Dr. Brad Wouters is Interim Director and a Senior Scientist at the Princess Margaret Cancer Centre, working on hypoxia signaling pathways in cancer.

  1. “Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium” J Enzyme Inhib Med Chem. 2014 Oct 27:1-33
    In a 5-year EU-wide project denoted METOXIA, hypoxia-related issues influencing cancer treatment were examined with an eye toward developing new methods and identifying novel therapies. The hypoxic regions of tumors harbor cell fractions that are resistant to radiotherapy as well as chemotherapy and which have a high likelihood to metastasize, so developing new therapy models can significantly improve the prognosis for many cancer patients. Projects within METOXIA investigating oxygen-regulated signaling cascades, HIF pathways, biological handling of reactive oxygen species and novel diagnostic methods are described in this review.

  2.  “Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER” NATURE COMMUNICATIONS 2014 5:5203

    Hypoxia promotes stemness in normal tissues and in cancer, and van den Beucken at al. provide data from breast cancer showing that one aspect effecting this phenotype transition may involve epigenetic suppression of DICER transcription. The DICER promoter is silenced at low oxygen tension, significantly reducing miRNA processing. DICER inhibition stimulates the epithelial-mesenchymal transition EMT and promotes the acquisition of stem cell phenotype, all of which drive metastasis in cancer.

The lab has a total of four HypOxystations and has been using versions of these hypoxic workstations for over 10 years. HypOxygen asked how the lab uses the workstation for hypoxic cell culture.

What is the focus of the research at your lab?

A: We have a number of different interests in our lab. One of our projects focusses on the unfolded protein response. Hypoxia influences protein folding in the endoplasmic reticulum, and this causes ER stress which activates a pathway called the unfolded protein response (UPR). This occurs as a consequence of the requirement for oxygen in disulfide bond formation and isomerization in the maturation and folding of proteins in the ER. Oxygen serves as a terminal electron acceptor and under hypoxia, these proteins can’t fold properly, instead they accumulate in the ER and in that situation, they activate the UPR. We are trying to elucidate the downstream signaling that mediates tolerance to hypoxia through activation of antioxidant defense mechanisms and through regulation of autophagy downstream of the UPR. We are also studying the effects of hypoxia on epigenetic regulation: oxygen is used by a family of dioxygenase enzymes, part of the same family of enzymes that mediate hydroxylation of HIF and HIF stability. These proteins are involved in a wide number of processes which include histone and DNA demethylation, which has a potential impact on regulation of epigenetic states. One of the genes affected by that regulation is DICER, which accumulates H3K27 trimethyl marks due to the inactivation of the histone demethylase, and this leads to DICER suppression, and impacts miRNA biogenesis. We have also been doing a number of functional genetic studies trying to identify vulnerabilities in hypoxic cells as potential therapeutic targets. These are studies using whole genome shRNA screens, which are carried out within the hypoxic workstation. There are many aspects of our research that require exposure of our cells to hypoxia in vitro, and as part of a much bigger hypoxia program at Princess Margaret, we have a lot of users of the chambers besides our own group.

How do you use the HypOxystation?

A: We have 2 H35 workstations, an H85, and our newest one, an H45. We started using these stations in my lab in Europe more than 10 years ago, which were originally from Don Whitley Scientific in the UK. I was one of the original users of this technology for mammalian cell culture. I spoke to the company a lot about developing a workstation for mammalian cells. We’ve evaluated many of these machines, and they work well. In my experience, at the very low oxygen levels we work at, it is always best to put secondary monitoring systems in place. We draw a gas sample periodically when an experiment starts, as a random check of the workstation atmosphere, and we find that the HypOxystation has been very stable. We assist many other groups on their studies in the workstations as Princess Margaret has a large community of researchers interested in this topic.

