Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Nitrogen-15 dynamic nuclear polarization of nicotinamide derivatives in biocompatible solutions.

Dissolution dynamic nuclear polarization (dDNP) increases the sensitivity of magnetic resonance imaging by more than 10,000 times, enabling in vivo metabolic imaging to be performed noninvasively in real time. Here, we are developing a group of dDNP polarized tracers based on nicotinamide (NAM). We synthesized 1-15N-NAM and 1-15N nicotinic acid and hyperpolarized them with dDNP, reaching (13.0 ± 1.9)% 15N polarization. We found that the lifetime of hyperpolarized 1-15N-NAM is strongly field- and pH-dependent, with T1 being as long as 41 s at a pH of 12 and 1 T while as short as a few seconds at neutral pH and fields below 1 T. The remarkably short 1-15N lifetime at low magnetic fields and neutral pH drove us to establish a unique pH neutralization procedure. Using 15N dDNP and an inexpensive rodent imaging probe designed in-house, we acquired a 15N MRI of 1-15N-NAM (previously hyperpolarized for more than an hour) in less than 1 s.

Insights into Gene Regulation under Temozolomide-Promoted Cellular Dormancy and Its Connection to Stemness in Human Glioblastoma.

The aggressive features of glioblastoma (GBM) are associated with dormancy. Our previous transcriptome analysis revealed that several genes were regulated during temozolomide (TMZ)-promoted dormancy in GBM. Focusing on genes involved in cancer progression, Chemokine (C-C motif) Receptor-Like (CCRL)1, Schlafen (SLFN)13, Sloan-Kettering Institute (SKI), Cdk5 and Abl Enzyme Substrate (Cables)1, and Dachsous Cadherin-Related (DCHS)1 were selected for further validation. All showed clear expression and individual regulatory patterns under TMZ-promoted dormancy in human GBM cell lines, patient-derived primary cultures, glioma stem-like cells (GSCs), and human GBM ex vivo samples. All genes exhibited complex co-staining patterns with different stemness markers and with each other, as examined by immunofluorescence staining and underscored by correlation analyses. Neurosphere formation assays revealed higher numbers of spheres during TMZ treatment, and gene set enrichment analysis of transcriptome data revealed significant regulation of several GO terms, including stemness-associated ones, indicating an association between stemness and dormancy with the involvement of SKI. Consistently, inhibition of SKI during TMZ treatment resulted in higher cytotoxicity, proliferation inhibition, and lower neurosphere formation capacity compared to TMZ alone. Overall, our study suggests the involvement of CCRL1, SLFN13, SKI, Cables1, and DCHS1 in TMZ-promoted dormancy and demonstrates their link to stemness, with SKI being particularly important.

Establishment of a Rodent Glioblastoma Partial Resection Model for Chemotherapy by Local Drug Carriers-Sharing Experience.

Local drug delivery systems (LDDS) represent a promising therapy strategy concerning the most common and malignant primary brain tumor glioblastoma (GBM). Nevertheless, to date, only a few systems have been clinically applied, and their success is very limited. Still, numerous new LDDS approaches are currently being developed. Here, (partial resection) GBM animal models play a key role, as such models are needed to evaluate the therapy prior to any human application. However, such models are complex to establish, and only a few reports detail the process. Here, we report our results of establishing a partial resection glioma model in rats suitable for evaluating LDDS. C6-bearing Wistar rats and U87MG-spheroids- and patient-derived glioma stem-like cells-bearing athymic rats underwent tumor resection followed by the implantation of an exemplary LDDS. Inoculation, tumor growth, residual tumor tissue, and GBM recurrence were reliably imaged using high-resolution Magnetic Resonance Imaging. The release from an exemplary LDDS was verified in vitro and in vivo using Fluorescence Molecular Tomography. The presented GBM partial resection model appears to be well suited to determine the efficiency of LDDS. By sharing our expertise, we intend to provide a powerful tool for the future testing of these very promising systems, paving their way into clinical application.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

A biopolymeric mesh enriched with PLGA microparticles loaded with AT101 for localized glioblastoma treatment.

