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

Reviewer #1 (Public Review):

Summary:
The authors have developed a zebrafish model of glioblastoma and characterized this, with a particular focus on the role of recruited myeloid cells in the tumours. Microglia/macrophages in the tumours are proposed to have an inflammatory phenotype and are engaged in phagocytosis. Knockout of Irf7 and Irf8 genes enhanced tumour initiation. Depleting mature myeloid cell types with chlodronate also enhanced tumour intitiation. It is proposed that in early-stage tumours, microglia/macrophages have tumour suppressive activity.

Strengths:
The authors have generated a novel glioblastoma model in zebrafish. Two key strengths of the zebrafish model are that early-stage tumours can be studied and in vivo visualization can be readily performed. The authors show a video of microglia/macrophages adopting the ameboid phenotype in tumours (as is observed in human tumours) and engaging in phagocytosis. Video 1 was very impressive in my opinion and shows the model is a very useful tool to study microglia/macrophage:glioblastoma cell interactions. The irf7/irf8 knockdown and the chlodronate experiments are consistent with a role for mature myeloid cells in suppressing tumour initiation, suggesting that the model may also be very valuable in understanding immune surveillance in glioblastoma initiation.

Weaknesses:
EGFRvIII is mainly associated with the classical subtype, so the mesenchymal subtype might be unexpected here. This could be commented on. Some more histologic characterization of the tumours would be helpful. Are they invasive, do larger tumours show necrosis and microvascular proliferation? This would help with understanding the full potential of the new model. Current thinking in established human glioblastoma is that the M1/M2 designations for macrophages are not relevant, with microglia macrophage populations showing a mixture of pre- and anti-inflammatory features. Ideally, there would be a much more detailed characterization of the intratumoral microglia/macrophage population here, as single markers can't be relied upon. Phagocytosis could have antitumour effects through the removal of live cancer cells, or could be cancer-promoting if apoptotic cancer cells are being rapidly cleared with concomitant activation of an immunosuppressive phenotype in the phagocytes (i.e. efferocytosis). It may be possible to distinguish between these two types of phagocytosis experimentally. Do the irf7/8 and chlodronate experiments distinguish between effects on microglia/macrophages and dendritic cells?

Reviewer #2 (Public Review):

Summary:
Glioblastoma is a common primary brain cancer, that is difficult to treat and has a low survival rate. The lack of genetically tractable and immunocompetent vertebrate animal models has prevented the discovery of new therapeutic targets and limited efforts for screening pharmaceutical agents for the treatment of the disease. Here Weiss et al., express oncogenic variants frequently observed in human glioblastoma within zebrafish lacking the tumor suppressor TP53 to generate a patient-relevant in vivo model. The authors demonstrate that loss of TP53 and overexpression of EGFR, PI3KCA, and mScarlet (p53EPS) in neural progenitors and radial glia leads to visible fluorescent brain lesions in live zebrafish. The authors performed RNA expression analysis that uncovered a molecular signature consistent with human mesenchymal glioblastoma and identified gene expression patterns associated with inflammation. Live imaging revealed high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. To define functional roles for regulators of inflammation on specific immune-related responses during tumorigenesis, transient CRISPR/Cas9 gene targeting was used to disrupt interferon regulator factor proteins and showed Inflammation-associated irf7 and irf8 are required to inhibit p53EPS tumor formation. Further, experiments to deplete the macrophages using clodronate liposomes suggest that macrophages contribute to the suppression of tumor engraftment following transplantation. The authors' conclusions are largely supported by the data and the experiments are thoroughly controlled throughout. Taken together, these results provide new insights into the regulation of glioblastoma initiation and growth by the surrounding microenvironment and provide a novel in vivo platform for the discovery of new molecular mechanisms and testing of therapeutics.

Strengths/Weaknesses:
The authors convincingly show that co-injection of activated human EGFRviii, PI3KCAH1047R, and mScarlet into TP53 null zebrafish promotes the formation of fluorescent brain lesions and glioblastoma-like tumor formation. The authors state that oncogenic MAPK/AKT pathway activation drives this glial-derived tumor formation. It would be important to include a wild-type or uninjected control for the pERK and pAKT staining shown in Fig1 I-K to aid in the interpretation of these results. Likewise, quantification of the pERK and pAKT staining would be useful to demonstrate the increase over WT, and would also serve to facilitate comparison with the similar staining in the KPG model (Supp Fig 2D).

The authors use a transplantation assay to further test the tumorigenic potential of dissociated cells from glial-derived tumors. Listing the percentage of transplants that generate fluorescent tumor would be helpful to fully interpret these data. Additionally, it was not clear based on the description in the results section that the transplantation assay was an "experimental surrogate" to model the relapse potential of the tumor cells. This is first mentioned in the discussion. The authors may consider adding a sentence for clarity earlier in the manuscript as it helps the reader better understand the logic of the assay.

The authors nicely show high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. However, a quantification of the emergence of macrophages over time in relation to tumor initiation and growth would provide significant support to the observations of tumor suppressive activity of the phagocytes. Along these lines, the inclusion of a statement about when leukocytes emerge during normal development would be informative for those not familiar with the zebrafish model.

From the data provided in Figure 4G and Supp Fig 7b, the authors suggest that "increased p53EPS tumor initiation following Ifr gene knock-down is a consequence of irf7 and irf8 loss-of-function in the TME". Given the importance of the local microenvironment highlighted in this study, spatial information in the form of in situ hybridization to identify the relevant location of the expression change would be important to support this conclusion.

The authors used neutral red staining that labels lysosomal-rich phagocytes to assess enrichment at the early stages of tumor initiation. The images in Figure 3 panel A should be labeled to denote the uninjected controls to aid in the interpretation of the data. In Supplemental Figure 6, the neutral red staining in the irf8 CRISPR-injected larvae looks to be increased, counter to the quantification. Can the authors comment if the image is perhaps not representative?