Analyzing the Impact of the Highest Expressed Epstein-Barr Virus-Encoded microRNAs on the Host Cell Transcriptome.

The Epstein-Barr virus (EBV) has a very high prevalence (>90% in adults), establishes a lifelong latency after primary infection, and exerts an oncogenic potential. This dsDNA virus encodes for various molecules, including microRNAs (miRs), which can be detected in the latent and lytic phases with different expression levels and affect, among others, immune evasion and malignant transformation. In this study, the different EBV miRs are quantified in EBV-positive lymphomas, and the impact on the host cell transcriptome of the most abundant EBV miRs will be analyzed using comparative RNA sequencing analyses. The EBV miRs ebv-miR-BART1, -BART4, -BART17, and -BHRF1-1 were most highly expressed, and their selective overexpression in EBV-negative human cells resulted in a large number of statistically significantly down- and up-regulated host cell genes. Functional analyses showed that these dysregulated target genes are involved in important cellular processes, including growth factor pathways such as WNT, EGF, FGF, and PDGF, as well as cellular processes such as apoptosis regulation and inflammation. Individual differences were observed between these four analyzed EBV miRs. In particular, ebv-miR-BHRF1-1 appears to be more important for malignant transformation and immune evasion than the other EBV miRs.

Elucidation of GPR55-Associated Signaling behind THC and LPI Reducing Effects on Ki67-Immunoreactive Nuclei in Patient-Derived Glioblastoma Cells.

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB1 and CB2 receptors, THC modulates cellular responses among others via GPR55. Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67+ nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gßγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids.

Time- and Concentration-Dependent Adverse Effects of Paclitaxel on Non-Neuronal Cells in Rat Primary Dorsal Root Ganglia.

Paclitaxel is a chemotherapeutic agent used to treat a wide range of malignant tumors. Although it has anti-tumoral properties, paclitaxel also shows significant adverse effects on the peripheral nervous system, causing peripheral neuropathy. Paclitaxel has previously been shown to exert direct neurotoxic effects on primary DRG neurons. However, little is known about paclitaxel's effects on non-neuronal DRG cells. They provide mechanical and metabolic support and influence neuronal signaling. In the present study, paclitaxel effects on primary DRG non-neuronal cells were analyzed and their concentration or/and time dependence investigated. DRGs of Wister rats (6-8 weeks old) were isolated, and non-neuronal cell populations were separated by the density gradient centrifugation method. Different concentrations of Paclitaxel (0.01 µM-10 µM) were tested on cell viability by MTT assay, cell death by lactate dehydrogenase (LDH) assay, and propidium iodide (PI) assay, as well as cell proliferation by Bromodeoxyuridine (BrdU) assay at 24 h, 48 h, and 72 h post-treatment. Furthermore, phenotypic effects have been investigated by using immunofluorescence techniques. Paclitaxel exhibited several toxicological effects on non-neuronal cells, including a reduction in cell viability, an increase in cell death, and an inhibition of cell proliferation. These effects were concentration- and time-dependent. Cellular and nuclear changes such as shrinkage, swelling of cell bodies, nuclear condensation, chromatin fragmentation, retraction, and a loss in processes were observed. Paclitaxel showed adverse effects on primary DRG non-neuronal cells, which might have adverse functional consequences on sensory neurons of the DRG, asking for consideration in the management of peripheral neuropathy.

MACC1-induced migration in tumors: Current state and perspective.

Malignant tumors are still a global, heavy health burden. Many tumor types cannot be treated curatively, underlining the need for new treatment targets. In recent years, metastasis associated in colon cancer 1 (MACC1) was identified as a promising biomarker and drug target, as it is promoting tumor migration, initiation, proliferation, and others in a multitude of solid cancers. Here, we will summarize the current knowledge about MACC1-induced tumor cell migration with a special focus on the cytoskeletal and adhesive systems. In addition, a brief overview of several in vitro models used for the analysis of cell migration is given. In this context, we will point to issues with the currently most prevalent models used to study MACC1-dependent migration. Lastly, open questions about MACC1-dependent effects on tumor cell migration will be addressed.