Current treatment strategies for glioblastoma (GBM) including surgical resection and adjuvant radio/chemotherapy result in a limited progression-free survival time of patients due to rapidly occurring tumor recurrences. The urgent need for more effective treatments has led to the development of different approaches for localized drug delivery systems (DDSs) offering the advantages of reduced systemic side effects. A promising candidate for the treatment of GBMs is AT101, the R-(-)-enantiomer of gossypol due to its ability to induce apoptosis or trigger autophagic cell death in tumor cells. Here, we present an alginate-based drug-releasing mesh ladened with AT101-loaded PLGA microspheres (AT101-GlioMesh). The AT101-loaded PLGA microspheres were fabricated using an oil-in-water emulsion solvent evaporation method obtaining a high encapsulation efficiency. The drug-loaded microspheres enabled the release of AT101 over several days at the tumor site. The cytotoxic effect of the AT101-loaded mesh was evaluated using two different GBM cell lines. Strikingly, encapsulation of AT101 in PLGA-microparticles and subsequent embedding in GlioMesh resulted in a sustained delivery and more efficient cytotoxic effect of AT101 on both GBM cell lines. Thus, such a DDS holds promise for GBM therapy likely by preventing the development of tumor recurrences.

Fabrication and Modelling of a Reservoir-Based Drug Delivery System for Customizable Release.

Localized therapy approaches have emerged as an alternative drug administration route to overcome the limitations of systemic therapies, such as the crossing of the blood-brain barrier in the case of brain tumor treatment. For this, implantable drug delivery systems (DDS) have been developed and extensively researched. However, to achieve an effective localized treatment, the release kinetics of DDS needs to be controlled in a defined manner, so that the concentration at the tumor site is within the therapeutic window. Thus, a DDS, with patient-specific release kinetics, is crucial for the improvement of therapy. Here, we present a computationally supported reservoir-based DDS (rDDS) development towards patient-specific release kinetics. The rDDS consists of a reservoir surrounded by a polydimethylsiloxane (PDMS) microchannel membrane. By tailoring the rDDS, in terms of membrane porosity, geometry, and drug concentration, the release profiles can be precisely adapted, with respect to the maximum concentration, release rate, and release time. The release is investigated using a model dye for varying parameters, leading to different distinct release profiles, with a maximum release of up to 60 days. Finally, a computational simulation, considering exemplary in vivo conditions (e.g., exchange of cerebrospinal fluid), is used to study the resulting drug release profiles, demonstrating the customizability of the system. The establishment of a computationally supported workflow, for development towards a patient-specific rDDS, in combination with the transfer to suitable drugs, could significantly improve the efficacy of localized therapy approaches.

Influence of Simulated Deep Brain Stimulation on the Expression of Inflammatory Mediators by Human Central Nervous System Cells In Vitro.

Deep brain stimulation (DBS) seems to modulate inflammatory processes. Whether this modulation leads to an induction or suppression of inflammatory mediators is still controversially discussed. Most studies of the influence of electrical stimulation on inflammation were conducted in rodent models with direct current stimulation and/or long impulses, both of which differ from the pattern in DBS. This makes comparisons with the clinical condition difficult. We established an in-vitro model that simulated clinical stimulation patterns to investigate the influence of electrical stimulation on proliferation and survival of human astroglial cells, microglia, and differentiated neurons. We also examined its influence on the expression of the inflammatory mediators C-X-C motif chemokine (CXCL)12, CXCL16, CC-chemokin-ligand-2 (CCL)2, CCL20, and interleukin (IL)-1ß and IL-6 by these cells using quantitative polymerase chain reaction. In addition, protein expression was assessed by immunofluorescence double staining. In our model, electrical stimulation did not affect proliferation or survival of the examined cell lines. There was a significant upregulation of CXCL12 in the astrocyte cell line SVGA, and of IL-1ß in differentiated SH-SY5Y neuronal cells at both messenger RNA and protein levels. Our model allowed a valid examination of chemokines and cytokines associated with inflammation in human brain cells. With it, we detected the induction of inflammatory mediators by electrical stimulation in astrocytes and neurons.

