A proteomic approach supports the clinical relevance of TAT-Cx43266-283 in glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer. The Src inhibitor, TAT-Cx43266-283, exerts antitumor effects in in vitro and in vivo models of GBM. Because addressing the mechanism of action is essential to translate these results to a clinical setting, in this study we carried out an unbiased proteomic approach. Data-independent acquisition mass spectrometry proteomics allowed the identification of 190 proteins whose abundance was modified by TAT-Cx43266-283. Our results were consistent with the inhibition of Src as the mechanism of action of TAT-Cx43266-283 and unveiled antitumor effectors, such as p120 catenin. Changes in the abundance of several proteins suggested that TAT-Cx43266-283 may also impact the brain microenvironment. Importantly, the proteins whose abundance was reduced by TAT-Cx43266-283 correlated with an improved GBM patient survival in clinical datasets and none of the proteins whose abundance was increased by TAT-Cx43266-283 correlated with shorter survival, supporting its use in clinical trials.

The Gap Junction Inhibitor Octanol Decreases Proliferation and Increases Glial Differentiation of Postnatal Neural Progenitor Cells.

Neural precursor cells (NPCs) that persist in the postnatal/adult subventricular zone (SVZ) express connexins that form hemichannels and gap junctions. Gap junctional communication plays a role in NPC proliferation and differentiation during development, but its relevance on postnatal age remains to be elucidated. In this work we aimed to evaluate the effect of the blockade of gap junctional communication on proliferation and cell fate of NPCs obtained from the SVZ of postnatal rats. NPCs were isolated and expanded in culture as neurospheres. Electron microscopy revealed the existence of gap junctions among neurosphere cells. Treatment of cultures with octanol, a broad-spectrum gap junction blocker, or with Gap27, a specific blocker for gap junctions formed by connexin43, produced a significant decrease in bromodeoxyuridine incorporation. Octanol treatment also exerted a dose-dependent antiproliferative effect on glioblastoma cells. To analyze possible actions on NPC fate, cells were seeded in the absence of mitogens. Treatment with octanol led to an increase in the percentage of astrocytes and oligodendrocyte precursors, whereas the percentage of neurons remained unchanged. Gap27 treatment, in contrast, did not modify the differentiation pattern of SVZ NPCs. Our results indicate that general blockade of gap junctions with octanol induces significant effects on the behavior of postnatal SVZ NPCs, by reducing proliferation and promoting glial differentiation.

Galectin-3 depletion tames pro-tumoural microglia and restrains cancer cells growth.

Galectin-3 (Gal-3) is a multifunctional protein that plays a pivotal role in the initiation and progression of various central nervous system diseases, including cancer. Although the involvement of Gal-3 in tumour progression, resistance to treatment and immunosuppression has long been studied in different cancer types, mainly outside the central nervous system, its elevated expression in myeloid and glial cells underscores its profound impact on the brain's immune response. In this context, microglia and infiltrating macrophages, the predominant non-cancerous cells within the tumour microenvironment, play critical roles in establishing an immunosuppressive milieu in diverse brain tumours. Through the utilisation of primary cell cultures and immortalised microglial cell lines, we have elucidated the central role of Gal-3 in promoting cancer cell migration, invasion, and an immunosuppressive microglial phenotypic activation. Furthermore, employing two distinct in vivo models encompassing primary (glioblastoma) and secondary brain tumours (breast cancer brain metastasis), our histological and transcriptomic analysis show that Gal-3 depletion triggers a robust pro-inflammatory response within the tumour microenvironment, notably based on interferon-related pathways. Interestingly, this response is prominently observed in tumour-associated microglia and macrophages (TAMs), resulting in the suppression of cancer cells growth.

Antiproliferative effect of boldine on neural progenitor cells and on glioblastoma cells.

Introduction: The subventricular zone (SVZ) is a brain region that contains neural stem cells and progenitor cells (NSCs/NPCs) from which new neurons and glial cells are formed during adulthood in mammals. Recent data indicate that SVZ NSCs are the cell type that acquires the initial tumorigenic mutation in glioblastoma (GBM), the most aggressive form of malignant glioma. NSCs/NPCs of the SVZ present hemichannel activity whose function has not yet been fully elucidated. In this work, we aimed to analyze whether hemichannel-mediated communication affects proliferation of SVZ NPCs and GBM cells. Methods and Results: For that purpose, we used boldine, an alkaloid derived from the boldo tree (Peumus boldus), that inhibits connexin and pannexin hemichannels, but without affecting gap junctional communication. Boldine treatment (50 µM) of rat SVZ NPCs grown as neurospheres effectively inhibited dye uptake through hemichannels and induced a significant reduction in neurosphere diameter and in bromodeoxyuridine (BrdU) incorporation. However, the differentiation pattern was not modified by the treatment. Experiments with specific blockers for hemichannels formed by connexin subunits (D4) or pannexin 1 (probenecid) revealed that probenecid, but not D4, produced a decrease in BrdU incorporation similar to that obtained with boldine. These results suggest that inhibition of pannexin 1 hemichannels could be partially responsible for the antiproliferative effect of boldine on SVZ NPCs. Analysis of the effect of boldine (25-600 µM) on different types of primary human GBM cells (GBM59, GBM96, and U87-MG) showed a concentration-dependent decrease in GBM cell growth. Boldine treatment also induced a significant inhibition of hemichannel activity in GBM cells. Discussion: Altogether, we provide evidence of an antimitotic action of boldine in SVZ NPCs and in GBM cells which may be due, at least in part, to its hemichannel blocking function. These results could be of relevance for future possible strategies in GBM aimed to suppress the proliferation of mutated NSCs or glioma stem cells that might remain in the brain after tumor resection.

