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Collective cell migration is fundamental for the development of organisms and in the adult, for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact-inhibition-of-locomotion (CIL), a local repulsive force, propels the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during Schwann cell collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin acts to traffic a repulsive Slit2/Slit3 signal to the cell-surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective SC migration, resulting in adherent, non-migratory cell clusters. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.

International audience; Glioblastoma is one of the most lethal forms of adult cancer with a median survival of around 15 months. A potential treatment strategy involves targeting glioblastoma stem-like cells (GSC), which constitute a cell autonomous reservoir of aberrant cells able to initiate, maintain, and repopulate the tumor mass. Here, we report that the expression of the paracaspase mucosa-associated lymphoid tissue l (MALT1), a protease previously linked to antigen receptor-mediated NF-jB activation and B-cell lymphoma survival, inversely correlates with patient probability of survival. The knockdown of MALT1 largely impaired the expansion of patient-derived stem-like cells in vitro, and this could be recapitulated with pharmacological inhibitors, in vitro and in vivo. Blocking MALT1 protease activity increases the endo-lysosome abundance, impairs autophagic flux, and culminates in lysosomal-mediated cell death, concomitantly with mTOR inactiva-tion and dispersion from endo-lysosomes. These findings place MALT1 as a new druggable target involved in glioblastoma and unveil ways to modulate the homeostasis of endo-lysosomes.

International audience; Glioblastoma multiforme (GBM) is a highly aggressive tumour of the central nervous system and is associated with an extremely poor prognosis. Within GBM exists a subpopulation of cells, glioblastoma-initiating cells (GIC), which possess the characteristics of progenitor cells, have the ability to initiate tumour growth and resist to current treatment strategies. We aimed at identifying novel specific inhibitors of GIC expansion through use of a large-scale chemical screen of approved small molecules. Here, we report the identification of the natural compound β-escin as a selective inhibitor of GIC viability. Indeed, β-escin was significantly cytotoxic in nine patient-derived GIC, whilst exhibiting no substantial effect on the other human cancer or control cell lines tested. In addition, β-escin was more effective at reducing GIC growth than current clinically used cytotoxic agents. We further show that β-escin triggers caspase-dependent cell death combined with a loss of stemness properties. However, blocking apoptosis could not rescue the β-escin-induced reduction in sphere formation or stemness marker activity, indicating that β-escin directly modifies the stem identity of GIC, independent of the induction of cell death. Thus, this study has repositioned β-escin as a promising potential candidate to selectively target the aggressive population of initiating cells within GBM.

It is not yet known how tumour cells traverse the blood-brain barrier (BBB) to form brain metastases. Substance P (SP) release is a key component of neurogenic inflammation which has been recently shown to increase the permeability of the BBB following CNS insults, making it a possible candidate as a mediator of tumour cell extravasation into the brain. This study investigated the properties of the BBB in the early stages of tumour cell invasion into the brain, and the possible involvement of SP. Male Wistar rats were injected with Walker 256 breast carcinoma cells via the internal carotid artery and euthanised at 1, 3, 6 and 9 days post tumour inoculation. Culture medium-injected animals served as controls at 1 and 9 days. Evidence of tumour cell extravasation across the BBB was first observed at 3 days post-inoculation, which corresponded with significantly increased albumin (p < 0.05) and SP immunoreactivity (p < 0.01) and significantly reduced endothelial barrier antigen labelling of microvessels when compared to culture medium control animals (p < 0.001). By day 9 after tumour cell inoculation, 100 % of animals developed large intracranial neoplasms that had significantly increased albumin in the peri-tumoral area (p < 0.001). The increased SP immunoreactivity and altered BBB properties at 3 days post-inoculation that coincided with early tumour invasion may be indicative of a mechanism for tumour cell extravasation into the brain. Thus, extravasation of tumour cells into the brain to form cerebral metastases may be a SP-mediated process. Refereed/Peer-reviewed

International audience; Glioblastoma are malignant highly vascularized brain tumours, which feature large oedema resulting from tumour-promoted vascular leakage. The pro-permeability factor Semaphorin3A (Sema3A) produced within glioblastoma has been linked to the loss of endothelial barrier integrity. Here, we report that extracellular vesicles (EVs) released by patient-derived glioblastoma cells disrupt the endothelial barrier. EVs expressed Sema3A at their surface, which accounted for in vitro elevation of brain endothelial permeability and in vivo vascular permeability, in both skin and brain vasculature. Blocking Sema3A or its receptor Neuropilin1 (NRP1) hampered EV-mediated permeability. In vivo models using ectopically and orthotopically xenografted mice revealed that Sema3A-containing EVs were efficiently detected in the blood stream. In keeping with this idea, sera from glioblastoma multiforme (GBM) patients also contain high levels of Sema3A carried in the EV fraction that enhanced vascular permeability, in a Sema3A/ NRP1-dependent manner. Our results suggest that EV-delivered Sema3A orchestrates loss of barrier integrity in glioblastoma and may be of interest for prognostic purposes.