Erroneous expression of NKG2D on granulocytes detected by phycoerythrin-conjugated clone 149810 antibody.

BACKGROUND: The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, γδ T cells and small subsets of CD4 T lymphocytes. During the course of an extensive flow cytometry phenotyping of immune cells in the peripheral blood of patients with glioblastoma multiforme (GBM) we noticed an unexpected expression of NKG2D receptor on granulocytes using the phycoerythrin (PE)-conjugated clone 149810 antibody. METHODS: Peripheral blood samples from 35 patients with GBM and 22 age-matched healthy control (HC) donors were analyzed using flow cytometry, imaging cytometry and real-time quantitative reverse transcription PCR to validate the observed expression of NKG2D receptor on myeloid cells. RESULTS: Reactivity with PE-149810 was mostly observed on granulocytes from GBM patients on dexamethasone treatment where it correlated with inferior survival rates. Surprisingly, such NKG2D expression on granulocytes was not observed using the allophycocyanin (APC)-conjugate of the same clone 149810 antibody or an indirect staining procedure with unconjugated clone 149810 antibody. Moreover, the PE-conjugate of a different anti-NKG2D clone (1D11) also did not stain granulocytes. Imaging cytometry indicated cell surface and intracellular localization of PE-149810 but not of PE-1D11 in granulocytes. CONCLUSION: Our results uncover an erroneous and false positive reactivity of PE-labeled (but not of APC-labeled or unconjugated) anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients and raise a note of caution for studies of NKG2D expression on non-lymphoid cells.

Fetal and Perinatal Expression Profiles of Proinflammatory Cytokines in the Neuroplacodes of Rats with Myelomeningoceles: A Contribution to the Understanding of Secondary Spinal Cord Injury in Open Spinal Dysraphism.

The cellular and molecular mechanisms that presumably underlie the progressive functional decline of the myelomeningocele (MMC) placode are not well understood. We previously identified key players in post-traumatic spinal cord injury cascades in human MMC tissues obtained during postnatal repair. In this study, we conducted experiments to further investigate these mediators in the prenatal time course under standardized conditions in a retinoic acid-induced MMC rat model. A retinoic acid MMC model was established using time-dated Sprague-Dawley rats, which were gavage-fed with all-trans retinoic acid (RA; 60 mg/kg) dissolved in olive oil at E10. Control animals received olive oil only. Fetuses from both groups were obtained at E16, E18, and E22. The spinal cords (SCs) of both groups were formalin-fixed or snap-frozen. Tissues were screened by real-time reverse transcription polymerase chain reaction for the expression of cytokines and chemokines known to play a role in the lesion cascades of the central nervous system after trauma. MMC placodes exhibited inflammatory cells and glial activation in the later gestational stages. At the messenger RNA (mRNA) level, interleukin-1 beta, tumor necrosis factor alpha, and tumor necrosis factor receptor type 1 exhibited significant induction at E22. interleukin-1 beta receptor type 1 mRNA was induced significantly at E16 and E22. Double labeling experiments confirmed the co-staining of these cytokines and their receptors with ionized calcium-binding adapter molecule 1 (i.e., inflammatory cells), vimentin, and nestin in different anatomical SC areas and neuronal nuclear protein in ventral horn neurons. C-X-C motif chemokine 12 mRNA was elevated in control and MMC animals at E16 compared with E18 and E22. C-X3-C motif ligand 1 mRNA was lower in MMC tissues than in control tissues on E16. The presented findings contribute to the concept that pathophysiological mechanisms, such as cytokine induction in the neuroplacode, in addition to the "first hit," promote secondary spinal cord injury with functional loss in the late fetal time course. Further, these mediators should be taken into consideration in the development of new therapeutic approaches for open spinal dysraphism.