Isolation and Purification of Fungal ß-Glucan as an Immunotherapy Strategy for Glioblastoma.

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor microenvironment. Immunotherapy has emerged as an effective strategy to turn the immune system response against tumor cells. Glioma-associated macrophages and microglia (GAMs) are major drivers of such anti-inflammatory scenarios. Therefore, enhancing the anti-cancerous response in GAMs may represent a potential co-adjuvant therapy to treat glioblastoma patients. In that vein, fungal ß-glucan molecules have long been known as potent immune modulators. Their ability to stimulate the innate immune activity and improve treatment response has been described. Those modulating features are partly attributed to their ability to bind to pattern recognition receptors, which, interestingly, are greatly expressed in GAMs. Thus, this work is focused on the isolation, purification, and subsequent use of fungal ß-glucans to enhance the tumoricidal response of microglia against glioblastoma cells. The mouse glioblastoma (GL261) and microglia (BV-2) cell lines are used to test the immunomodulatory properties of four different fungal ß-glucans extracted from mushrooms heavily used in the current biopharmaceutical industry: Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus, and Ganoderma lucidum. To test these compounds, co-stimulation assays were performed to measure the effect of a pre-activated microglia-conditioned medium on the proliferation and apoptosis activation in glioblastoma cells.

Purinergic Receptor Blockade with Suramin Increases Survival of Postnatal Neural Progenitor Cells In Vitro.

Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.

Connexin43 Region 266-283, via Src Inhibition, Reduces Neural Progenitor Cell Proliferation Promoted by EGF and FGF-2 and Increases Astrocytic Differentiation.

Neural progenitor cells (NPCs) are self-renewing cells that give rise to the major cells in the nervous system and are considered to be the possible cell of origin of glioblastoma. The gap junction protein connexin43 (Cx43) is expressed by NPCs, exerting channel-dependent and -independent roles. We focused on one property of Cx43-its ability to inhibit Src, a key protein in brain development and oncogenesis. Because Src inhibition is carried out by the sequence 266-283 of the intracellular C terminus in Cx43, we used a cell-penetrating peptide containing this sequence, TAT-Cx43266-283, to explore its effects on postnatal subventricular zone NPCs. Our results show that TAT-Cx43266-283 inhibited Src activity and reduced NPC proliferation and survival promoted by epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). In differentiation conditions, TAT-Cx43266-283 increased astrocyte differentiation at the expense of neuronal differentiation, which coincided with a reduction in Src activity and ß-catenin expression. We propose that Cx43, through the region 266-283, reduces Src activity, leading to disruption of EGF and FGF-2 signaling and to down-regulation of ß-catenin with effects on proliferation and differentiation. Our data indicate that the inhibition of Src might contribute to the complex role of Cx43 in NPCs and open new opportunities for further research in gliomagenesis.

Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43266-283.

BACKGROUND: Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43266-283 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43266-283. METHODS: Metabolic impairment induced by the c-Src inhibitor TAT-Cx43266-283 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence. FINDINGS: TAT-Cx43266-283 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266-283 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43266-283 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3. INTERPRETATION: The reduced ability of TAT-Cx43266-283-treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43266-283. FUNDING: Spanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).

Connexin43 peptide, TAT-Cx43266-283, selectively targets glioma cells, impairs malignant growth, and enhances survival in mouse models in vivo.

BACKGROUND: Malignant gliomas are the most frequent primary brain tumors and remain among the most incurable cancers. Although the role of the gap junction protein, connexin43 (Cx43), has been deeply investigated in malignant gliomas, no compounds have been reported with the ability to recapitulate the tumor suppressor properties of this protein in in vivo glioma models. METHODS: TAT-Cx43266-283 a cell-penetrating peptide which mimics the effect of Cx43 on c-Src inhibition, was studied in orthotopic immunocompetent and immunosuppressed models of glioma. The effects of this peptide in brain cells were also analyzed. RESULTS: While glioma stem cell malignant features were strongly affected by TAT-Cx43266-283, these properties were not significantly modified in neurons and astrocytes. Intraperitoneally administered TAT-Cx43266-283 decreased the invasion of intracranial tumors generated by GL261 mouse glioma cells in immunocompetent mice. When human glioma stem cells were intracranially injected with TAT-Cx43266-283 into immunodeficient mice, there was reduced expression of the stemness markers nestin and Sox2 in human glioma cells at 7 days post-implantation. Consistent with the role of Sox2 as a transcription factor required for tumorigenicity, TAT-Cx43266-283 reduced the number and stemness of human glioma cells at 30 days post-implantation. Furthermore, TAT-Cx43266-283 enhanced the survival of immunocompetent mice bearing gliomas derived from murine glioma stem cells. CONCLUSION: TAT-Cx43266-283 reduces the growth, invasion, and progression of malignant gliomas and enhances the survival of glioma-bearing mice without exerting toxicity in endogenous brain cells, which suggests that this peptide could be considered as a new clinical therapy for high-grade gliomas.

