Correlation of MR-Based Metabolomics and Molecular Profiling in the Tumor Microenvironment of Temozolomide-Treated Orthotopic GL261 Glioblastoma in Mice.

The tumor microenvironment in glioblastoma (GB) is considered to be "cold", i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent "imprinting" mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage-tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8.

Effects of Caloric Restriction on Spatial Object Recognition Memory, Hippocampal Neuron Loss and Neuroinflammation in Aged Rats.

Age-related neurobiological changes significantly affect hippocampal structure and function, such that the main cognitive impairments associated with aging are related to the integrity of this brain structure, including the deterioration in spatial object recognition (SOR) memory. Previous studies have shown that intrinsic factors such as neuroinflammation, as well as lifestyle factors such as diet, can affect aging-associated brain functions and cognitive performance. In this regard, caloric restriction (CR) produces beneficial effects on health and life expectancy, although its ability to slow down age-dependent effects on cognitive decline and hippocampus (HPC) functioning remains unclear. Therefore, we set out to evaluate the effects of CR on SOR memory in aged male Wistar rats, as well as those on hippocampal neuron loss, neurogenesis and inflammation. The data show that CR in aged rats attenuates the decline in SOR memory, age-associated hippocampal neuron loss, and age-dependent microglial activation. Furthermore, we found a significant reduction in neurogenesis in the dentate gyrus of the old animals relative to adult rats. These findings support the positive effect of CR on SOR memory, suggesting that it dampens hippocampal neuronal loss and reduces proinflammatory activity.

Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells.

Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.

Three-dimensional vascular microenvironment landscape in human glioblastoma.

The cellular complexity of glioblastoma microenvironments is still poorly understood. In-depth, cell-resolution tissue analyses of human material are rare but highly necessary to understand the biology of this deadly tumor. Here we present a unique 3D visualization revealing the cellular composition of human GBM in detail and considering its critical association with the neo-vascular niche. Our images show a complex vascular map of human 3D biopsies with increased vascular heterogeneity and altered spatial relationship with astrocytes or glioma-cell counterparts. High-resolution analysis of the structural layers of the blood brain barrier showed a multilayered fenestration of endothelium and basement membrane. Careful examination of T cell position and migration relative to vascular walls revealed increased infiltration corresponding with tumor proliferation. In addition, the analysis of the myeloid landscape not only showed a volumetric increase in glioma-associated microglia and macrophages relative to GBM proliferation but also revealed distinct phenotypes in tumor nest and stroma. Images and data sets are available on demand as a resource for public access.

Ursolic Acid Inhibits Collective Cell Migration and Promotes JNK-Dependent Lysosomal Associated Cell Death in Glioblastoma Multiforme Cells.

Ursolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM) and offers a promising starting point in drug discovery; however, understanding the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrate that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM collective cell migration in a time dependent manner that is independent of the JNK signalling pathway. Cytotoxicity induced by UA results in the formation of acidic vesicle organelles (AVOs), speculating the activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs. Confocal microscopy and isosurface visualisation determined co-localisation of lysosomes with the previously identified AVOs, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death. Collectively, our data identify that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM collective cell migration.

Phagocytic glioblastoma-associated microglia and macrophages populate invading pseudopalisades.

Hypoxic pseudopalisades are a pathological hallmark of human glioblastoma, which is linked to tumour malignancy and aggressiveness. Yet, their function and role in the tumour development have scarcely been explored. It is thought that pseudopalisades are formed by malignant cells escaping from the hypoxic environment, although evidence of the immune component of pseudopalisades has been elusive. In the present work, we analyse the immunological constituent of hypoxic pseudopalisades using high-resolution three-dimensional confocal imaging in tissue blocks from excised tumours of glioblastoma patients and mimic the hypoxic gradient in microfluidic platforms in vitro to understand the cellular motility. We visualize that glioblastoma-associated microglia and macrophages abundantly populate pseudopalisades, displaying an elongated kinetic morphology across the pseudopalisades, and are oriented towards the necrotic focus. In vitro experiments demonstrate that under hypoxic gradient, microglia show a particular motile behaviour characterized by the increase of cellular persistence in contrast with glioma cells. Importantly, we show that glioblastoma-associated microglia and macrophages utilize fibres of glioma cells as a haptotactic cue to navigate along the anisotropic structure of the pseudopalisades and display a high phagocytic activity at the necrotic border of the pseudopalisades. In this study, we demonstrate that glioblastoma-associated microglia and macrophages are the main immune cells of pseudopalisades in glioblastoma, travelling to necrotic areas to clear the resulting components of the prothrombotic milieu, suggesting that the scavenging features of glioblastoma-associated microglia and macrophages at the pseudopalisades serve as an essential counterpart for glioma cell invasion.

Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells.

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

The MTOC/Golgi Complex at the T-Cell Immunological Synapse.