Palmitoylethanolamide Mitigates Paclitaxel Toxicity in Primary Dorsal Root Ganglion Neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of several chemotherapeutic agents, such as Paclitaxel. The main symptoms of CIPN are pain and numbness in the hands and feet. Paclitaxel is believed to accumulate in the dorsal root ganglia and free nerve endings. Novel therapeutic agents might help to mitigate or prevent Paclitaxel toxicity on dorsal root ganglion (DRG) neurons. Thus, we used primary DRG neurons as a model to investigate the potential neuroprotective effects of the endocannabinoid-like substance, palmitoylethanolamide (PEA). DRG neurons were isolated from cervical to sacral segments of spinal nerves of Wister rats (6-8 weeks old). After isolation and purification of neuronal cell populations, different concentrations of Paclitaxel (0.01-10 µM) or PEA (0.1-10 µM) or their combination were tested on cell viability by MTT assay at 24 h, 48, and 72 h post-treatment. Furthermore, morphometric analyses of neurite length and soma size for DRG neurons were performed. Adverse Paclitaxel effects on cell viability were apparent at 72 h post-treatment whereas Paclitaxel significantly reduced the neurite length in a concentration-dependent manner nearly at all investigated time points. However, Paclitaxel significantly increased the size of neuronal cell bodies at all time windows. These phenotypic effects were significantly reduced in neurons additionally treated with PEA, indicating the neuroprotective effect of PEA. PEA alone led to a significant increase in neuron viability regardless of PEA concentrations, apparent improvements in neurite outgrowth as well as a significant decrease in soma size of neurons at different investigated time points. Taken together, PEA showed promising protective effects against Paclitaxel-related toxicity on DRG neurons.

Glioblastoma-Derived Three-Dimensional Ex Vivo Models to Evaluate Effects and Efficacy of Tumor Treating Fields (TTFields).

Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients' tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.

MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics.

Metastasis-associated in colon cancer 1 (MACC1) is a marker for metastasis, tumor cell migration, and increased proliferation in colorectal cancer (CRC). Tumors with high MACC1 expression show a worse prognosis and higher invasion into neighboring structures. Yet, many facets of the pro-migratory effects are not fully understood. Atomic force microscopy and single cell live imaging were used to quantify biomechanical and migratory properties in low- and high-MACC1-expressing CRC cells. Furthermore, collective migration and expansion of small, cohesive cell colonies were analyzed using live cell imaging and particle image velocimetry. Lastly, the impact of proliferation on collective migration was determined by inhibition of proliferation using mitomycin. MACC1 did not affect elasticity, cortex tension, and single cell migration of CRC cells but promoted collective migration and colony expansion in vitro. Measurements of the local velocities in the dense cell layers revealed proliferation events as regions of high local speeds. Inhibition of proliferation via mitomycin abrogated the MACC1-associated effects on the collective migration speeds. A simple simulation revealed that the expansion of cell clusters without proliferation appeared to be determined mostly by single cell properties. MACC1 overexpression does not influence single cell biomechanics and migration but only collective migration in a proliferation-dependent manner. Thus, targeting proliferation in high-MACC1-expressing tumors may offer additional effects on cell migration.

Surgical Anatomy of the Radial Nerve at the Dorsal Region of the Humerus: A Cadaveric Study.