International audience; Glioblastoma multiforme (GBM) constitutes the most common and the most aggressive type of human tumors affecting the central nervous system. Prognosis remains dark due to the inefficiency of current treatments and the rapid relapse. Paralleling other human tumors, GBM contains a fraction of tumor initiating cells with the capacity to self-renew, initiate and maintain the tumor mass. These cells were found in close proximity to brain vasculature, suggesting functional interactions between brain tumor-initiating cells (BTICs) and endothelial cells within the so-called vascular niche. However, the mechanisms by which these cells impact on the endothelium plasticity and function remain unclear. Using culture of BTICs isolated from a cohort of 14 GBM patients, we show that BTICs secretome promotes brain endothelial cell remodeling in a VEGF-independent manner. Gene array analysis unmasked that BTICs-released factors drove the expression of Ptch2 in endothelial cells. Interestingly, BTICs produce desert hedgehog (DHH) ligand, enabling a paracrine DHH/Ptch2 signaling cascade that conveys elevated permeability and angiogenesis. Finally, DHH silencing in BTICs dramatically reduced tumor growth, as well as vascularization and intra-tumor permeability. Collectively, our data unveil a role for DHH in exacerbated tumor angiogenesis and permeability, which may ultimately favor glioblastoma growth, and thus place the DHH/Ptch2 nexus as a molecular target for novel therapies.

International audience; Glioblastoma multiforme are mortifying brain tumors that contain a subpopulation of tumor cells with stem-like properties, termed as glioblastoma stem-like cells (GSCs). These GSCs constitute an autonomous reservoir of aberrant cells able to initiate, maintain, and repopulate the tumor mass. A new therapeutic strategy would consist of targeting the GSC population. The GSCs are situated in perivascular niches, closely associated with brain microvascular endothelial cells thereby involved in bidirectional molecular and cellular interactions. In this scenario, the endothelium not only supplies oxygen and necessary nutrients but also seeds a protective microenvironment for tumor growth. Although GSCfate, plasticity, and survival are regulated by external cues emanating from endothelial cells, the nature of such angiocrine signals remains unknown. Our laboratory conclusively demonstrated that brain endothelial cells positively control the expansion of GSCs. Notably, we found that GSCs are addicted to the hormonal peptide apelin (APLN) secreted by surrounding endothelial cells, and identified the APLN/APLNR nexus as a promising druggable network in glioblastoma.

Evidence for the involvement of the Substance P (SP)/NK1 receptor system in the development and progression of cancer strongly supports its potential as a therapeutic target in malignancies. Novel strategies for approaching cancer treatment are urgently required particularly with regard to tumours of the central nervous system (CNS), which are notoriously difficult to effectively treat and associated with extremely poor prognosis for many patients. This is due, in part, to the presence of the highly specialised blood-brain barrier, which is known to restrict common treatments such as chemotherapy and hinder early tumour diagnosis. Additionally, tumours of the CNS are difficult to surgically resect completely, often contributing to the resurgence of the disease many years later. Interestingly, despite the presence of the blood-brain barrier, circulating tumour cells are able to gain entry to the brain and form secondary brain tumours; however, the underlying mechanisms of this process remain unclear. Tachykinins, in particular Substance P, have been implicated in early blood-brain barrier disruption via neurogenic inflammation in a number of other CNS pathologies. Recent evidence also suggests that Substance P may play a central role in the development of CNS tumours. It has been well established that a number of tumour cells express Substance P, NK1 receptors and mRNA for the tachykinin NK1 receptor. This increase in the Substance P/NK1 receptor system is known to induce proliferation and migration of tumour cells as well as stimulate angiogenesis, thus contributing to tumour progression. Accordingly, the NK1 receptor antagonist presents a novel target for anti-cancer therapy for which a number of patents have been filed. This review will examine the role of Substance P in the development of CNS tumours and its potential application as an anti-cancer agent.