Cannabidiol converts NF-κB into a tumor suppressor in glioblastoma with defined antioxidative properties.

BACKGROUND: The transcription factor NF-κB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma [GBM]). Precise therapeutic modulation of NF-κB activity can suppress central oncogenic signaling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive. METHODS: In a pharmacogenomics study with a panel of transgenic glioma cells, we observed that NF-κB can be converted into a tumor suppressor by the non-psychotropic cannabinoid cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study, we performed pharmacological assays, gene expression profiling, biochemical, and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM datasets. RESULTS: We found that CBD promotes DNA binding of the NF-κB subunit RELA and simultaneously prevents RELA phosphorylation on serine-311, a key residue that permits genetic transactivation. Strikingly, sustained DNA binding by RELA-lacking phospho-serine 311 was found to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen species (ROS), while high ROS content in other tumors blocked CBD-induced hGSC death. Consequently, ROS levels served as a predictive biomarker for CBD-sensitive tumors. CONCLUSIONS: This evidence demonstrates how a clinically approved drug can convert NF-κB into a tumor suppressor and suggests a promising repurposing option for GBM therapy.

Effects of the Anti-Tumorigenic Agent AT101 on Human Glioblastoma Cells in the Microenvironmental Glioma Stem Cell Niche.

Glioblastoma (GBM) is a barely treatable disease due to its profound chemoresistance. A distinct inter- and intratumoral heterogeneity reflected by specialized microenvironmental niches and different tumor cell subpopulations allows GBMs to evade therapy regimens. Thus, there is an urgent need to develop alternative treatment strategies. A promising candidate for the treatment of GBMs is AT101, the R(-) enantiomer of gossypol. The present study evaluates the effects of AT101, alone or in combination with temozolomide (TMZ), in a microenvironmental glioma stem cell niche model of two GBM cell lines (U251MG and U87MG). AT101 was found to induce strong cytotoxic effects on U251MG and U87MG stem-like cells in comparison to the respective native cells. Moreover, a higher sensitivity against treatment with AT101 was observed upon incubation of native cells with a stem-like cell-conditioned medium. This higher sensitivity was reflected by a specific inhibitory influence on the p-p42/44 signaling pathway. Further, the expression of CXCR7 and the interleukin-6 receptor was significantly regulated upon these stimulatory conditions. Since tumor stem-like cells are known to mediate the development of tumor recurrences and were observed to strongly respond to the AT101 treatment, this might represent a promising approach to prevent the development of GBM recurrences.

Evaluation of a Brown Seaweed Extract from Dictyosiphon foeniculaceus as a Potential Therapeutic Agent for the Treatment of Glioblastoma and Uveal Melanoma.

Ingredients of brown seaweed like fucoidans are often described for their beneficial biological effects, that might be interesting for a medical application. In this study, we tested an extract from Dictyosiphon foeniculaceus (DF) to evaluate the effects in glioblastoma and uveal melanoma, looking for a possible anti-cancer treatment. We investigated toxicity, VEGF (vascular endothelial growth factor) secretion and gene expression of tumor and non-tumor cells. SVGA (human fetal astrocytes), the human RPE (retinal pigment epithelium) cell line ARPE-19, the tumor cell line OMM-1 (human uveal melanoma), and two different human primary glioblastoma cultures (116-14 and 118-14) were used. Tests for cell viability were conducted with MTS-Assay (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and the proliferation rate was determined with cell counting. VEGF secretion was assessed with ELISA (enzyme-linked immunosorbent assay). The gene expression of VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2) and VEGF-A was determined with real-time qPCR (quantitative polymerase chain reaction). DF lowered the cell viability of OMM-1. Proliferation rates of ARPE-19 and OMM-1 were decreased. The VEGF secretion was inhibited in ARPE-19 and OMM-1, whereas it was increased in SVGA and 116-14. The expression of VEGFR1 was absent and not influenced in OMM-1 and ARPE-19. VEGFR2 expression was lowered in 116-14 after 24 h, whereas VEGF-A was increased in 118-14 after 72 h. The extract lowered cell viability slightly and was anti-proliferative depending on the cell type investigated. VEGF was heterogeneously affected. The results in glioblastoma were not promising, but the anti-tumor properties in OMM-1 could make them interesting for further research concerning cancer diseases in the human eye.

