Research by an expert lab at the University of Pittsburgh Medical School led by Prof. Paul A Johnston shines new light on the importance of moving to 3D cell cultures from 2D. They explored well established HNSCC cell lines for hypoxic experience, regional patterns and microenvironments.
Hypoxia is correlated with poor outcomes in head and neck cancers (HNC) and is therefore of notable importance in both drug development and therapeutic decision-making. 3D microtissues such as multicellular tumor spheroids (MCTS) recapitulate a better physiological representation of avascular tumor regions and it follows that their hypoxic experience should be more informative than 2D cell culture alternatives.
To study the hypoxic experience of MCTS in a time-lapse, continuous mode over several days the far-red hypoxia reporter HypoxiTRAK™ was utilised, showing excellent correlation to both Hif-1ɑ and pimonidazole but with the direct readout of fluorescence from the reporter rather than the need for an end-point readout requiring fixation, permeabilization and antibody staining - time-consuming and expensive.
HypoxiTRAK™ was simply added to the culture medium, when convenient, for example at the aggregation stage or after a few days for spheroid formation, and then present throughout the experiment thereafter to accumulate in cells that have metabolically responded to hypoxic stress. Importantly, HypoxiTRAK™ caused no apparent toxicity or growth limitation compared to controls yet completely penetrated MCTS to report hypoxia at full depth. It should be noted the latter was not the case for pimonidazole - seen as a reference for hypoxia - and which might be attributed to either poor penetrance of the drug (not previously reported) or inability of the antibody to penetrate and label adducts (unlikely since Hif-1ɑ was unaffected) but more likely the propensity of nitroimidazole-based chemistries to create hypoxia as a consequence of futile redox cycling and thereby limiting any adduct formation to cell layers near the surface.
HypoxiTRAK™ is simple to use, requiring little optimisation and persists stably in culture medium over many days. Red wavelength excitation limits phototoxicity and enables multi-colour examination at end-point with Hoechst 33342 or visible-wavelength chromophores. The direct fluorescence readout enables time-lapse capture of the hypoxic experience of MCTS of cultures over several days and is fully compatible with high content imaging and high sample throughput.
Reference:
Close, David A., and Paul A. Johnston. "Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines." SLAS Discovery 27.1 (2022): 39-54.
Additional links:
Blog: Imaging hypoxia in 2D & 3D
Notes:
HypoxiTRAK™ is also compatible with the flow cytometric detection of the accumulated hypoxic experience, cell-by-cell. Full details are available in the technical datasheet on the product's webpage or contact our technical support for advice in your specific application (email: enquiry@biostatus.com).