T cells effectively explore the tissue in search for antigens. When activated, they dedicate a big amount of energy and resources to arrange a complex structure called immunological synapse (IS), containing a particular distribution of molecules defined as supramolecular activation clusters (SMACs), and become polarized toward the target cell in a manner that channels the information specifically. This arrangement is symmetrical and requires the polarization of the MTOC and the Golgi to be operational, especially for the proper delivery of lytic granules and the recycling of molecules three dimensionally segregated at the clustered interface. Alternatively, after the productive encounter, T cells need to rearrange again to newly navigate through the tissue, changing back to a motile state called immunological kinapse (IK). In this IK state, the MTOC and the Golgi apparatus are repositioned and recruited at the back of the T cell to facilitate motility, while the established symmetry of the elements of the SMACs is broken and distributed in a different pattern. Both states, IS and IK, are interchangeable and are mainly orchestrated by the MTOC/Golgi complex, being critical for an effective immune response.

Imbalance of immunological synapse-kinapse states reflects tumor escape to immunity in glioblastoma.

Since the proper activation of T cells requires the physical interaction with target cells through the formation of immunological synapses (IS), an alteration at this level could be a reason why tumors escape the immune response. As part of their life cycle, it is thought that T cells alternate between a static phase, the IS, and a dynamic phase, the immunological kinapse (IK), depending on high or low antigen sensing. Our investigation performed in tissue samples of human glioma shows that T cells are able to establish synapsing interactions not only with glioma tumorigenic cells, but also with stromal myeloid cells. Particularly, the IS displaying a T cell receptor-rich (TCR-rich) central supramolecular activation cluster (cSMAC) is preferentially established with stromal cells, as opposed to malignant cells. Conversely, T cells in the malignant areas showed distinct morphometric parameters compared with nonneoplastic tissue - the former characterized by an elongated shape, well-suited to kinaptic dynamics. Importantly, high-resolution 3-dimensional analyses demonstrated the existence of bona-fide IK preferentially arranged in malignant areas of the tumor. This imbalance of IS/IK states between these 2 microenvironments reveals the low antigenic sensing of T cells when patrolling tumorigenic cells and reflects the immunoevasive environment of the tumor.

Cold Atmospheric Plasma Induces ATP-Dependent Endocytosis of Nanoparticles and Synergistic U373MG Cancer Cell Death.

Gold nanoparticles (AuNP) have potential as both diagnostic and therapeutic vehicles. However, selective targeting and uptake in cancer cells remains challenging. Cold atmospheric plasma (CAP) can be combined with AuNP to achieve synergistic anti-cancer cytotoxicity. To explore synergistic mechanisms, we demonstrate both rate of AuNP uptake and total amount accumulated in U373MG Glioblastoma multiforme (GBM) cells are significantly increased when exposed to 75 kV CAP generated by dielectric barrier discharge. No significant changes in the physical parameters of AuNP were caused by CAP but active transport mechanisms were stimulated in cells. Unlike many other biological effects of CAP, long-lived reactive species were not involved, and plasma-activated liquids did not replicate the effect. Chemical effects induced by direct and indirect exposure to CAP appears the dominant mediator of enhanced uptake. Transient physical alterations of membrane integrity played a minor role. 3D-reconstruction of deconvoluted confocal images confirmed AuNP accumulation in lysosomes and other acidic vesicles, which will be useful for future drug delivery and diagnostic strategies. Toxicity of AuNP significantly increased by 25-fold when combined with CAP. Our data indicate that direct exposure to CAP activates AuNP-dependent cytotoxicity by increasing AuNP endocytosis and trafficking to lysosomes in U373MG cells.

Studying the T Cell-Astrocyte Immune Synapse.

In this chapter, we describe the technical details to visualize and analyze effector immunological synapses between T cells and astrocytes in the brain with high-resolution confocal imaging. This procedure is critical for the optimal and even penetration of labeling antibodies within the nerve tissue to obtain accurate staining and allow a uniform three-dimensional analysis of the T cell-astrocyte interactions. We emphasize here the comprehensive exploration of the tissue and analysis with confocal microscope as well as the display of microanatomical details of the three-dimensional reconstruction for interface visualization (including peripheral and central supramolecular activation clusters, effector molecules, and other organelles such as microtubule organizing centers (MTOCs) and Golgi apparatus).

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. METHODS: We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. RESULTS: We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. CONCLUSIONS: Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy.

Kupfer-type immunological synapses in vivo: Raison D'être of SMAC.

T cells engage with antigen-presenting cells to form immunological synapses. These intimate contacts are characterized by the complex arrangement of molecules at the intercellular interface, which has been described as the supramolecular activation cluster (SMAC). However, due to T cells functioning without SMAC formation and the difficulties of studying these complex arrangements in vivo, its biological importance has been questioned. In light of recent data, we focus this review on the putative functionality of SMACs in T-cell synaptic contacts in vivo and emphasize the therapeutic potential of SMAC manipulation in immune-driven diseases.