AT101-Loaded Cubosomes as an Alternative for Improved Glioblastoma Therapy.

INTRODUCTION: AT101, the R-(-)-enantiomer of the cottonseed-derived polyphenol gossypol, is a promising drug in glioblastoma multiforme (GBM) therapy due to its ability to trigger autophagic cell death but also to facilitate apoptosis in tumor cells. It does have some limitations such as poor solubility in water-based media and consequent low bioavailability, which affect its response rate during treatment. To overcome this drawback and to improve the anti-cancer potential of AT101, the use of cubosome-based formulation for AT101 drug delivery has been proposed. This is the first report on the use of cubosomes as AT101 drug carriers in GBM cells. MATERIALS AND METHODS: Cubosomes loaded with AT101 were prepared from glyceryl monooleate (GMO) and the surfactant Pluronic F-127 using the top-down approach. The drug was introduced into the lipid prior to dispersion. Prepared formulations were then subjected to complex physicochemical and biological characterization. RESULTS: Formulations of AT101-loaded cubosomes were highly stable colloids with a high drug entrapment efficiency (97.7%) and a continuous, sustained drug release approaching 35% over 72 h. Using selective and sensitive NMR diffusometry, the drug was shown to be efficiently bound to the lipid-based cubosomes. In vitro imaging studies showed the high efficiency of cubosomal nanoparticles uptake into GBM cells, as well as their marked ability to penetrate into tumor spheroids. Treatment of GBM cells with the AT101-loaded cubosomes, but not with the free drug, induced cytoskeletal rearrangement and shortening of actin fibers. The prepared nanoparticles revealed stronger in vitro cytotoxic effects against GBM cells (A172 and LN229 cell lines), than against normal brain cells (SVGA and HMC3 cell lines). CONCLUSION: The results indicate that GMO-AT101 cubosome formulations are a promising basic tool for alternative approaches to GBM treatment.

Intratumoral Distribution of Lactate and the Monocarboxylate Transporters 1 and 4 in Human Glioblastoma Multiforme and Their Relationships to Tumor Progression-Associated Markers.

(1) Background: Metabolic reprogramming has been postulated to be one of the hallmarks of cancer, thus representing a promising therapeutic target also in glioblastoma multiforme (GBM). Hypoxic tumor cells produce lactate, and monocarboxylate transporters (MCTs) play an important role in its distribution; (2) Methods: We examined the distribution of lactate by multi voxel magnetic resonance spectroscopic imaging and ELISA in glioblastoma multiforme (GBM) patients. In addition, we investigated the expression and cellular localization of MCT1, MCT4, and of several markers connected to tumor progression by quantitative PCR and immunofluorescence double-staining in human GBM ex vivo tissues; (3) Results: The highest lactate concentration was found at the center of the vital parts of the tumor. Three main GBM groups could be distinguished according to their regional gene expression differences of the investigated genes. MCT1 and MCT4 were found on cells undergoing epithelial to mesenchymal transition and on tumor stem-like cells. GBM cells revealing an expression of cellular dormancy markers, showed positive staining for MCT4; (4) Conclusion: Our findings indicate the existence of individual differences in the regional distribution of MCT1 and MCT4 and suggest that both transporters have distinct connections to GBM progression processes, which could contribute to the drug resistance of MCT-inhibitors.

