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

Glioblastoma cell fate is differentially regulated by the microenvironments of the tumour bulk and infiltrative margin

Glioblastoma recurrence originates from invasive cells at the tumour margin that escape surgical debulking, but their biology remains poorly understood. Here we generated three somatic mouse models recapitulating the main glioblastoma driver mutations to characterise margin cells. We find that, regardless of genetics, tumours converge on a common set of neural- like cellular states. However, bulk and margin display distinct neurogenic patterns and immune microenvironments. The margin is immune-cold and preferentially follows developmental-like trajectories to produce astrocyte-like cells. In contrast, injury-like programmes dominate in the bulk, are associated with immune infiltration and generate lowly-proliferative injured neural progenitor-like (iNPCs) cells. In vivo label-retention approaches further demonstrate that iNPCs account for a significant proportion of dormant glioblastoma cells and are induced by interferon signalling within T-cell niches. These findings indicate that tumour region is a major determinant of glioblastoma cell fate and therapeutic vulnerabilities identified in bulk may not extend to the margin residuum

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

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

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

Spatial distribution of podoconiosis in relation to environmental factors in Ethiopia: a historical review

BACKGROUND An up-to-date and reliable map of podoconiosis is needed to design geographically targeted and cost-effective intervention in Ethiopia. Identifying the ecological correlates of the distribution of podoconiosis is the first step for distribution and risk maps. The objective of this study was to investigate the spatial distribution and ecological correlates of podoconiosis using historical and contemporary survey data. METHODS Data on the observed prevalence of podoconiosis were abstracted from published and unpublished literature into a standardized database, according to strict inclusion and exclusion criteria. In total, 10 studies conducted between 1969 and 2012 were included, and data were available for 401,674 individuals older than 15 years of age from 229 locations. A range of high resolution environmental factors were investigated to determine their association with podoconiosis prevalence, using logistic regression. RESULTS The prevalence of podoconiosis in Ethiopia was estimated at 3.4% (95% CI 3.3%-3.4%) with marked regional variation. We identified significant associations between mean annual Land Surface Temperature (LST), mean annual precipitation, topography of the land and fine soil texture and high prevalence of podoconiosis. The derived maps indicate both widespread occurrence of podoconiosis and a marked variability in prevalence of podoconiosis, with prevalence typically highest at altitudes >1500 m above sea level (masl), with >1500 mm annual rainfall and mean annual LST of 19-21°C. No (or very little) podoconiosis occurred at altitudes 24°C. CONCLUSION Podoconiosis remains a public health problem in Ethiopia over considerable areas of the country, but exhibits marked geographical variation associated in part with key environmental factors. This is work in progress and the results presented here will be refined in future work

AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons

Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMC alpha 2KO and AgRP alpha 2KO mice lacking AMPK alpha 2 in proopiomelanocortin- (POMC-) and agouti-related protein-expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMC alpha 2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. in contrast, AgRPa2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPK alpha 2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMC alpha 2KO and AgRP alpha 2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus