HypOxystation user Brad Wouters at the Princess Margaret Cancer Center in Toronto was a collaborator in a recent publication on “A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients“ (Rodenhizer et al., Nature Materials 15, 227–234, 2016). Dr. Wouters told HypOxygen that “we describe a new device that enables us to create naturally occurring oxygen gradients, such as the ones found in tumors. We use the HypOxystation to establish a baseline, as a control, on unrolled TRACER membranes …We can set the external concentration to a fixed oxygen level and look at the resulting gradients and metabolites and so on, too… The external level is what we define. That could be 20% oxygen, but it could also be 1% or 2%, and in that case we can have the rolled-up TRACER inside the hypoxia chamber. We have tried out various oxygen levels in the hypoxia workstation and the oxygen gradients in the TRACER are very different, as are the metabolite gradients.“
The authors investigated respiration-induced gradients in cell layers growing on a scaffold membrane that is rolled up and cultured, allowing rapid disassembly and various assays on spatially defined cell regions which mimic the three-dimensional structure of a tumor. These “engineered tumors” developed metabolite and oxygen gradients as a result of metabolism, re-creating the situation in the progressively less well perfused tumor. The cellular behavior of the ovarian cancer lines used in the research was consistent with that of cells in vivo with regard to cell death, proliferation and cellular response to drugs, many of which properties were attributed to increasing hypoxia in the inner TRACER layers, comparable with poor oxygen and nutrient availability in tumors. Deep layers of the TRACER were severely hypoxic, corresponding to oxygen levels of less than 0.1%. Responses of HIF (hypoxia inducible factor) and UPR (unfolded protein response) target genes were monitored over time and revealed a gradual adaptation effect signifying decreased oxygen consumption in the deeper layers of the TRACER. Layer-specific liquid chromatography-mass spectrometry-based metabolomics analysis revealed distinct, oxygen-dependent metabolic signatures across the cell layers. The authors concluded that spatial microenvironmental metabolic reprogramming occurs based on oxygen availability and can be comprehensively investigated in this engineered tumor model.
Dr. Wouters says, “For historical reasons, people culture cells at 20% oxygen, although there are very few cells in the body that experience that level of oxygenation…When you change conditions, what you observe … is sometimes just a recognition of the adaption that’s going on. Each enzymatic system that uses oxygen has different dependencies, different affinities for oxygen, different Km’s and kinetics, so as you change oxygen concentrations, oxygen sensitive signaling pathways are differentially influenced.” Dr. Wouters currently uses four HypOxystations in his lab at the Princess Margaret Cancer Center in Toronto, where his group investigates the tumor microenvironment, with the aim of understanding the cellular and molecular responses to hypoxia in the context of the biological behavior of tumors.