Glial cell responses on tetrapod-shaped graphene oxide and reduced graphene oxide 3D scaffolds in brain in vitro and ex vivo models of indirect contact.

Brain implants are promising instruments for a broad variety of nervous tissue diseases with a wide range of applications, e.g. for stimulation, signal recording or local drug delivery. Recently, graphene-based scaffold materials have emerged as attractive candidates as neural interfaces, 3D scaffolds, or drug delivery systems due to their excellent properties like flexibility, high surface area, conductivity, and lightweight. To date, however, there is a lack of appropriate studies of the foreign body response, especially by glial cells, towards graphene-based materials. In this work, we investigated the effects of macroscopic, highly porous (>99.9%) graphene oxide (GO) and reduced graphene oxide (rGO) (conductivity ∼1 S m-1) scaffolds with tailorable macro- and microstructure on human astrocyte and microglial cell viability and proliferation as well as expression of neuroinflammation and astrogliosis associated genes in an indirect contact approach. In this in vitro model, as well as ex vivo in organotypic murine brain slices, we could demonstrate that both GO and rGO based 3D scaffolds exert slight effects on the glial cell populations which are the key players of glial scar formation. These effects were in most cases completely abolished by curcumin, a known anti-inflammatory and anti-fibrotic drug that could in perspective be applied to brain implants as a protectant.

Liposomal Encapsulated Curcumin Effectively Attenuates Neuroinflammatory and Reactive Astrogliosis Reactions in Glia Cells and Organotypic Brain Slices.

INTRODUCTION: The polyphenolic spice and food coloring ingredient curcumin has beneficial effects in a broad variety of inflammatory diseases. Amongst them, curcumin has been shown to attenuate microglia reaction and prevent from glial scar formation in spinal cord and brain injuries. METHODS: We developed a protocol for the efficient encapsulation of curcumin as a model for anti-inflammatory drugs yielding long-term stable, non-toxic liposomes with favorable physicochemical properties. Subsequently, we evaluate the effects of liposomal curcumin in experimental models for neuroinflammation and reactive astrogliosis. RESULTS: We could show that liposomal curcumin can efficiently reduce the reactivity of human microglia and astrocytes and preserve tissue integrity of murine organotypic cortex slices. DISCUSSION AND PERSPECTIVE: In perspective, we want to administer this curcumin formulation in brain implant coatings to prevent neuroinflammation and glial scar formation as foreign body responses of the brain towards implanted materials.

Expression Patterns of Hypoxia-Inducible Factors, Proinflammatory, and Neuroprotective Cytokines in Neuroepithelial Tissues of Lumbar Spinal Lipomas-A Pilot Study.

OBJECTIVE: Lumbosacral lipomas (LSLs), one form of closed spinal dysraphism, are congenital disorders of the terminal spinal cord (SC). Delayed neurologic deterioration often occurs in the subsequent developmental course of the patient. Identifying the cellular and molecular factors underlying the progressive damage to neural structures is a prerequisite for developing treatment strategies for LSLs. METHODS: Nine LSL specimens obtained from the SC/lipoma interface during surgical resection were examined. Normal SC tissue served as a control. Clinical characteristics were obtained, and spinal magnetic resonance imaging was re-evaluated. Cellular marker profiles were established. Immunoreactivity (IR) of hypoxia-inducible factor 1α (HIF-1α/-2α), erythropoietin (Epo)/erythropoietin receptor (EpoR), interleukin-1ß (IL-1ß)/IL-1R1, and tumor necrosis factor α/tumor necrosis factor receptor type 1 were analyzed qualitatively and semiquantitatively by densitometry. Colabeling with cellular markers was determined by multifluorescence labeling. Cytokines were further analyzed by real-time reverse transcription polymerase chain reaction. RESULTS: LSL specimens showed significant gliosis. HIF-1α/HIF-2α-IR and Epo/Epo-IR were found at significantly higher levels in the LSL specimens, as were IL-1ß-/IL-1ß receptor type 1 (IL1-R1) and tumor necrosis factor α/tumor necrosis factor receptor type 1 (P < 0.001), than were the controls. At the messenger RNA level, cytokines appeared partially induced. Double immunofluorescence labeling confirmed the costaining of these factors with inflammatory and glial markers. CONCLUSIONS: The expression of hypoxia-related and inflammatory mediators was shown for the first time in LSL specimens. These factors might play a role in multifactorial secondary lesion cascades underlying further damage to the neural placode in closed dysraphism.