Red/near-infrared irradiation therapy (R/NIR-IT) delivered by laser or light-emitting diode (LED) has improved functional outcomes in a range of CNS injuries. However, translation of R/NIR-IT to the clinic for treatment of neurotrauma has been hampered by lack of comparative information regarding the degree of penetration of the delivered irradiation to the injury site and the optimal treatment parameters for different CNS injuries. We compared the treatment efficacy of R/NIR-IT at 670 nm and 830 nm, provided by narrow-band LED arrays adjusted to produce equal irradiance, in four in vivo rat models of CNS injury: partial optic nerve transection, light-induced retinal degeneration, traumatic brain injury (TBI) and spinal cord injury (SCI). The number of photons of 670 nm or 830 nm light reaching the SCI injury site was 6.6% and 11.3% of emitted light respectively. Treatment of rats with 670 nm R/NIR-IT following partial optic nerve transection significantly increased the number of visual responses at 7 days after injury (P ≤ 0.05); 830 nm R/NIR-IT was partially effective. 670 nm R/NIR-IT also significantly reduced reactive species and both 670 nm and 830 nm R/NIR-IT reduced hydroxynonenal immunoreactivity (P ≤ 0.05) in this model. Pre-treatment of light-induced retinal degeneration with 670 nm R/NIR-IT significantly reduced the number of Tunel+ cells and 8-hydroxyguanosine immunoreactivity (P ≤ 0.05); outcomes in 830 nm R/NIR-IT treated animals were not significantly different to controls. Treatment of fluid-percussion TBI with 670 nm or 830 nm R/NIR-IT did not result in improvements in motor or sensory function or lesion size at 7 days (P>0.05). Similarly, treatment of contusive SCI with 670 nm or 830 nm R/NIR-IT did not result in significant improvements in functional recovery or reduced cyst size at 28 days (P>0.05). Outcomes from this comparative study indicate that it will be necessary to optimise delivery devices, wavelength, intensity and duration of R/NIR-IT individually for different CNS injury types.

The neuropeptide substance P (SP) has been implicated in the disruption of the blood-brain barrier (BBB) and development of cerebral edema in acute brain injury. Cerebral edema accumulates rapidly around brain tumors and has been linked to several tumor-associated deficits. Currently, the standard treatment for peritumoral edema is the corticosteroid dexamethasone, prolonged use of which is associated with a number of deleterious side effects. As SP is reported to increase in many cancer types, this study examined whether SP plays a role in the genesis of brain peritumoral edema. A-375 human melanoma cells were injected into the right striatum of male Balb/c nude mice to induce brain tumor growth, with culture medium injected in animals serving as controls. At 2, 3 or 4 weeks following tumor cell inoculation, non-treated animals were perfusion fixed for immunohistochemical detection of Albumin, SP and NK1 receptor. A further subgroup of animals was treated with a daily injection of the NK1 antagonist Emend (3 mg/kg), dexamethasone (8 mg/kg) or saline vehicle at 3 weeks post-inoculation. Animals were sacrificed a week later to determine BBB permeability using Evan's Blue and brain water content. Non-treated animals demonstrated a significant increase in albumin, SP and NK1 receptor immunoreactivity in the peritumoral area as well as increased perivascular staining in the surrounding brain tissue. Brain water content and BBB permeability was significantly increased in tumor-inoculated animals when compared to controls (p

Recent research has investigated the expression and secretion of neuropeptides by tumors, and the potential of these peptides to facilitate tumor growth and spread. In particular, substance P (SP) and its receptor NK1 have been implicated in tumor cell growth and evasion of apoptosis, although few studies have examined this relationship in vivo. The present study used both in vitro and in vivo models to characterize the role of SP in tumor pathogenesis. Immunohistochemical assessment of human primary and secondary brain tumor tissue demonstrated a marked increase in SP and its NK1 receptor in all tumor types investigated. Of the metastatic tumors, melanoma demonstrated particularly elevated SP and NK1 receptor staining. Subsequently, A-375 human melanoma cell line was examined in vitro and found to express both SP and the NK1 receptor. Treatment with the NK1 receptor antagonist Emend IV resulted in decreased cell viability and an increase in cell death in this cell line in vitro. An animal model of brain tumors using the same cell line was employed to assess the effect of Emend IV on tumor growth in vivo. Administration of Emend IV was found to decrease tumor volume and decrease cellular proliferation indicating that SP may play a role in tumor pathogenesis within the brain. We conclude that SP may provide a novel therapeutic target in the treatment of certain types of brain tumors, with further research required to determine whether the role of SP in cancer is tumor-type dependent.

