Live Imaging of Cell Invasion Using a Multicellular Spheroid Model and Light-Sheet Microscopy.

Three-dimensional cellular assays are becoming increasingly popular as a fundamental tool to bridge the gap between tissue culture systems and in vivo tissue. In particular, spheroids are recognised today as a necessary intermediate model between testing in monolayer cultures and testing in animals. This chapter describes a straightforward protocol, from sample preparation to image acquisition and initial post-processing, based on one of most widely used commercial light-sheet fluorescence microscopy platform, the Zeiss Lightsheet Z.1.

Use of infrared microspectroscopy to elucidate a specific chemical signature associated with hypoxia levels found in glioblastoma.

Hypoxia is a common feature of solid tumours and is associated with poor prognosis, resistance to radio- and chemotherapy, and tumour aggressiveness. For predictive purposes as well as for improved therapeutic intervention, it is increasingly needed to have direct and specific diagnostic tools in order to measure the extent of, and changes in, tumour hypoxia. In this article, we have investigated the potential of Fourier Transform Infrared (FTIR) microspectroscopy, at cellular and subcellular resolution, for detecting hypoxia-induced metabolic changes in brain tumour (glioblastoma) cell lines and in short term primary cultures derived from patient samples. The most prominent and common changes observed were the increase in glycogen (specifically in the U87MG cell line) and lipids (all cell lines studied). Additionally, each cell line presented specific individual metabolic fingerprints. The metabolic changes did not evolve markedly with time (from 1 to 5 days hypoxic incubation), and yet were harder to detect under chronic hypoxic conditions, which is consistent with cellular adaptation occurring upon long term changes in the microenvironment. The metabolic signature was similar regardless of the severity of the hypoxic insult and was replicated by the hypoxia mimetic drug dimethyloxalylglycine (DMOG). To investigate any specific changes at subcellular levels and to improve the sensitivity of the detection method, spectra were recorded separately in the cytoplasm and in the nucleus of D566 glioblastoma cells, thanks to the use of a synchrotron source. We show that this method provides improved detection in both cell compartments. Whilst there was a high spectral variability between cell lines, we show that FTIR microspectroscopy allowed the detection of the common metabolic changes triggered by hypoxia regardless of cell type, providing a potential new approach for the detection of hypoxic tumours.

Cell cycle progression in glioblastoma cells is unaffected by pathophysiological levels of hypoxia.

Hypoxia is associated with the increased malignancy of a broad range of solid tumours. While very severe hypoxia has been widely shown to induce cell cycle arrest, the impact of pathophysiological hypoxia on tumour cell proliferation is poorly understood. The aim of this study was to investigate the effect of different oxygen levels on glioblastoma (GBM) cell proliferation and survival. GBM is an extremely aggressive brain tumour with a heterogeneous oxygenation pattern. The effects of a range of oxygen tensions on GBM cell lines and primary cells were assessed using flow cytometry. Results indicate that cell cycle distribution and viability are unaffected by long term exposure (24-96 h) to pathophysiological levels of oxygen (1-8% O2). Both transient cell cycle arrest and small amounts of cell death could only be detected when cells were exposed to severe hypoxia (0.1% O2). No significant changes in p21 protein expression levels were detected. These findings reinforce the importance of using physiologically relevant oxygen tensions when investigating tumour hypoxia, and help to explain how solid tumours can be both hypoxic and highly proliferative, as is the case with GBM.