Glioblastoma heterogeneity and the tumour microenvironment: implications for preclinical research and development of new treatments.

Glioblastoma is the deadliest form of brain cancer. Aside from inadequate treatment options, one of the main reasons glioblastoma is so lethal is the rapid growth of tumour cells coupled with continuous cell invasion into surrounding healthy brain tissue. Significant intra- and inter-tumour heterogeneity associated with differences in the corresponding tumour microenvironments contributes greatly to glioblastoma progression. Within this tumour microenvironment, the extracellular matrix profoundly influences the way cancer cells become invasive, and changes to extracellular (pH and oxygen levels) and metabolic (glucose and lactate) components support glioblastoma growth. Furthermore, studies on clinical samples have revealed that the tumour microenvironment is highly immunosuppressive which contributes to failure in immunotherapy treatments. Although technically possible, many components of the tumour microenvironment have not yet been the focus of glioblastoma therapies, despite growing evidence of its importance to glioblastoma malignancy. Here, we review recent progress in the characterisation of the glioblastoma tumour microenvironment and the sources of tumour heterogeneity in human clinical material. We also discuss the latest advances in technologies for personalised and in vitro preclinical studies using brain organoid models to better model glioblastoma and its interactions with the surrounding healthy brain tissue, which may play an essential role in developing new and more personalised treatments for this aggressive type of cancer.

Review: Synthetic scaffolds to control the biochemical, mechanical, and geometrical environment of stem cell-derived brain organoids.

Stem cell-derived brain organoids provide a powerful platform for systematic studies of tissue functional architecture and the development of personalized therapies. Here, we review key advances at the interface of soft matter and stem cell biology on synthetic alternatives to extracellular matrices. We emphasize recent biomaterial-based strategies that have been proven advantageous towards optimizing organoid growth and controlling the geometrical, biomechanical, and biochemical properties of the organoid's three-dimensional environment. We highlight systems that have the potential to increase the translational value of region-specific brain organoid models suitable for different types of manipulations and high-throughput applications.

IGF-1 receptor regulates dynamic changes in neuronal polarity during cerebral cortical migration.

During cortical development, neurons undergo polarization, oriented migration and layer-type differentiation. The biological and biochemical mechanisms underlying these processes are not completely understood. In neurons in culture we showed that IGF-1 receptor activation is important for growth cone assembly and axonal formation. However, the possible roles of the insulin like growth factor-1 receptor (IGF-1R) on neuronal differentiation and polarization in vivo in mammals have not yet been studied. Using in utero electroporation, we show here that the IGF-1R is essential for neocortical development. Neurons electroporated with a shRNA targeting IGF-1 receptor failed to migrate to the upper cortical layers and accumulated at the ventricular/subventricular zones. Co-electroporation with a constitutively active form of PI3K rescued migration. The change of the morphology from multipolar to bipolar cells was also attenuated. Cells lacking the IGF-1 receptor remain arrested as multipolar forming a highly disorganized tissue. The typical orientation of the migrating neurons with the Golgi complex oriented toward the cortical upper layers was also affected by electroporation with shRNA targeting IGF-1 receptor. Finally, cells electroporated with the shRNA targeting IGF-1 receptor were unable to form an axon and, therefore, neuron polarity was absent.

The physiological role of the amyloid precursor protein as an adhesion molecule in the developing nervous system.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell-surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over-expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition. Read the Editorial Highlight for this article on page 9. Cover Image for this issue: doi. 10.1111/jnc.13817.

The Motor KIF5C Links the Requirements of Stable Microtubules and IGF-1 Receptor Membrane Insertion for Neuronal Polarization.

Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity.

Protein interacting with NIMA (never in mitosis A)-1 regulates axonal growth cone adhesion and spreading through myristoylated alanine-rich C kinase substrate isomerization.

Axonal growth cone motility requires precise regulation of adhesion to navigate the complex environment of the nervous system and reach its target. Myristoylated alanine-rich C kinase substrate (MARCKS) protein is enriched in the developing brain and plays an important, phosphorylation-dependent role in the modulation of axonal growth cone adhesion. The ratio of phospho-MARCKS (MARCKS-P) to total MARCKS controls adhesion modulation and spreading of the axonal growth cone. Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that recognizes and binds to phosphorylated serine/threonine residues preceded by a proline (pSer/Thr-Pro) is also expressed in the developing brain. Here, we show that Pin1 is present in the growth cone, interacts with MARCKS-P, and regulates its dephosphorylation. We also described morphological alterations in the corpus callosum and cerebral cortex fibers of the Pin1 knockout mouse brain that may be caused by the misregulation of MARCKS-P and alterations of neuronal adhesion. We have shown that MARCKS, a critical protein in the movement of neuronal growth cones, is in turn regulated by both phosphorylation and cis-trans peptidyl isomerization mediated by Pin1. In the absence of Pin1, MARCKS is hyperphosphorylated, leading to loss of adhesions, and collapse of the growth cone. The Pin1 KO mice exhibited disturbed neuronal projections from the cerebral cortex and reduced white matter tracks such as the corpus callosum. This study highlights a novel function of Pin1 in neurodevelopment.

Selected SNARE proteins are essential for the polarized membrane insertion of igf-1 receptor and the regulation of initial axonal outgrowth in neurons.

The establishment of polarity necessitates initial axonal outgrowth and, therefore, the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles (PPVs) primarily at the neuronal growth cone. Little is known about the SNAREs family proteins involved in the regulation of PPV fusion with the neuronal plasmalemma at early stages of differentiation. We show here that five SNARE proteins (VAMP2, VAMP4, VAMP7, Syntaxin6 and SNAP23) were expressed by hippocampal pyramidal neurons before polarization. Expression silencing of three of these proteins (VAMP4, Syntaxin6 and SNAP23) repressed axonal outgrowth and the establishment of neuronal polarity, by inhibiting IGF-1 receptor exocytotic polarized insertion, necessary for neuronal polarization. In addition, stimulation with IGF-1 triggered the association of VAMP4, Syntaxin6 and SNAP23 to vesicular structures carrying the IGF-1 receptor and overexpression of a negative dominant form of Syntaxin6 significantly inhibited exocytosis of IGF-1 receptor containing vesicles at the neuronal growth cone. Taken together, our results indicated that VAMP4, Syntaxin6 and SNAP23 functions are essential for regulation of PPV exocytosis and the polarized insertion of IGF-1 receptor and, therefore, required for initial axonal elongation and the establishment of neuronal polarity.

Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway.

Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k, and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7.