Emend, an NK1 antagonist, and dexamethasone are used to treat complications associated with metastatic brain tumours and their treatment. It has been suggested that these agents exert anticancer effects apart from their current use. The effects of the NK1 antagonists, Emend and N-acetyl-L-tryptophan, and dexamethasone on tumour growth were investigated in vitro and in vivo at clinically relevant doses. For animal experiments, a stereotaxic injection model of Walker 256 rat breast carcinoma cells into the striatum of Wistar rats was used. Emend treatment led to a decrease in tumour cell viability in vitro, although this effect was not replicated by N-acetyl-L-tryptophan. Dexamethasone did not decrease tumour cell viability in vitro but decreased tumour volume in vivo, likely to be through a reduction in tumour oedema, as indicated by the increase in tumour cell density. None of the agents investigated altered tumour cell replication or apoptosis in vivo. Inoculated animals showed increased glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 immunoreactivity indicative of astrocytes and microglia in the peritumoral area, whereas treatment with Emend and dexamethasone reduced the labelling for both glial cells. These results do not support the hypothesis that NK1 antagonists or dexamethasone exert a cytotoxic action on tumour cells, although these conclusions may be specific to this model and cell line. Refereed/Peer-reviewed

Background Metastatic brain tumours are a common end stage of breast cancer progression, with significant associated morbidity and high mortality. Walker 256 is a rat breast carcinoma cell line syngeneic to Wistar rats and commonly used to induce secondary brain tumours. Previously there has been the assumption that the same cancer cell line from different cell banks behave in a similar manner, although recent studies have suggested that cell lines may change their characteristics over time in vitro. Methods In this study internal carotid artery injection and direct cerebral inoculation models of secondary brain tumours were used to determine the tumorigenicity of Walker 256 cells obtained from two cell banks, the American Type Culture Collection (ATCC), and the Cell Resource Centre for Medical Research at Tohoku University (CRCTU). Results Tumour incidence and volume, plus immunoreactivity to albumin, IBA1 and GFAP, were used as indicators of tumorigenicity and tumour interaction with the host brain microenvironment. CRCTU Walker 256 cells showed greater incidence, larger tumour volume, pronounced blood–brain barrier disruption and prominent glial response when compared to ATCC cell line. Conclusions These findings indicate that immortalised cancer cell lines obtained from different cell banks may have diverse characteristics and behaviour in vivo.

Glioblastoma (GBM) is the most malignant and lethal brain tumor harboring glioma stem cells (GSCs) that promote tumor propagation and therapeutic resistance. GSCs preferentially express several critical cell surface molecules that regulate the pro-survival signaling for maintaining the stem cell-like phenotype. Tetraspanin CD9 has recently been reported as a GSC biomarker that is relevant to the GSC maintenance. However, the underlying molecular mechanisms of CD9 in maintaining GSC property remain elusive. Herein, we report that CD9 stabilizes the IL-6 receptor glycoprotein 130 (gp130) by preventing its ubiquitin-dependent lysosomal degradation to facilitate the STAT3 activation in GSCs. CD9 is preferentially expressed in GSCs of human GBM tumors. Mass spectrometry analysis identified gp130 as an interacting protein of CD9 in GSCs, which was confirmed by immunoprecipitation and immunofluorescent analyses. Disrupting CD9 or gp130 by shRNA significantly inhibited the self-renewal and promoted the differentiation of GSCs. Moreover, CD9 disruption markedly reduced gp130 protein levels and STAT3 activating phosphorylation in GSCs. CD9 stabilized gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote the BMX-STAT3 signaling in GSCs. Importantly, targeting CD9 potently inhibited GSC tumor growth in vivo, while ectopic expression of the constitutively activated STAT3 (STAT3-C) restored the tumor growth impaired by CD9 disruption. Collectively, we uncovered a critical regulatory mechanism mediated by tetraspanin CD9 to maintain the stem cell-like property and tumorigenic potential of GSCs.

