A syngeneic spontaneous zebrafish model oftp53-deficient, EGFRviii, and PI3KCAH1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapsein vivo
To build a patient-relevantin vivomodel of human glioblastoma, we expressed common oncogenic variants including activated human EGFRviii or KRAS,and PI3KCAH1047Runder the control of the radial glial-specific promoterher4.1in syngeneictp53loss-of-function mutant zebrafish.Robust tumor formation was observed prior to 45 days of life, with a gene expression signature similar to human glioblastoma of the mesenchymal subtype, along with a strong inflammatory component. Within early stage tumor lesions, and in an intact and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells bympeg1.1:GFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiationin vivo. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factorsirf7andirf8led to increased tumor formation in the primary context, while suppression of microglial/macrophage activity led to enhanced tumor cell engraftment following transplantation into otherwise immune competent zebrafish hosts. Altogether, we developed a genetically-relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor suppressive roles for the innate immune system on glioblastoma initiation, with important future significance for therapeutic discovery and optimizations. Overall design: To capture broad transcriptomic differences in tumor-burdened brains versus normal brains, we performed bulk RNA sequencing and gene expression analysis of whole brains and sorted cells.  EGFRviii derived tumors were induced by microinjection of EGFRviii, PI3KCAH1047R, and mScarlet constructs (defined as EPS). KRAS derived tumors were induced by microinjection of KRAS, PI3KCAH1047R, GFP constructs (defined as KPG). Three whole brains positive for fluorescent tumors (EPS_1-3, KPG_1-3), alongside three control brains from injected siblings who did not develop tumors (CTRL_1-3), were dissected for each condition for bulk RNA sequencing. We also performed fluorescent activated cell sorting on pooled dissociated brains from the EPS condition, resulting in enrichment for mScarlet postive tumor cells (CS_Pos) and mScarlet negative cells (CS_Neg) for comparative analysis.