Interactions Between Neural Progenitor Cells and Microglia in the Subventricular Zone: Physiological Implications in the Neurogenic Niche and After Implantation in the Injured Brain.

The adult subventricular zone (SVZ) of the mammalian brain contains neural progenitor cells (NPCs) that continuously produce neuroblasts throughout life. These neuroblasts migrate towards the olfactory bulb where they differentiate into local interneurons. The neurogenic niche of the SVZ includes, in addition to NPCs and neuroblasts, astrocytes, ependymal cells, blood vessels and the molecules released by these cell types. In the last few years, microglial cells have also been included as a key component of the SVZ neurogenic niche. Microglia in the SVZ display unique phenotypic features, and are more densely populated and activated than in non-neurogenic regions. In this article we will review literature reporting microglia-NPC interactions in the SVZ and the role of this bilateral communication in microglial function and in NPC biology. This interaction can take place through the release of soluble factors, extracellular vesicles or gap junctional communication. In addition, as NPCs are used for cell replacement therapies, they can establish therapeutically relevant crosstalks with host microglia which will also be summarized throughout the article.

A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity.

The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266-283, inhibits Src activity, we investigated the effect of TAT-Cx43266-283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266-283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266-283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266-283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266-283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266-283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266-283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266-283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266-283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation.

Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells.

The postnatal subventricular zone (SVZ) lining the walls of the lateral ventricles contains neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. Communication via gap junctions between cells in the SVZ is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. SVZ NPCs can be expanded in vitro in the form of neurospheres that can be used for transplantation purposes after brain injury. We have previously reported that neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when they are implanted after mechanical injury. To analyze functionality of NPC-glial cell gap junctions we performed dye coupling experiments in co-cultures of SVZ NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, Cx45 and pannexin 1 (Panx1). Dye coupling experiments revealed that gap junctional communication occurred among neurosphere cells (incidence of coupling: 100%). Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap junction blocker. Altogether, these results suggest the existence of functional hemichannels and gap junction channels in postnatal SVZ neurospheres. In addition, they demonstrate that SVZ-derived NPCs can establish functional gap junctions with astrocytes or microglia. Therefore, cell-cell communication via gap junctions and hemichannels with host glial cells might subserve a role in the functional integration of NPCs after implantation in the damaged brain.

Neural progenitor cell implants in the lesioned medial longitudinal fascicle of adult cats regulate synaptic composition and firing properties of abducens internuclear neurons.

Transplants of neural progenitor cells (NPCs) into the injured CNS have been proposed as a powerful tool for brain repair, but, to date, few studies on the physiological response of host neurons have been reported. Therefore, we explored the effects of NPC implants on the discharge characteristics and synaptology of axotomized abducens internuclear neurons, which mediate gaze conjugacy for horizontal eye movements. NPCs were isolated from the subventricular zone of neonatal cats and implanted at the site of transection in the medial longitudinal fascicle of adult cats. Abducens internuclear neurons of host animals showed a complete restoration of axotomy-induced alterations in eye position sensitivity, but eye velocity sensitivity was only partially regained. Analysis of the inhibitory and excitatory components of the discharge revealed a normal re-establishment of inhibitory inputs, but only partial re-establishment of excitatory inputs. Moreover, their inhibitory terminal coverage was similar to that in controls, indicating that there was ultimately no loss of inhibitory synaptic inputs. Somatic coverage by synaptophysin-positive contacts, however, showed intermediate values between control animals and animals that had undergone axotomy, likely due to partial loss of excitatory inputs. We also demonstrated that severed axons synaptically contacted NPCs, most of which were VEGF immunopositive, and that abducens internuclear neurons expressed the VEGF receptor Flk1. Together, our results suggest that VEGF neurotrophic support might underlie the increased inhibitory-to-excitatory balance observed in the postimplant cells. The noteworthy improvement of firing properties of injured neurons following NPC implants indicates that these cells might provide a promising therapeutic strategy after neuronal lesions.

Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants.

Neural progenitor cell implants modulate vascular endothelial growth factor and brain-derived neurotrophic factor expression in rat axotomized neurons.

Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might contribute to the restorative effects of these implants.