Entry and exit of chemotherapeutically-promoted cellular dormancy in glioblastoma cells is differentially affected by the chemokines CXCL12, CXCL16, and CX3CL1.

Glioblastoma multiforme (GBM) is a malignant brain tumor that evades therapy regimens. Since cellular dormancy is one strategy for surviving, and since chemokines determine the environmental conditions in which dormancy occurs, we investigated how chemokines affect temozolomide (TMZ)-promoted cellular dormancy entry and exit in GBM cells. TMZ administration over ten days promoted cellular dormancy entry, whereas discontinuing TMZ for a further 15 days resulted in resumption of proliferation. Co-administration of a chemokine cocktail containing CXCL12, CXCL16, and CX3CL1 resulted in both delayed entry and exit from cellular dormancy. A microarray-based transcriptome analysis in LN229 GBM cells revealed that cellular dormancy entry was characterized by an increased expression of CCL2 and SAA2, while THSD4, FSTL3, and VEGFC were upregulated during dormancy exit. Co-stimulation with the chemokine cocktail reduced upregulation of identified genes. After verifying the appearance of identified genes in human GBM primary cultures and ex vivo samples, we clarified whether each chemokine alone impacts cellular dormancy mechanisms using specific antagonists and selective CRISPR/Cas9 clones. While expression of CCL2 and SAA2 in LN229 cells was altered by the CXCL12-CXCR4-CXCR7 axis, CXCL16 and CX3CL1 contributed to reduced upregulation of THSD4 and, to a weaker extent, of VEGFC. The influence on FSTL3 expression depended on the entire chemokine cocktail. Effects of chemokines on dormancy entry and exit-associated genes were detectable in human GBM primary cells, too, even if in a more complex, cell-specific manner. Thus, chemokines play a significant role in the regulation of TMZ-promoted cellular dormancy in GBMs.

Dormancy and NKG2D system in brain metastases: Analysis of immunogenicity.

Patients with breast cancer (BC) and lung cancer (LC) are prone to developing brain metastases, which are associated with devastating prognoses. Dormant tumor cells, a population of non­apoptotic quiescent cells and immunological escape mechanisms, including the Natural Killer Group 2 member D (NKG2D) receptor­ligand system, represent potential mechanisms of tumor recurrence. To date, the immunological characteristics of dormant tumor cells concerning the NKG2D system in cerebral malignancies are mostly unknown. In the present study, an extensive characterization of dormant and NKG2D ligand (NKG2DL)+ cells in cerebral metastases was performed. The expression profiles and localization patterns of various NKG2DL and several dormancy markers were analyzed in solid human brain metastases from patients with BC and LC using immunostaining and reverse transcription­quantitative polymerase chain reaction analyses. Statistical analysis was performed using Student's t­test and Bravais­Pearson correlation analysis. Not only 'peripheral', but also 'central' dormancy markers, which had been previously described in primary brain tumors, were identified in all cerebral metastases at detectable levels at protein and mRNA levels. Notably, the majority of NKG2DL+ cells were also positive for 'central' dormancy markers, but not 'peripheral' dormancy markers in both patient groups. This cell population may represent a promising future therapeutic target.