UV Laser Stimulation of Ca2+ Transients in Aggressive Glioblastoma Brain Cancer Cells.

Glioblastoma (GBM) is a lethal astrocytoma being the most common highest-grade adult brain cancer. GBM tumours are highly invasive and display rapid growth to surrounding areas of the brain. Despite treatment, diagnosed patients continue to have poor prognosis with average survival time of 8 months. Calcium (Ca2+) is a main communication channel used in GBM and its understanding holds the potential to unlock new approaches to treatment. The aim of this work is to provide a first step to accurately evoking Ca2+ transients in GBM cells using single UV nanosecond laser pulses in vitro such that this communication pathway can be more reliably studied from the single-cell to the network level.

Patterning Networks of Grade IV Glioblastoma on Silicon Chip.

Glioblastoma (GBM) is the most aggressive high-grade brain cancer with a median survival time of <15 months. Due to GBMs fast and infiltrative growth patient prognosis is poor with recurrence after treatment common. Investigating GBMs ability to communicate, specifically via Ca2+ signaling, within its functional tumour networks may unlock new therapeutics to reduce the rapid infiltration and growth which currently makes treatment ineffective. This work aims to produce patterned networks of GBM cells such that the Ca2+ communication at a network level can be repeatedly and reliably investigated.

Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cellsin vitro.

Objective.Glioblastoma (GBM) is the most common and lethal type of high-grade adult brain cancer. The World Health Organization have classed GBM as an incurable disease because standard treatments have yielded little improvement with life-expectancy being 6-15 months after diagnosis. Different approaches are now crucial to discover new knowledge about GBM communication/function in order to establish alternative therapies for such an aggressive adult brain cancer. Calcium (Ca2+) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca2+behaves and modulates activity in GBM at the single-cell level is relatively unexplored but holds the potential to yield opportunities for new therapeutic strategies and approaches for cancer treatment.Approach.In this article we establish a spatially and temporally precise method for stimulating Ca2+transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca2+communication can be studied from single-cell to larger network scales. We demonstrate that this is possible by administering a single optimized ultra-violet (UV) nanosecond laser pulse to trigger GBM Ca2+transients.Main results.We determine that 1.58µJµm-2is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca2+transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca2+communication in a larger network of GBM cells.Significance.Using adult patient-derived mesenchymal GBM brain cancer cell-lines, the most aggressive form of GBM cancer, this work is the first of its kind as it provides a new effective modality of which to stimulate GBM cells at the single-cell level in an accurate, repeatable, and reliable manner; and is a first step toward Ca2+communication in GBM brain cancer cells and their networks being more effectively studied.

Critical Spatial-Temporal Dynamics and Prominent Shape Collapse of Calcium Waves Observed in Human hNT Astrocytes in Vitro.

Networks of neurons are typically studied in the field of Criticality. However, the study of astrocyte networks in the brain has been recently lauded to be of equal importance to that of the neural networks. To date criticality assessments have only been performed on networks astrocytes from healthy rats, and astrocytes from cultured dissociated resections of intractable epilepsy. This work, for the first time, presents studies of the critical dynamics and shape collapse of calcium waves observed in cultures of healthy human astrocyte networks in vitro, derived from the human hNT cell line. In this article, we demonstrate that avalanches of spontaneous calcium waves display strong critical dynamics, including power-laws in both the size and duration distributions. In addition, the temporal profiles of avalanches displayed self-similarity, leading to shape collapse of the temporal profiles. These findings are significant as they suggest that cultured networks of healthy human hNT astrocytes self-organize to a critical point, implying that healthy astrocytic networks operate at a critical point to process and transmit information. Furthermore, this work can serve as a point of reference to which other astrocyte criticality studies can be compared.

Disruption of transverse-tubular network reduces energy efficiency in cardiac muscle contraction.

AIM: Altered organization of the transverse-tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t-tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation-contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t-tubular network has a direct effect on cardiac mechano-energetics. Our aim was to understand the fundamental link between t-tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event. METHODS: We exploited the "formamide-detubulation" technique to acutely disrupt the t-tubular network in rat left-ventricular trabeculae. We assessed the energy utilization by cellular Ca2+ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured. RESULTS: Following structural disorganization from detubulation, muscle heat output associated with Ca2+ cycling was reduced, indicating impaired intracellular Ca2+ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force-length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced. CONCLUSIONS: These results reveal the direct energetic consequences of disrupted t-tubular network, linking the energy disturbance and the t-tubular remodelling typically observed in heart failure.