Despite recent advances in cancer treatment and diagnosis, the prognosis for patients with CNS tumours remains extremely poor. This is, in part, due to the difficulty in completely removing tumours surgically, and also because of the presence of the blood brain barrier, which can prevent the entry of chemotherapeutic agents typically used in cancer treatment. Despite the presence of the blood brain barrier, tumour cells are capable of entering and colonising the brain to form secondary brain tumours. Additionally, tumour related disruption of the blood brain barrier is associated with the clinical presentation of many patients, with accompanying increases in intracranial pressure due, in part, to the development of vasogenic oedema. Vasogenic oedema results because the newly formed angiogenic vessels within brain tumours do not retain the highly selective properties of the blood brain barrier, and thus allow for the extravasation of plasma proteins and water into the brain parenchyma. Tachykinins, and in particular substance P, have been implicated in blood brain barrier disruption and the genesis of cerebral oedema in other CNS insults via a process known as neurogenic inflammation. Recent evidence suggests that substance P may play a similar role in CNS tumours. It has been well established that an upregulation of substance P and its receptors occurs in a number of different cancer types, including CNS neoplasms. In addition to disrupting blood brain barrier permeability, substance P and the NK1 receptors facilitate promotion of tumour growth and the development of cerebral oedema. Accordingly, recent patents describe the potential of NK1 receptor antagonists as anti-cancer agents suggesting that substance P may provide a novel cancer treatment target. This review will examine the role of substance P in the development of CNS tumours. Refereed/Peer-reviewed

Dexamethasone, the standard treatment for peritumoral brain oedema, inhibits classical inflammation. Neurogenic inflammation, which acts via substance P (SP), has been implicated in vasogenic oedema in animal models of CNS injury. SP is elevated within and outside CNS tumours. This study investigated the efficacy of NK1 receptor antagonists, which block SP, compared with dexamethasone treatment, in a rat model of tumorigenesis. Dexamethasone reverted normal brain water content and reduced Evans blue and albumin extravasation, while NK1 antagonists did not ameliorate oedema formation. We conclude that classical inflammation rather than neurogenic inflammation drives peritumoral oedema in this brain tumour model. Refereed/Peer-reviewed

Cancers of the brain are intrinsically more complicated to treat than systemic malignancies due to the unique anatomical features of the brain. The blood-brain barrier prevents chemotherapeutic agents from reaching brain neoplasms, and angiogenesis occurs as the metabolic needs of the tumour increase, thus further complicating treatment. The newly formed blood vessels form the blood-tumour barrier and are distinct from the blood-brain barrier in that they are more permeable. Being more permeable, these abnormal blood vessels lead to the formation of peri-tumoural edema, which is the cause of much morbidity and mortality associated with central nervous system neoplasms. While the cause of the increased permeability is unclear, kinins have been implicated in regulating the permeability of normal vasculature. Kinins are also known to exert many inflammatory actions affecting both normal and angiogenic blood vessels, as well as tumour cells. The vasodilatory and vascular permeabilizing effects of kinins, and particularly bradykinin and substance P, have been investigated with regard to delivery of chemotherapeutic agents to neoplastic brain tissue through both vascular barriers. In contrast, kinin receptor antagonists have been found to exert effects on tumour cells that result in decreased angiogenesis, tumour cell motility and growth. Thus, many recent patents describe kinin activity on brain vasculature, which may play an integral role in the development of treatments for malignancies in the central nervous system through amelioration of angiogenesis. In conjunction, patents that discuss the ability of kinins to decrease tumour cell migration and proliferation demonstrate that kinins may offer novel approaches to brain tumour therapy in the future. Refereed/Peer-reviewed

Angiotensin-converting enzyme (ACE) inhibitors are widely used as blood pressure medications in hypertensive individuals. However, ACE inhibitors also play an integral role in the breakdown of neuronal substance P, which has been recently implicated in the development of functional deficits following traumatic brain injury (TBI). The present study therefore examined the effects of ACE inhibitors on histological and motor outcome following TBI. Male Sprague-Dawley rats were treated with Captopril, Enalapril or equal volume saline for 7 days prior to the induction of diffuse TBI using the impact acceleration model. At 5 h post-injury, animals administered Captopril demonstrated significantly increased substance P immunoreactivity compared to vehicle controls (p < 0.01), and increased dark cell change that persisted to 7 days post-trauma. Captopril also resulted in exacerbated motor deficits compared to vehicle treated animals (p < 0.05) as assessed by the rotarod test over a 7-day post-traumatic period. Administration of the alternative ACE inhibitor, Enalapril, likewise exacerbated motor deficits, confirming a class effect of ACE inhibitors rather than a compound effect specific to Captopril. We conclude that ACE inhibitors are deleterious to outcome following TBI, presumably by impairing the degradation of substance P and increasing substance P mediated neuronal injury. Refereed/Peer-reviewed