Janos Kriston-Vizi, Scheilly Tsilova, Yinghui Xu, Nicole Bata, Robin Ketteler
University College London (UCL), MRC Laboratory for Molecular Cell Biology, London, UK
A growing tumour encounters a variety of stresses, including nutrient limitation, hypoxia and metabolic adaptation. These factors are in part mitigated by an upregulation of the cellular stress response, autophagy, resulting in an extension of survival and the ability to cope with these stress factors through transcriptional, metabolic and angiogenic responses. The interplay between autophagy and hypoxia in the context of a growing tumour is not well understood and better model systems to assay these pathways are required. Here, we present a three-dimensional model system that spontaneously forms a robust spheroid structure with a hypoxic core in a 96-well format. These structures are amenable to high-content screening and produce spheroids of reproducible sizes, thus enabling higher throughput applications. We have introduced the tandem mCherry-EGFPLC3B reporter to monitor autophagic flux in these cells and applied image analysis algorithms to determine the levels of autophagy within sub-structures of the spheroid. We find that these spheroids show high levels of hypoxia in the core region, accompanied by core cell death, and an increase in local autophagy in cells surrounding the core. Treatment with autophagy enhancing compounds reduces core cell death and the acute autophagy seen in surrounding cells. We have used this system in a proof-of-concept high-content screen to identify small molecule compounds that effectively rescue cell death and autophagy in the spheroid core region.
Keywords: 3D HCS, 3D screening, autophagy, high-content screening, hypoxia, spheroid.