Hypoxia - is this the key to rubbing-out cancer

Two groups of scientists working to eradicate cancer have been investigating how the disease behaves in low oxygen conditions

Creating the right environmental conditions is critical to the success of research experiments. Two independent cancer research laboratories in Scotland investigating behaviour mechanisms of tumour cells face the challenge of creating low oxygen, or hypoxic, conditions for their ultimate research goal of identifying new avenues of cancer treatment and drug development.

At the Beatson Institute for Cancer Research in Glasgow, Professor Eyal Gottlieb leads a research group studying apoptosis (cell death) and tumour metabolism. The group is investigating the relationship between cancer, cell metabolism and cell death. Prof Gottlieb’s team noted that rapid tumour growth surpasses the blood supply needed to support this growth and cell division, exposing cancer cells to extreme conditions of metabolic deficits and stress. In response to these metabolic conditions, cancer cells have evolved defence mechanisms to support both proliferation and endurance under severe stress, becoming more aggressive in the process. Targeting these survival mechanisms could help curb cancer growth and ultimately induce cancer cell death¹.    

Also investigating tumour behaviour mechanisms, a laboratory within the Breakthrough Breast Cancer Research Unit (BBCRU) in Edinburgh targets the changes in a tumour’s microenvironment with the aim of making them more responsive to current cancer treatments. Unlike Prof Gottlieb’s laboratory, the research team led by Dr Carol Ward focuses on the tumour cells’ response to treatment, and the molecular mechanisms driving resistance to chemotherapy and radiation therapy. The team’s research objective is to improve preventative care and develop new treatments for patients whose tumours do not respond to traditional endocrine therapies. By undertaking comprehensive genetic analyses to develop molecular profiles of breast tumours, the researchers aim to characterise the underlying mechanisms and changes that occur during drug treatment. Dr Ward’s team uses breast cancer cell lines to compare proliferation and cell death between samples in 21% and 0.5% oxygen, as most of the molecular changes only happen in hypoxic conditions.

Hypoxic tumour regions often produce the most aggressive and therapy-resistant cells, causing tumours to spread (metastasis). In a 2010 study², Prof Gottlieb examined how cell behaviour is affected by the hypoxia inducible transcription factor (HIF), an important mediator of metabolic adaptation of cancer cells. HIF is regulated by a family of enzymes known as HIF prolyl hydroxylases (PHDs) which sense oxygen in cells as part of a tumour cell’s metabolic adaptation to hypoxia. These PHD enzymes become functionally inactivated under hypoxia. However, it has also been shown that PHDs can be reactivated under hypoxia, creating a metabolic defect. This has led the Beatson Institute group to investigate whether reactivation of PHD enzymes can inhibit tumour progression. The team observed that by keeping PHD active under hypoxia and hence preventing the hypoxia-mediated metabolic changes, tumours experience a metabolic crisis and undergo cell death.

In order to observe the PHDs’ activation status in vitro it is important to accurately recreate the cyclic exposure to hypoxia and reoxygenation that takes place in tumours in vivo. The activation of PHDs under hypoxia reverses metabolic adaptation, ultimately leading to cell death and the inhibition of tumour growth¹. This influence on tumour survival and progression validates PHDs as potentially druggable targets for cancer².

For both the Beatson Institute and BBCRU research groups, recreating a tumour-like hypoxic environment is essential to enable the teams to study the specific cell behaviour of interest. Many solid tumours consist of large areas of poorly perfused cells leading to areas of low oxygen throughout the tumour mass². However, many laboratory cell culture systems can only expose cells to 21% oxygen – more than double the physiological concentration of oxygen found in most cells in vivo. As cells are known to behave differently in hypoxic conditions, researchers such as Prof Gottlieb and Dr Ward rely on being able to recreate and maintain a controlled low oxygen environment in order to more accurately reflect the cellular interaction within tumours.  

Dr Ward’s breast cancer research group uses a specialised hypoxic workstation to treat and manipulate the cells while keeping tight control of the required low oxygen concentration. When working under hypoxic conditions any introduction of atmospheric oxygen invalidates the environment, resulting in reoxygenation-based signalling in the cells instead of hypoxia per se. Specialised hypoxic workstations, for example Don Whitley Scientific’s H35 Hypoxystation, allow researchers to monitor and record the consistently low oxygen levels as well as measuring the alterations in tumour cell metabolism. The ability to generate and precisely control a hypoxic environment offers researchers peace of mind, ensuring that the required internal conditions remain constant at all times.  Environmental parameters such as oxygen and carbon dioxide concentrations, temperature and humidity can all be controlled and monitored via a touch screen interface.

The accuracy of Dr Ward’s methodology also depends on the ability to manipulate cells under controlled hypoxia without altering the incubation environment – seal the sample plates, examine, radiate and photograph them. It is equally important to be able to return the cell samples back into the controlled environment to continue an experiment, with the oxygen status remaining uncompromised. Precision in environmental parameters is vital for cell biology research that often has to be performed over a comprehensive range of oxygen tension. The requisite low oxygen conditions can only be recreated in vitro with the help of specialised laboratory equipment.

Hypoxia workstations featuring a ‘rapid-airlock’ porthole system enable the research groups to save time when transferring cellware to and from the workstation environment. The inherent flexibility offered by specialised hypoxic workstations means additional laboratory equipment can be used throughout an experiment without the risk of cell reoxygenation. This can be facilitated by gloveless instant access portholes that prevent fluctuations in oxygen when accessing the workstation. Portholes can also be used to add drugs or media and enable manipulation of cells in situ without disturbing other cells and/or experiments that are running concurrently.

Experimenting with oxygen levels in the tumour environment has helped Prof Gottlieb’s team to identify a new approach – direct targeting of PHDs by addressing hypoxia in general rather than targeting HIF only. PHDs in most tumours are functionally silenced by the prevailing hypoxia. The research has demonstrated that reactivating these enzymes under hypoxia may be a suitable approach for cancer treatment¹.

The hypoxia workstations are designed to allow the oxygen content to be tightly controlled in 0.1% increments from 0.1% to 20%. This enables researchers to carry out a comprehensive and more accurate ex vivo analysis of hypoxic tumours that are known to be more aggressive, with high metastatic potential and higher resistance to therapy. These often do not respond to the endocrine therapy or may acquire resistance to treatment.

Furthermore, unique PIN number user access control features mean that even if in use by several individuals or groups, the parameters set for ongoing experiments cannot be changed in error. Workstations with a removable front panel can also be advantageous as they permit the use of additional laboratory equipment, such as mini-centrifuges, inside the workstation.

The success of Dr Ward’s study has attracted the attention of other laboratories in BBCRU that will be interested in using a hypoxic workstation to refine processes for characterising tumours and predicting their response to therapy. Meanwhile, Prof Gottlieb’s research team continues to investigate the metabolic transformation process that cancer cells undergo in order to survive and grow. Future studies at the Beatson Institute will continue to focus on the unique metabolic traits of cancer cells and how the survival mechanism is regulated through the inhibition of PHDs. A recent report has shown that PHD1 expression is increased across a range of breast cancers, indicating the need for further research into PHD regulation in tumours³ using hypoxic workstations.