A syngeneic spontaneous zebrafish model of tp53-deficient, EGFR^viii, and PI3KCA^H1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapse in vivo

Alex Weiss
Cassandra D’Amata
Bret J. Pearson author has email address
Madeline N. Hayes author has email address

Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Canada
Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, 97239, USA
Department of Pediatrics, Papé Research Institute, Oregon Health & Science University, Portland, Oregon, 97239, USA
Department of Molecular Genetics, University of Toronto, Toronto, Canada

https://doi.org/10.7554/eLife.93077.1

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Figures and data

her4.1-driven over-expression of patient-relevant oncogenes drives glial-derived brain tumor formation in syngeneic tp53 loss-of-function mutant zebrafish.
A) Schematic of modeling strategy where linearized transgene vectors with the zebrafish her4.1 promoter driving human EGFR^viii, human PI3KCA^H1047R, and mScarlet fluorescent protein are co-injected at the one-cell stage into syngeneic (CG1) tp53^-/- mutant zebrafish embryos. Starting at 15 days post fertilization (dpf), mosaic-injected zebrafish are screened for CNS tumor formation, indicated by mScarlet expression in the brain region of live zebrafish. Co-injection of gfap:GFP linearized transgene is used to assess glial-specific cell fate specification in vivo. (B) her4.1:mScarlet and gfap:GFP expression in the anterior CNS of mosaic-injected syngeneic (CG1) tp53^-/- zebrafish at 30dpf. (C) Whole brain dissected from a p53EPS mosaic-injected zebrafish at 30dpf. (D) Cumulative frequencies of mScarlet+ CNS lesions in syngeneic tp53^-/- mutant (CG1tp53^-/-) and wild-type (CG1) zebrafish injected at the one cell stage with her4.1:EGFR^viii (E), her4.1:PI3KCA^H1047R (P), and/or her4.1:mScarlet (S). (E) Syngeneic (CG1) zebrafish at 30dpf engrafted with her4.1:mScarlet+/gfap:GFP+ brain tumor cells, following primary transplantation (1T) at 2dpf into the embryonic brain ventricle. (F) Whole brain dissected from engrafted syngeneic host (CG1) zebrafish at 30dpf. (G) FACS plot of bulk syngeneic host brain following primary transplant (1T) of her4.1:EGFR^viii + her4.1:PI3KCA^H1047R + her4.1:mScarlet + gfap:GFP brain tumor cells. (H) Hematoxylin and eosin (H&E) staining of brain region of p53EPS mosaic-injected zebrafish at 30dpf. Inset highlights tumor region. Scale bars represent 200μm and 20μm, respectively. (I-K) Immunohistochemical staining of proliferating cell nuclear antigen (PCNA, I), phosphorylated ERK (p-ERK, J), and phosphorylated AKT (p-AKT, K) on tumor section. Scare bars represent 50μm.

RNA expression analysis establishes enrichment of mesenchymal glioblastoma and inflammation signatures in p53EPS model.
(A) Principal component analysis (PCA) of mRNA sequencing from whole control injected brains (CTRL), p53EPS and p53KPS tumor-burdened brains. (B) Heatmap of normalized counts for genes upregulated in p53EPS tumor-burdened brains (log2FoldChange > 2, padj < 0.05), compared to whole control injected brains (CTRL). A selected list of upregulated transcripts is indicated. (C) Gene set enrichment analysis (GSEA) plots of published gene signatures for mesenchymal subtype glioblastoma for genes differentially regulated in p53EPS compared to control injected brains^6,19. Normalized Enrichment Scores (NES) and False Discovery Rates (FDR) are indicated. (D) Bar plot of Normalized Enrichment Score from Gene Set Enrichment Analysis (GSEA) of Hallmark gene sets³³. (E) Volcano plots of differentially expressed genes between sorted mScarlet+ p53EPS tumor cells and control injected whole brain tissue (CTRL WB), and sorted mScarlet-negative cells from p53EPS tumor-burdened brains and control injected whole brains (CTRL WB). (F) her4.1:mScarlet and rag2:EGFP expression in live zebrafish with a p53EPS tumor at 30 dpf. (G) FACS plot of p53EPS brain with rag2:EGFP co-expression from (F). (H, I) her4.1:mScarlet and mpeg1.1:EGFP expression in live zebrafish with a p53EPS tumor at 30 dpf.

p53EPS recruits activated microglia/macrophages at early stages of tumor initiation.
(A) Neutral red staining of mScarlet-negative and mScarlet+ p53EPS injected brains at 10 dpf. Neutral red foci in early-stage lesions are highlighted with arrows and are indicative of phagocytic cells. (B) Whole brain with p53EPS-induced tumor in a transgenic Tg(tnfa:EGFP) zebrafish at 20 dpf. Black arrows indicate tnfa:EGFP+ punctae associated with mScarlet+ brain tumor lesion. (C) Z-stack projections of three independent p53EPS brains at 10-13 dpf in transgenic Tg(mpeg1.1:GFP) background. (D) Quantification of tumor-associated mpeg1.1:GFP+ cells with overlapping and/or internalized her4.1:mScarlet+ punctae (n=3).