BACKGROUND: Surgery for humeral shaft fractures is associated with a high risk of iatrogenic radial nerve palsy (RNP). Plausible causes are difficult anatomical conditions and variants. METHODS: We performed a cadaveric study with 23 specimens (13 female and 10 male Caucasian donors) to assess the course and anatomy of the radial nerve (RN) with its branches alongside the humeral shaft. The accuracy of identification of the RN in the surgical field was analyzed by measuring the location, course, diameter, and form of each nerve and vessel of interest. RESULTS: The RN is not a single structure running alongside the humeral shaft; at least 4 parallel structures crossed the dorsal humerus in all subjects. The RN was accompanied by 2 vessels and at least 1 other nerve, which we named the musculocutaneous branch (MCB). With an oval profile and an average diameter of 3.1 mm (range, 2.6 to 3.8 mm), the MCB was thinner but, in some cases, close to the average diameter of 4.7 mm (range, 4.0 to 5.2 mm) of the RN, which had a round profile. Both accompanying vessels had similar diameters: 3.5 mm (range, 2.6 to 4.2 mm) for the radial collateral artery and 4.0 mm (range, 2.9 to 4.4 mm) for the medial collateral artery. In 20 (87%) of the cases, the RN ran proximal to and in 3 (13%) of the cases, distal to the MCB. Furthermore, a distal safe zone of at least 110 mm (range, 110 to 160 mm) was found, measured from the radial (lateral) epicondyle proximally. CONCLUSIONS: The RN does not cross the dorsal humerus alone, as often stated in anatomical textbooks, but runs parallel to vessels and at least 1 nerve branch with a similar appearance. Thus, for reliable preservation of the RN, we recommend identification and protection of all crossing structures in posterior humeral surgeries 110 mm proximal to the radial epicondyle.

Interaction of Glia Cells with Glioblastoma and Melanoma Cells under the Influence of Phytocannabinoids.

Brain tumor heterogeneity and progression are subject to complex interactions between tumor cells and their microenvironment. Glioblastoma and brain metastasis can contain 30-40% of tumor-associated macrophages, microglia, and astrocytes, affecting migration, proliferation, and apoptosis. Here, we analyzed interactions between glial cells and LN229 glioblastoma or A375 melanoma cells in the context of motility and cell-cell interactions in a 3D model. Furthermore, the effects of phytocannabinoids, cannabidiol (CBD), tetrahydrocannabidiol (THC), or their co-application were analyzed. Co-culture of tumor cells with glial cells had little effect on 3D spheroid formation, while treatment with cannabinoids led to significantly larger spheroids. The addition of astrocytes blocked cannabinoid-induced effects. None of the interventions affected cell death. Furthermore, glial cell-conditioned media led to a significant slowdown in collective, but not single-cell migration speed. Taken together, glial cells in glioblastoma and brain metastasis micromilieu impact the tumor spheroid formation, cell spreading, and motility. Since the size of spheroid remained unaffected in glial cell tumor co-cultures, phytocannabinoids increased the size of spheroids without any effects on migration. This aspect might be of relevance since phytocannabinoids are frequently used in tumor therapy for side effects.

Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension.

Collective behavior of cells emerges from coordination of cell-cell-interactions and is important to wound healing, embryonic and tumor development. Depending on cell density and cell-cell interactions, a transition from a migratory, fluid-like unjammed state to a more static and solid-like jammed state or vice versa can occur. Here, we analyze collective migration dynamics of astrocytes and glioblastoma cells using live cell imaging. Furthermore, atomic force microscopy, traction force microscopy and spheroid generation assays were used to study cell adhesion, traction and mechanics. Perturbations of traction and adhesion were induced via ROCK or myosin II inhibition. Whereas astrocytes resided within a non-migratory, jammed state, glioblastoma were migratory and unjammed. Furthermore, we demonstrated that a switch from an unjammed to a jammed state was induced upon alteration of the equilibrium between cell-cell-adhesion and tension from adhesion to tension dominated, via inhibition of ROCK or myosin II. Such behavior has implications for understanding the infiltration of the brain by glioblastoma cells and may help to identify new strategies to develop anti-migratory drugs and strategies for glioblastoma-treatment.

THC Reduces Ki67-Immunoreactive Cells Derived from Human Primary Glioblastoma in a GPR55-Dependent Manner.