Lipoic acid treatment after brain injury: study of the glial reaction.

After trauma brain injury, oxidative substances released to the medium provoke an enlargement of the initial lesion, increasing glial cell activation and, occasionally, an influx of immune cells into the central nervous system, developing the secondary damage. In response to these stimuli, microglia are activated to perform upregulation of intracellular enzymes and cell surface markers to propagate the immune response and phagocytosis of cellular debris. The phagocytosis of debris and dead cells is essential to limit the inflammatory reaction and potentially prevent extension of the damage to noninjured regions. Lipoic acid has been reported as a neuroprotectant by acting as an antioxidant and anti-inflammatory agent. Furthermore, angiogenic effect promoted by lipoic acid has been recently shown by our group as a crucial process for neural regeneration after brain injury. In this work, we focus our attention on the lipoic acid effect on astroglial and microglial response after brain injury.

Glial-mediated inflammation underlying parkinsonism.

The interest in studying neuroimmune interactions is increasing in the scientific community, and for many researchers, immunity is becoming a crucial factor in the understanding of the physiology of the normal brain as well as the biology underlying neurodegenerative diseases. Mounting data over the last two decades point toward immune and inflammatory alterations as important mediators of the progressive dopaminergic degeneration in Parkinson's disease. The purpose of this review is to address, under a historical perspective, as well as in the light of recent reports, the glial-mediated inflammatory and immune responses that occur in Parkinsonism. In line with this, this review also evaluates and highlights available anti-inflammatory drugs and putative targets for Parkinson's disease therapy for the near future.

Persistent phagocytic characteristics of microglia in the substantia nigra of long-term Parkinsonian macaques.

Patients with Parkinson's disease show persistent microglial activation in the areas of the brain where the degeneration of dopaminergic neurons takes place. The reason for maintaining this activated state is still unknown, but it is thought that this persistent microglial activation may contribute to the degeneration of dopaminergic neurons. In this study, we report the microanatomical details of microglia and the relationship between microglia and neurons in the substantia nigra pars compacta of Parkinsonian monkeys years after insult with MPTP. We observed that microglial cells appear polarized toward dopaminergic neurons in MPTP-treated macaques compared to untreated animals and present clear phagocytic characteristics, such as engulfing gliaptic contacts, an increase in Golgi apparatus protein machinery and ball-and-chain phagocytic buds. These results demonstrate that activated microglia maintain phagocytic characteristics years after neurotoxin insult, and phagocytosis may be a key contributor to the neurodegenerative process.

Imaging the microanatomy of astrocyte-T-cell interactions in immune-mediated inflammation.

The role of astrocytes in the immune-mediated inflammatory response in the brain is more prominent than previously thought. Astrocytes become reactive in response to neuro-inflammatory stimuli through multiple pathways, contributing significantly to the machinery that modifies the parenchymal environment. In particular, astrocytic signaling induces the establishment of critical relationships with infiltrating blood cells, such as lymphocytes, which is a fundamental process for an effective immune response. The interaction between astrocytes and T-cells involves complex modifications to both cell types, which undergo micro-anatomical changes and the redistribution of their binding and secretory domains. These modifications are critical for different immunological responses, such as for the effectiveness of the T-cell response, for the specific infiltration of these cells and their homing in the brain parenchyma, and for their correct apposition with antigen-presenting cells (APCs) to form immunological synapses (ISs). In this article, we review the current knowledge of the interactions between T-cells and astrocytes in the context of immune-mediated inflammation in the brain, based on the micro-anatomical imaging of these appositions by high-resolution confocal microscopy and three-dimensional rendering. The study of these dynamic interactions using detailed technical approaches contributes to understanding the function of astrocytes in inflammatory responses and paves the way for new therapeutic strategies.

ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo.

The role of microglial motility in the context of adult neurodegeneration is poorly understood. In the present work, we investigated the microanatomical details of microglia-neuron interactions in an experimental mouse model of Parkinson's disease following the intraperitoneal injection of MPTP. The specific intoxication of dopaminergic neurons induces the cellular polarization of microglia, leading to the formation of body-to-body neuron-glia contacts, called gliapses, which precede neuron elimination. Inhibiting ROCK/Cdc42-mediated microglial motility in vivo blocks the activating features of microglia, such as increased cell size and number of filopodia and diminishes their phagocyting/secreting domains, as the reduction of the Golgi apparatus and the number of microglia-neuron contacts has shown. High-resolution confocal images and three-dimensional rendering demonstrate that microglia engulf entire neurons at one-to-one ratio, and the microglial cell body participates in the formation of the phagocytic cup, engulfing and eliminating neurons in areas of dopaminergic degeneration in adult mammals.