Inflammation-associated irf7 and irf8 inhibit p53EPS formation in vivo.
(A-C) Primary (1°) control (A), irf7 CRISPR/Cas9 (B), and irf8 CRISPR/Cas9 (C) injected p53EPS at 30 dpf. (D) p53EPS incidence at 30 dpf in control (n=3 independent experiments, 108 zebrafish), irf7 CRISPR/Cas9 (***p<0.0001, Fisher’s exact test, n=2 independent experiments, 31 zebrafish), and irf8 CRISPR/Cas9 (*p=0.0155, Fisher’s exact test, n= 2 independent experiments, 36 total injected zebrafish). (E) Tg(her4.1:Cas9-2A-EGFP) expression at 30 dpf. (F) mScarlet+ p53EPS at 30 dpf in Tg(her4.1:Cas9-2A-EGFP) injected with irf7 gRNAs at the one-cell stage. (G) p53EPS incidence at 30 dpf in Tg(her4.1:Cas9-2A-EGFP)-negative gRNA-injected control siblings, and Tg(her4.1:Cas9-2A-EGFP) zebrafish injected at the one-cell stage with irf7 or irf8 gRNAs. n.s. not significant, Fisher’s exact test.

Inflammation-associated microglia/macrophages inhibit p53EPS tumor engraftment.
(A,B) CG1 syngeneic host zebrafish at 20 dpf engrafted with p53EPS tumor cells transplanted with vehicle control (A) or Clodronate liposomes (B) at 2 dpf. (C-E) Quantification of p53EPS control engrafted and p53EPS tumors engrafted into CG1 host embryos with (C) Clodronate liposomes (p=0.0048, Fisher’s exact test, n=2 independent experiments, total 56 transplanted vehicle control and 50 transplanted Clodrosome-injected hosts), (D) engrafted into irf8 CRISPR/Cas9-injected into CG1 syngeneic host embryos (p=0.0002, Fisher’s exact test, n=2 independent experiments, total 100 transplanted control and 74 transplanted irf8 CRISPR/Cas9-injected hosts).

Inter-tumoral heterogeneity in p53EPS induced tumors. Sample screens of p53EPS injected fish from three different cohorts showing distinct tumor initiation sites and varying degrees of mScarlet-fluorescent intensity, with intense fluorescence indicative of tumorigenesis. Tumors predominantly arise in the optic tectum/mesencephalon region (21/29) with a distinctive subset appearing in the telencephalon/diencephalon region (8/29). Distinct tumors did not arise in the cerebellum/rhombencephalon regions over the course of our experiments.

(A) her4.1:mScarlet and gfap:GFP expression in the anterior CNS of 30dpf syngeneic (CG1) tp53^-/-zebrafish injected at the one-cell stage with linearized her4.1:KRAS^G12D + her4.1:PI3KCA^H1047R + her4.1:GFP + gfap:mScarlet transgenes. (B) Whole brain dissected from mosaic-injected her4.1:KRAS^G12D + her4.1:PI3KCA^H1047R + her4.1:GFP zebrafish at 30dpf. (C) Cumulative frequencies of GFP+ brain lesions in syngeneic tp53^-/- mutant (CG1tp53^-/-) and wild-type (CG1) zebrafish injected at the one cell stage with her4.1:KRAS^G12D (K), her4.1:PI3KCA^H1047R (P), and/or her4.1:GFP (G). (D) Immunohistochemical staining of proliferating cell nuclear antigen (PCNA), phosphorylated ERK (p-ERK), and phosphorylated AKT (p-AKT) on her4.1:KRAS^G12D + her4.1:PI3KCA^H1047R + her4.1:GFP tumor section. Scale bars represent 50μm.

Gene set enrichment analysis (GSEA) plots of published gene signatures for molecular subtypes of human glioblastoma and medulloblastoma for genes differentially regulated in p53EPS compared to control injected brains^6,19,32. Normalized Enrichment Scores (NES) and False Discovery Rates (FDR) are indicated.

Quantitative real-time PCR analysis of neural stem cell (NSC) genes and genes associated with published inflammatory gene signatures identified using bulk RNA sequencing. Gene expression in pooled her4.1:mScarlet+/gfap:GFP+ FACS-sorted cells relative to non-tumor control brain tissue. *p<0.01, **p<0.001, n=3, Student’s t-test.

Quantitative real-time PCR analysis of irf7 CRISPR/Cas9-injected (A) and irf8 CRISPR/Cas9-injected p53EPS (B). Gene expression is represented relative to control p53EPS tumor-burdened whole brains. At least 5 tumor-burdened brains were pooled for each cohort and expression was normalized to mScarlet mRNA expression to account for differences in tumor size. *p<0.01, Student’s t-test.

Neutral red (phagocyte) foci in control and irf8 CRISPR/Cas9-injected zebrafish larvae at 8 dpf. *p<0.01, n=4 fish for each cohort, Student’s t-test.

(A) Quantitative real-time PCR analysis of neural stem cell (NSC) genes and inflammation genes in non-tumor control brains, pooled p53EPS-burdened brains and pooled brains engrafted with p53EPS tumor cells at 20 dpf. *p<0.01 compared to non-tumor control whole brain tissue, Student’s t-test. (B) Quantitative real-time PCR analysis of neural stem cell (NSC) genes and inflammation genes at 20 dpf in primary transplanted (1T) p53EPS-burdened brains injected with vehicle control liposomes (1T p53EPS) and Clodrosomes at 2 dpf. At least 5 tumor-burdened brains were pooled for each cohort and expression was normalized to mScarlet mRNA expression to account for differences in tumor size. *p<0.01 compared to 1T p53EPS, Student’s t-test.

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