Glioblastoma (GBM) is the most frequent malignant tumor of the central nervous system in humans with a median survival time of less than 15 months. ∆9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the best-characterized components of Cannabis sativa plants with modulating effects on cannabinoid receptors 1 and 2 (CB1 and CB2) and on orphan receptors such as GPR18 or GPR55. Previous studies have demonstrated anti-tumorigenic effects of THC and CBD in several tumor entities including GBM, mostly mediated via CB1 or CB2. In this study, we investigated the non-CB1/CB2 effects of THC on the cell cycle of GBM cells isolated from human tumor samples. Cell cycle entry was measured after 24 h upon exposure by immunocytochemical analysis of Ki67 as proliferation marker. The Ki67-reducing effect of THC was abolished in the presence of CBD, whereas CBD alone did not cause any changes. To identify the responsible receptor for THC effects, we first characterized the cells regarding their expression of different cannabinoid receptors: CB1, CB2, GPR18, and GPR55. Secondly, the receptors were pharmacologically blocked by application of their selective antagonists AM281, AM630, O-1918, and CID16020046 (CID), respectively. All examined cells expressed the receptors, but only in presence of the GPR55 antagonist CID was the THC effect diminished. Stimulation with the GPR55 agonist lysophosphatidylinositol (LPI) revealed similar effects as obtained for THC. The LPI effects were also inhibited by CBD and CID, confirming a participation of GPR55 and suggesting its involvement in modifying the cell cycle of patient-derived GBM cells.

Preparation and Culture of Organotypic Hippocampal Slices for the Analysis of Brain Metastasis and Primary Brain Tumor Growth.

Brain metastasis is a major challenge for therapy and defines the end stage of tumor progression with a very limited patients' prognosis. Experimental setups that faithfully mimic these processes are necessary to understand the mechanism of brain metastasis and to develop new improved therapeutic strategies. Here, we describe an in vitro model, which closely resembles the in vivo situation. Organotypic hippocampal brain slice cultures (OHSCs) prepared from 3- to 8-day-old mice are well suited for neuro-oncology research including brain metastasis. The original morphology is preserved in OHSCs even after culture periods of several days to weeks. Tumor cells or cells of metastatic origin can be seeded onto OHSCs to evaluate micro-tumor formation, tumor cell invasion, or treatment response. We describe preparation and culture of OHSCs including the seeding of tumor cells. Finally, we show examples of how to treat the OHSCs for life-dead or immunohistochemical staining.

MACC1 driven alterations in cellular biomechanics facilitate cell motility in glioblastoma.

BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) is an established marker for metastasis and tumor cell migration in a multitude of tumor entities, including glioblastoma (GBM). Nevertheless, the mechanism underlying the increased migratory capacity in GBM is not comprehensively explored. METHODS: We performed live cell and atomic force microscopy measurements to assess cell migration and mechanical properties of MACC1 overexpressing GBM cells. We quantified MACC1 dependent dynamics of 3D aggregate formation. For mechanistic studies we measured the expression of key adhesion molecules using qRT-PCR, and MACC1 dependent changes in short term adhesion to fibronectin and laminin. We then determined changes in sub-cellular distribution of integrins and actin in dependence of MACC1, but also in microtubule and intermediate filament organization. RESULTS: MACC1 increased the migratory speed and elastic modulus of GBM cells, but decreased cell-cell adhesion and inhibited the formation of 3D aggregates. These effects were not associated with altered mRNA expression of several key adhesion molecules or altered short-term affinity to laminin and fibronectin. MACC1 did neither change the organization of the microtubule nor intermediate filament cytoskeleton, but resulted in increased amounts of protrusive actin on laminin. CONCLUSION: MACC1 overexpression increases elastic modulus and migration and reduces adhesion of GBM cells thereby impeding 3D aggregate formation. The underlying molecular mechanism is independent on the organization of microtubules, intermediate filaments and several key adhesion molecules, but depends on adhesion to laminin. Thus, targeting re-organization of the cytoskeleton and cell motility via MACC1 may offer a treatment option to impede GBM spreading. Video Abstract.

Abnormal Cannabidiol Affects Production of Pro-Inflammatory Mediators and Astrocyte Wound Closure in Primary Astrocytic-Microglial Cocultures.

