Can ECIS Biosensor Technology Be Used to Measure the Cellular Responses of Glioblastoma Stem Cells?

Glioblastoma is considered the most aggressive and lethal form of brain cancer. Glioblastoma tumours are complex, comprising a spectrum of oncogenically transformed cells displaying distinct phenotypes. These can be generated in culture and are called differentiated-glioblastoma cells and glioblastoma stem cells. These cells are phenotypically and functionally distinct, where the stem-like glioblastoma cells give rise to and perpetuate the tumour. Electric cell-substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method for the analysis of cellular behaviour, based on cellular adhesion. Therefore, we asked the question of whether ECIS was suitable for, and capable of measuring the adhesion of glioblastoma cells. The goal was to identify whether ECIS was capable of measuring glioblastoma cell adhesion, with a particular focus on the glioblastoma stem cells. We reveal that ECIS reliably measures adhesion of the differentiated glioblastoma cells on various array types. We also demonstrate the ability of ECIS to measure the migratory behaviour of differentiated glioblastoma cells onto ECIS electrodes post-ablation. Although the glioblastoma stem cells are adherent, ECIS is substantially less capable at reliably measuring their adhesion, compared with the differentiated counterparts. This means that ECIS has applicability for some glioblastoma cultures but much less utility for weakly adherent stem cell counterparts.

Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes.

Cannabinoid receptor 2 (CB2) is a promising therapeutic target for immunological modulation. There is, however, a deficit of knowledge regarding CB2 signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB2-selective ligand (HU308). We observed a "lag" phase of unchanged cAMP concentration prior to development of classically expected Gαi-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gαi effects by concurrent Gαs activation. Monitoring downstream signaling events showed that activation of p38 was mediated by Gαi, whereas ERK1/2 and Akt phosphorylation were mediated by Gαi-coupled ßγ. Activation of CREB integrated multiple components; Gαs and ßγ mediated ∼85% of the response, while ∼15% was attributed to Gαi. Responses to HU308 had an important functional outcome-secretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1α, and TNF-α were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB2. Additionally, signaling and functional effects were completely blocked by a CB2-selective inverse agonist, giving additional evidence for CB2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB2 ligands as immunomodulatory therapeutics.

The Importance of Multifrequency Impedance Sensing of Endothelial Barrier Formation Using ECIS Technology for the Generation of a Strong and Durable Paracellular Barrier.

In this paper, we demonstrate the application of electrical cell-substrate impedance sensing (ECIS) technology for measuring differences in the formation of a strong and durable endothelial barrier model. In addition, we highlight the capacity of ECIS technology to model the parameters of the physical barrier associated with (I) the paracellular space (referred to as Rb) and (II) the basal adhesion of the endothelial cells (α, alpha). Physiologically, both parameters are very important for the correct formation of endothelial barriers. ECIS technology is the only commercially available technology that can measure and model these parameters independently of each other, which is important in the context of ascertaining whether a change in overall barrier resistance (R) occurs because of molecular changes in the paracellular junctional molecules or changes in the basal adhesion molecules. Finally, we show that the temporal changes observed in the paracellular Rb can be associated with changes in specific junctional proteins (CD144, ZO-1, and catenins), which have major roles in governing the overall strength of the junctional communication between neighbouring endothelial cells.

Cannabinoid Receptors: A brief history and what not

Our understanding of the complexity of the endocannabinoid system has evolved considerably since the cloning of the receptors in the early 1990s. Since then several endogenous ligands have been identified and their respective biosynthetic pathways unravelled. This research has revealed the involvement of the cannabinoid system in a number of important physiological processes including the regulation of neurotransmitter release, pain and analgesia, energy homeostasis, and control of immune cell function. All of these events are mediated by two similar receptors, CB1 and CB2, which were initially thought to possess mutually exclusive expression profiles. Recent advances have begun to dissolve such absolutes with the discovery of CB2 in brain tissue and identification of a range of functions for CB1 in peripheral tissues. With improved understanding of the cannabinoid system comes the illumination of various roles in disease pathologies and identification of potential therapeutic targets. This review provides an overview of the current understanding of the endocannabinoid system, and then focuses on recent discoveries that we believe are likely to shape the future directions of the field.