images are representative of at least three independent experiments. (d) Immunohistochemical staining of GFAP in GBM-like neoCORs. Images are representative of two independent renal implantations. Dotted black lines indicate the boundary between implanted neoCORs and murine kidney. Dotted red line indicates the renal tubule. Arrowheads indicate invaded tumour cells. (e) Hierarchical clustering analysis of GBM invasiveness-relevant genes from four-month-old organoids (n=3 for CTRL organoids; n=4 for MYC^OE, n=4 for GBM-1, n=4 for GBM-2, and n=3 for GBM-3 neoCORs, from three independent cultures for each group). The heatmap was created from log2(TPM) transformed data that was row (gene) normalized using the “Median Center Genes/Rows” and “Normalize Genes/Rows” functions to report data as relative expression between samples. (f) Representative immunofluorescence staining of neoCORs from GBM-1 group for the indicated mesenchymal marker; GFP is also shown. Images are representative of two independent experiments. Scale bar: a, 1000 mm; b and c, 200 mm; d, 25 μm; f: 100 μm.

Figure 6. NeoCORs are suitable for preclinical investigations.

(a) Schematic of the drug treatment and FACS analysis using neoCORs. (b-e) Images and FACS quantification of cells from neoCORs after the indicated treatment. The percentage of GFP⁺ cells from drug-treated groups was normalized to the percentage of GFP⁺ cells from DMSO-treated neoCORs. Afatinib diminished tumour cells in GBM-1 (b; n=6 from one experiment; p=0.0005) and GBM-3 (c; n=3 from one experiment; p=0.0004) neoCORs, but not in MYC^OE (d; n=8 from one experiment; p=0.5261) and GBM-2 (e; n=5 from one experiment; p=0.7916) groups. The experiments were performed twice independently with similar results. Statistical analysis was performed using unpaired two-tailed Student’s t-test. Data were presented as mean±SD, details of sample size and values are provided in Source Data. ***, p<0.001. Scale bar: b-e, 1000 μm.