Abnormal cannabidiol (abn-CBD) exerts neuroprotective effects in vivo and in vitro. In the present study, we investigated the impact of abn-CBD on the glial production of proinflammatory mediators and scar formation within in vitro models. Primary astrocytic-microglial cocultures and astrocytic cultures from neonatal C57BL/6 mice and CB2 receptor knockout mice were stimulated with lipopolysaccharide (LPS), and the concentrations of tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and nitrite were determined. Furthermore, we performed a live cell microscopy-based scratch-wound assay. After LPS stimulation, TNFα, IL-6 and nitrite production was more strongly increased in cocultures than in isolated astrocytes. Abn-CBD treatment attenuated the LPS-induced production of TNFα and nitrite in cocultures, while IL-6 production remained unaltered. In isolated astrocytes, only LPS-induced TNFα production was reduced by abn-CBD. Similar effects were observed after abn-CBD application in cocultures of CB2 knockout mice. Interestingly, LPS-induced TNFα and nitrite levels were far lower in CB2 knockout cultures compared to wildtypes, while IL-6 levels did not differ. In the scratch-wound assay, treatment with abn-CBD decelerated wound closure when microglial cells were present. Our data shows a differential role of abn-CBD for modulation of glial inflammation and astrocytic scar formation. These findings provide new explanations for mechanisms behind the neuroprotective potential of abn-CBD.

On the importance of the innervation of the human cervical longitudinal ligaments at vertebral level.

PURPOSE: In our aging society, the prevalence of degenerative spinal diseases rose drastically within the last years. However, up till now, the origin of cervical pain is incompletely understood. While animal and small cadaver studies indicate that a complex system of sensory and nociceptive nerve fibers in the anterior (ALL) and posterior longitudinal ligament (PLL) at the level of the intervertebral disc might be involved, there is a lack of data exploring whether such a network exists and is equally distributed within the cervical vertebrae (VB). We, therefore, aimed to investigate the spatial distribution of the mentioned nerve networks in human tissue. METHODS: We performed macroscopic (Sihler staining, Spalteholz technique, and Plastination) and microscopic (immunohistochemistry for PGP 9.5 and CGRP) studies to characterize spatial differences in sensory and nociceptive innervation patterns. Therefore, 23 human body donors were dissected from level C3-C6. RESULTS: We could show that there is a focal increase in sensory and nociceptive nerve fibers at the level of C4 and C5 for both ALL and PLL, while we observed less nerve fiber density at the level of C3 and C6. An anatomical vicinity between nerve and vessels was observed. CONCLUSION: To our knowledge, these findings for the first time report spatial differences in sensory and nociceptive nerve fibers in the human cervical spine at VB level. The interconnection between nerves and vessels supports the importance of the perivascular plexus. These findings might be of special interest for clinical practice as many patients suffer from pain after cervical spine surgery.

Radiosensitization and a Less Aggressive Phenotype of Human Malignant Glioma Cells Expressing Isocitrate Dehydrogenase 1 (IDH1) Mutant Protein: Dissecting the Mechanisms.

The presence of an isocitrate dehydrogenase 1 (IDH1) mutation is associated with a less aggressive phenotype, increased sensitivity to radiation, and increased overall survival in patients with diffuse glioma. Based on in vitro experimentations in malignant glioma cell lines, the consequences on cellular processes of IDH1R132H expression were analyzed. The results revealed that IDH1R132H expression enhanced the radiation induced accumulation of residual γH2AX foci and decreased the amount of glutathione (GSH) independent of the oxygen status. In addition, expression of the mutant IDH1 caused a significant increase of cell stiffness and induced an altered organization of the cytoskeleton, which has been shown to reinforce cell stiffness. Furthermore, IDH1R132H expression decreased the expression of vimentin, an important component of the cytoskeleton and regulator of the cell stiffness. The results emphasize the important role of mutant IDH1 in treatment of patients with diffuse gliomas especially in response to radiation. Hence, detection of the genetic status of IDH1 before therapy massively expands the utility of immunohistochemistry to accurately distinguish patients with a less aggressive and radiosensitive IDH1-mutant diffuse glioma suitable for radiotherapy from those with a more aggressive IDH1-wildtype diffuse glioma who might benefit from an individually intensified therapy comprising radiotherapy and alternative medical treatments.