We are well aware of the importance of dissolved oxygen: we are very careful to create the conditions where we understand the issue of dissolved versus atmospheric oxygen. It is very hard to work at low oxygen in standard culture conditions with confluent cells, because the oxygen sink from metabolism is huge, and changes with time. It all comes down to oxygen gradients that form in the media; oxygen in the media will equilibrate with the atmosphere at the surface, and gradients develop because the cells at the bottom of the dish are consuming oxygen at a higher rate than O2 can diffuse into the media. This is similar to gradients forming in tissue and in tumors, and the best way to avoid that is to disrupt the gradient by gentle agitation of the dish. We place a small orbital shaker inside the workstation and that helps the media move back and forth and gets rid of the gradients. A lot of our experiments are carried out at very low oxygen, even close to anoxia, but when you are working at .5 or 1% oxygen, gradients that form after a time in the direct vicinity of the cells are changing that immediate oxygen environment. We have one of our HypOxystations set up to provide anaerobic conditions using pre-mixed gas and a catalyst.

What would you say are the advantages of the workstation as compared to

  • working at ambient O2 in a CO2 incubator
  • working in a low oxygen incubator at hypoxia

A: The most important aspect is that we can control the oxygen atmosphere: we can go in and out without disturbing it. Oftentimes we’ll add compounds, or drugs, or other agents at different periods of time; sometimes we’ll do time course experiments where we take some dishes in and out. An incubator just isn’t suitable for that, because whenever the door is opened, the oxygen concentration changes dramatically and the cells will go through waves of deoxygenation and oxygenation. Even if temperature and humidity and ph changes are definitely an issue in incubators as cells are taken in and out, I think the oxygen level is really the most crucial.

In a workstation, you can manipulate the cells while they are hypoxic. For some assays we lyse the cells inside the workstation, we do pulse-chase radioactivity experiments there, we add compounds to the cells while they are still hypoxic, we can passage cultures inside. Sometimes we have cultures inside the workstation for 3-4 weeks looking for genetic changes in continuous culture at low oxygen. The continuous hypoxia we achieve in the workstation is a prerequisite for studies with hypoxia-activated drugs used in cancer therapy strategies. Incubators are only useful down to 5% oxygen at best, anything else is not a good idea.

How do you precondition your media and do you feel you need to know the level of the dissolved oxygen?

A: If we want to change the oxygen concentration rapidly then we’ll pre-equilibrate the media. If that’s not important we just use the media as is.  The rate of equilibration is proportional to the area of the liquid surface relative to its volume, so if we want to equilibrate quickly we use large dishes with a thin film of media. Preconditioning a whole bottle of media to low oxygen would take days, if it worked at all. It would be very useful to have a dissolved oxygen monitor to assess the amount of oxygen in the media.  

Do you ever place animals inside the workstation or do you anticipate the need to do so?

A: Not in my workstations, but we have an older workstation that’s used at 7% oxygen for animal work. The only reason to work at lower oxygen would be to acutely change oxygen distribution and measure the effects; normally we would use a mask and a bottle with defined oxygen concentration to create a very low hypoxia situation.

How are you using the Incucyte inside the workstation?

A: The Incucyte doesn’t fit very well inside the H35, we had to modify it ourselves. We can’t use one of the doors, and we built a platform to fit over the Incucyte so we can place dishes on top of the imager, to maximize our usage of the space there. We are placing it inside the new H45 now so it will fit in there much better. It is a very useful system for live-cell imaging: the conditions inside the Incucyte are the same as the surrounding atmosphere, so we can follow growth and proliferation of our cells in that hypoxic environment. We perform various assays while the cells are in the workstation, to assess cell death, apoptosis, migration; there are a lot of aspects where we need to collect images over time at low oxygen.

Do you ever use the oxygen profiling/cycling option to simulate hypoxia/reperfusion or slow deoxygenation processes?

A: We have used the profiling option, and the speed with which the atmosphere changes is reasonable given the large volume of the workstation.

Do you have plans to do metabolism studies using a Seahorse XF Analyzer?

A: We have two Seahorses and I have considered placing them in the workstation. It is very difficult to measure oxygen consumption in cells equilibrated at 1%, so I would be interested in trying this out for myself. It is definitely useful to measure oxygen consumption at more relevant oxygen concentrations, even at around 5% instead of 20% ambient. We do a lot of Seahorse measurements of cells that have been exposed to hypoxia and then reoxygenated, but we haven’t been able to do the experiments at hypoxia yet.

Which conferences do you and your colleagues visit?

We go to the AACR annual meeting, Keystone metabolism meetings and anything related to tumor hypoxia.

See more of what Brad Wouters is doing here: