Generation of immunocompetent somatic glioblastoma mouse models through in situ transformation of subventricular zone neural stem cells

Disease-relevant in vivo tumor models are essential tools for both discovery and translational research. Here, we describe a highly genetically tractable technique for generating immunocompetent somatic glioblastoma (GBM) mouse models using piggyBac transposition and CRISPR-Cas9-mediated gene editing in wild-type mice. We describe steps to deliver plasmids into subventricular zone endogenous neural stem cells by injection and electroporation, leading to the development of adult tumors that closely recapitulate the histopathological, molecular, and cellular features of human GBM. For complete details on the use and execution of this protocol, please refer to Garcia-Diaz et al.1

Injury programs shape glioblastoma

Glioblastoma is the most common and aggressive primary brain cancer in adults and is almost universally fatal due to its stark therapeutic resistance. During the past decade, although survival has not substantially improved, major advances have been made in our understanding of the underlying biology. It has become clear that these devastating tumors recapitulate features of neurodevelopmental hierarchies which are influenced by the microenvironment. Emerging evidence also highlights a prominent role for injury responses in steering cellular phenotypes and contributing to tumor heterogeneity. This review highlights how the interplay between injury and neurodevelopmental programs impacts on tumor growth, invasion, and treatment resistance, and discusses potential therapeutic considerations in view of these findings

Diet suppresses tumour initiation by maintaining quiescence of mutation-bearing neural stem cells

Glioblastoma is thought to originate from neural stem cells (NSCs) of the subventricular zone that acquire genetic alterations. In the adult brain, NSCs are largely quiescent, suggesting that deregulation of quiescence maintenance may be a pre-requisite for tumour initiation. Although inactivation of the tumour suppressor p53 is a frequent event in gliomagenesis, whether, or how, it affects quiescent NSCs (qNSCs) remains unclear. Here we show that p53 maintains quiescence by inducing fatty acid oxidation (FAO) and that acute p53 deletion in qNSCs results in their premature activation to a proliferative state. Mechanistically, this occurs through direct transcriptional induction of PPARGC1a, which in turn activates PPARα to upregulate FAO genes. Strikingly, dietary supplementation with fish oil containing omega-3 fatty acids, natural PPARα ligands, fully restores quiescence of p53-deficient NSCs and delays tumour initiation in a glioblastoma mouse model. Thus, diet can silence glioblastoma driver mutations, with important implications for cancer prevention

Diet suppresses glioblastoma initiation in mice by maintaining quiescence of mutation-bearing neural stem cells

Glioblastoma is thought to originate from neural stem cells (NSCs) of the subventricular zone that acquire genetic alterations. In the adult brain, NSCs are largely quiescent, suggesting that deregulation of quiescence maintenance may be a prerequisite for tumor initiation. Although inactivation of the tumor suppressor p53 is a frequent event in gliomagenesis, whether or how it affects quiescent NSCs (qNSCs) remains unclear. Here, we show that p53 maintains quiescence by inducing fatty-acid oxidation (FAO) and that acute p53 deletion in qNSCs results in their premature activation to a proliferative state. Mechanistically, this occurs through direct transcriptional induction of PPARGC1a, which in turn activates PPARα to upregulate FAO genes. Dietary supplementation with fish oil containing omega-3 fatty acids, natural PPARα ligands, fully restores quiescence of p53-deficient NSCs and delays tumor initiation in a glioblastoma mouse model. Thus, diet can silence glioblastoma driver mutations, with important implications for cancer prevention

Injury primes mutation-bearing astrocytes for dedifferentiation in later life

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis

p53 restricts injury-induced plasticity in cortical astrocytes

Following injury, cortical astrocytes acquire neurogenic potential when explanted in vitro, but remain restricted to their own lineage in vivo. This lineage barrier can be fully transgressed in tumourigenesis, however the mechanisms that restrict normal astrocyte plasticity or how they are subverted by oncogenic insults are poorly defined. In this thesis, I have demonstrated that loss of the tumour suppressor gene p53 is sufficient to relax astrocyte identity. I found that in vitro, loss of p53 fully dedifferentiates astrocytes to neural stem-like cells in the presence of mitogens. Using single cell RNA sequencing I described the biological changes that underlie this process, revealing that dedifferentiating astrocytes undergo actin remodelling, followed by enhanced ribosomal biogenesis before reaching a highly proliferative, stem-like state. ChIP-sequencing of wildtype astrocytes indicated that p53 does not supress dedifferentiation by directly repressing a neurogenic program or maintaining glial fate. Instead, pathway analysis and loss-of-function experiments revealed that p53 null astrocytes increase expression of EGFR and its downstream signalling sensitising them to EGF and leading to ERK-dependent dedifferentiation. In vivo, p53 loss was induced in cortical astrocytes by stereotaxic injection, and led to some astrocytes retracting their processes and downregulating lineage markers, indicative of a change in cellular identity. Interestingly, these non-astrocytic cells were located very close to the injection wound site, suggesting that, as in vitro, extrinsic injury signals cooperate with p53 loss to drive this phenotype. Consistent with this, increasing EGF levels at the injury site through local infusion, resulted in complete dedifferentiation of p53-deficient astrocytes to a proliferative, stem-like state. Thus, injury signals cooperate with tumour-initiating mutations to increase astrocyte plasticity. This work suggests a possible mechanism of tumour initiation in the adult brain, with important implications for the aetiology and treatment of brain cancer