HypOxygen's Blog - Latest News from HypOxygen

Jumping Off the Redox Cliff

Increased production of ROS, which is both a symptom and a driver of cancer Hallmarks, can push cancer cells over the cliff of oxygen homeostasis. Compounds adding oxidative pressure can thus be utilized for selective tumor therapy. In their paper “Triggering apoptosis in cancer cells with an analogue of cribrostatin 6 that elevates intracellular ROS” , HypOxystation users Asby et al. describe their approach to chemically modifying a natural compound from marine sponges, cribrostatin 6, to enhance its cytotoxic potential. They synthesized a modified molecule 8-phenylcribrostatin 6 (8PC6) that was both more potent and more selective for breast cancer cells.  Co-author Ali Tavassoli from the University of Southampton says, "We study HIF-1, so working in a hypoxic environment is critical. Besides culturing our cells in the H35, we also harvest proteins and collect RNA inside the chamber. The H35 is very easy to use; the touch screen controls are straightforward and intuitive. We have used the workstation to incubate cells in hypoxia for ~5 days, and the atmosphere remains stable over time."

Annexin V/7-AAD staining indicated that 8PC6 induces apoptosis in cancer cells. Treatment of MCF7 cancer cells with ROS-sensing dyes and siRNA to knock down ROS-protective TIGAR demonstrated that 8PC6 increases intracellular reactive oxygen species, upsetting the delicate redox balance in highly susceptible cancer cells and leading to cell death. Hypothesizing that reduction of the cribrostatin analogue yields a semi-quinone that reacts with molecular oxygen to generate superoxide, Asby’s group decided to withdraw oxygen from the equation by incubating the MCF7 cells in the HypOxystation at 1% O2. Pre-incubation and subsequent incubation with increasing doses of 8PC6 in normoxia versus hypoxia showed that, indeed, the IC50 was increased up to 46-fold in hypoxia due to lack of oxygen. The HypOxystation’s closed workstation format and rigorous control of oxygen, CO2, temperature and humidity facilitates authentic cell behavior as in vivo conditions are replicated. Thus, hypoxia in the workstation equated to significant reduction in the intracellular availability of oxygen for the generation of ROS. For research being conducted on highly hypoxic tumors, the workstation atmosphere represents a close approximation of the actual conditions cells encounter. 

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Creating Healthy Environments for Cell Culture

We at HypOxygen hope you had a constructive World Heart Day! The World Heart Federation prompts us to find ways to create a heart-healthy environment by exercising more and eating more healthily. Work by our HypOxystation users demonstrates the benefits of creating healthy environments for cell culture, by providing more physiological, low oxygen conditions that let cells breathe. The closed culture workstation controls oxygen, CO2, temperature and humidity throughout the duration of culture and manipulation, ensuring that ambient air does not compromise the physiology and growth of the cells. Cardiovascular researchers all around the world are culturing their cells at physioxia in the HypOxystation.

In his 2016 paper on post-MI remodeling, HypOxystation user Li et al. investigated the role of miR-7a/b in cardiomyocytes responding to oxidative stress/hypoxia. Similarly to the conditions during and after MI, miR-7a/b and their target molecules PARP-1 and Sp1 protect cardiomyocytes from apoptosis and reduce fibrosis in an oxygen-dependent manner. The authors postulate that hypoxia-induced injury to the heart can be mitigated by modified miRNA expression, helping to prevent heart failure. As with Li’s work, the results obtained by another HypOxystation user, Jim Uniacke at University of Guelph, show “the importance of oxygen as a cell culture parameter when making physiological inferences.”

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World Heart Day

September 29, 2016 is World Heart Day, when organizations globally advocate “creating heart-healthy environments”, for example by eating more healthily and exercising 30 minutes a day. Healthy environments are also a priority for Don Whitley Scientific and HypOxygen, whose HypOxystation is used globally by cardiovascular researchers to mimic the in vivo environment, so cells behave in a physiologically relevant manner. Temperature, CO2, oxygen and humidity are controlled in the HypOxystation, and cells are cultured and manipulated in that physiological environment without ever experiencing the “oxygen shock” of the bench atmosphere. The in vivo environment recreated in the HypOxystation is healthy with regard to metabolism, proliferation, signaling, and drug responsiveness, to name a few.

Hear Dr. Michael Cross, Molecular and Clinical Pharmacology Department, University of Liverpool, speak about his work with cardiac spheroids to assess cardiovascular drug toxicity. “The H35 allows us to generate oxygen levels that reflect the in vivo physiology these cells would be exposed to. We chose the Hypoxystation with its oxygen profiling feature, which allows us to recreate cycles of ischemia, where oxygen levels typically sink to 1-3%”.

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HypOxystation Gas Consumption Studies

Patrick Frost, Professor at UCLA and the West Los Angeles Medical Center, investigates how targeting angiogenesis and the adaptive hypoxic response in multiple myeloma cells affects tumor progression. He hopes his studies will provide insight into the pathology and chemotherapy resistance of this incurable disease, which occurs with a significant frequency in the Veteran population. He has been using the H35 HypOxystation for almost 3 years now, and he does not miss bygone days in the lab: “We started out with just a Plexiglas box, with some valves in it, with a front cover just held on magnetically, placed inside an incubator. We would burn through a 50 L tank of nitrogen in 48 hours. I was never convinced that we had the correct level of oxygen in there”.

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HypOxystation and the TRACER Project

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.“

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Keystone Symposia "New Frontiers in Understanding Tumor Metabolism" in Banff

At this week’s Keystone Symposia “New Frontiers in Understanding Tumor Metabolism” joint with “Immunometabolism in Immune Function and Inflammatory Disease”, taking place in the dramatic mountain panorama of Banff National Park in Canada, almost 600 researchers have come together to present and discuss their newest data. Certainly, there is a common theme of metabolism to all these talks and posters, so rushing from one session to the other is very much the thing to do here. HypOxygen is at the conference (poster exhibit hall Q7) exhibiting the Don Whitley Scientific HypOxystation for low oxygen cell culture.

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