The Cytoskeleton-A Complex Interacting Meshwork.

The cytoskeleton of animal cells is one of the most complicated and functionally versatile structures, involved in processes such as endocytosis, cell division, intra-cellular transport, motility, force transmission, reaction to external forces, adhesion and preservation, and adaptation of cell shape. These functions are mediated by three classical cytoskeletal filament types, as follows: Actin, microtubules, and intermediate filaments. The named filaments form a network that is highly structured and dynamic, responding to external and internal cues with a quick reorganization that is orchestrated on the time scale of minutes and has to be tightly regulated. Especially in brain tumors, the cytoskeleton plays an important role in spreading and migration of tumor cells. As the cytoskeletal organization and regulation is complex and many-faceted, this review aims to summarize the findings about cytoskeletal filament types, including substructures formed by them, such as lamellipodia, stress fibers, and interactions between intermediate filaments, microtubules and actin. Additionally, crucial regulatory aspects of the cytoskeletal filaments and the formed substructures are discussed and integrated into the concepts of cell motility. Even though little is known about the impact of cytoskeletal alterations on the progress of glioma, a final point discussed will be the impact of established cytoskeletal alterations in the cellular behavior and invasion of glioma.

Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner.

The current treatment of glioblastoma is not sufficient, since they are heterogeneous and often resistant to chemotherapy. Earlier studies demonstrated effects of specific cannabinoid receptor (CB) agonists on the invasiveness of glioblastoma cell lines, but the exact mechanism remained unclear. Three human glioblastoma cell lines were treated with synthetic CB ligands. The effect of cannabinoids on microRNAs (miRs), Akt, and on the expression of proliferation and apoptosis markers were analyzed. Furthermore, in a model of organotypic hippocampal slice cultures cannabinoid mediated changes in the invasiveness were assessed. MicroRNAs and the activation of Akt which are related to cell migration, apoptosis, and proliferation were evaluated and found not to be associated with changes in the invasiveness after treatment with CB ligands. Also proliferation and/or apoptosis were not altered after treatment. The effects of cannabinoids on invasiveness could be blocked by the application of receptor antagonists and are likely mediated via CB1/CB2. In conclusion, our results suggest that cannabinoids can influence glioblastoma cell invasion in a receptor and cell type specific manner that is independent of proliferation and apoptosis. Thus, cannabinoids can potentially be used in the future as an addition to current therapy.

On the influence of cannabinoids on cell morphology and motility of glioblastoma cells.

The mechanisms behind the anti-tumoral effects of cannabinoids by impacting the migratory activity of tumor cells are only partially understood. Previous studies demonstrated that cannabinoids altered the organization of the actin cytoskeleton in various cell types. As actin is one of the main contributors to cell motility and is postulated to be linked to tumor invasion, we tested the following hypothesizes: 1) Can cannabinoids alter cell motility in a cannabinoid receptor dependent manner? 2) Are these alterations associated with reorganizations in the actin cytoskeleton? 3) If so, what are the underlying molecular mechanisms? Three different glioblastoma cell lines were treated with specific cannabinoid receptor 1 and 2 agonists and antagonists. Afterwards, we measured changes in cell motility using live cell imaging and alterations of the actin structure in fixed cells. Additionally, the protein amount of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), focal adhesion kinases (FAK) and phosphorylated FAK (pFAK) over time were measured. Cannabinoids induced changes in cell motility, morphology and actin organization in a receptor and cell line dependent manner. No significant changes were observed in the analyzed signaling molecules. Cannabinoids can principally induce changes in the actin cytoskeleton and motility of glioblastoma cell lines. Additionally, single cell motility of glioblastoma is independent of their morphology. Furthermore, the observed effects seem to be independent of p44/42 MAPK and pFAK pathways.

Correction: The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

[This corrects the article DOI: 10.1371/journal.pone.0189514.].