Patrick Frost is at the Department of Hematology-Oncology at the VA Hospital in Los Angeles. He has been using an H35 HypOxystation for almost 3 years, and just in March published his most recent findings on multiple myeloma cells cultured in the HypOxystation. At lower than 1% oxygen, the atmosphere inside the chamber mimics the hypoxic niche in the bone marrow microenvironment. Patrick Frost’s group investigated the effects of a polyamide capable of binding to the hypoxia response element HRE of genes such as VEGF that are up-regulated under low oxygen conditions. They found a significant cytotoxic effect of the polyamide against MM tumor cells which they traced to an inhibition of HIF-mediated gene transcription and decreased resistance to hypoxia-induced apoptosis. These results are described in “A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity” (Mysore et al., 2016, Mol Cancer Res; 14(3); 253-66.)

The focus of our lab’s research is multiple myeloma, specifically with the aim of targeting the adaptive hypoxic response in myeloma cells. We have a number of interesting compounds, and we are studying HIF regulation to understand the mechanisms of what happens in these cells treated with the compounds. We have a polyamide, a synthetic molecule that recognizes the hypoxia response element HRE and binds to the minor groove of the DNA. We have to use relatively high concentrations of this compound but it is a good model to block the hypoxic response. In order to characterize the effect of the molecule, we used it in some reporter assays, in reporter cells, then we tested it to see whether it could overcome their resistance to hypoxia-mediated killing. There is some thought that hypoxia confers resistance to drugs, which we did not find in our particular experiments. We found that the drugs and the hypoxia actually synergize. I think there is a need to differentiate more between resistance and cell cycle arrest in this regard, as opposed to killing the cells. The HypOxystation is very good for placing the cells at hypoxic conditions, at different levels, and sampling at different time points. The time points especially are very useful with this chamber, because with other systems such as boxes, once the cells are inside and you start doing anything with them, you’ve re-set the system; it doesn’t take very long for those media to get re-oxygenated. So we can set up to do a 24 hour assay, using the interlock to get in and out, and take a sample of the growing cells every 24 hours. That really helps to get a nice time course. Once we have the cells inside the chamber, we can assay apoptosis, gene expression, protein expression, anything. The in vitro models are really very useful, we can cover a lot of ground quickly.

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, and there is no way to confirm the gas levels inside. Although we assumed that 1% oxygen would be suitable, we saw no effect at all, and we couldn’t reliably go lower than 1%. But we really need to decrease the oxygen to low levels before we see any effects, and actually stay at those low levels for 72 hours.  We work at 0.1-0.2% oxygen now in the HypOxystation, and the cells start to show effects at 48-72 hours. These myeloma cell lines are resistant to begin with, while myeloma cells from patients are notoriously difficult to culture, dying within 96 hours, even at the hypoxia they come from in the bone marrow. Patient samples are always difficult to culture, they just don’t survive very long.

Frost H35Hepa2 webWhen we got our HypOxygen workstation, things started moving, it was so much easier. Where before, in the box, we could only set up for 48 hours, with no sampling at 24 hours or any other times, in the workstation we can sample at different points in time without disturbing the atmosphere around the cells.

Our HypOxystation has been working without glitches for about 3 years here. It’s reliable and easy to use, and very well-suited for the types of timed experiments we do. I would highly recommend the system. We love it. Another group uses our workstation at times, they also conduct 24-hour timed culture experiments, examining adipose tissue.

We have put instrumentation inside the chamber, a cell shaker and a centrifuge, so we can collect cells and put them on ice immediately. I worry that the cells will quickly reset in ambient oxygen, especially if you are looking at HIF. We did an experiment in which the cells were placed in hypoxia and then reoxygenated, and within 30 minutes HIF had disappeared. If that type of granularity and precision is needed, then by all means, harvest and spin down the cells inside the workstation.

We will soon be looking at acidification of the media, as hypoxia in tumors leads to a change in pH based on lactate production as metabolism changes. The same sort of process that drives tumorigenisis, and progression of malignancy, and changes in resistance in tumor cells in response to hypoxia, may also be driven by changes in pH. Metabolism, pH and hypoxia are all linked.

We precondition our media by placing the media flask inside the workstation for approx. 24 hours. If the endpoint of our assay is 72 hours, we aren’t too concerned about the media equilibrating to the atmospheric oxygen level, we just assume the actual time at hypoxia is shorter. That’s another advantage of the HypOxystation over the old box system: in there, deoxygenating any volume of media was not possible, the space was just big enough for the plates. The exact level of the media oxygen isn’t too important to me at the beginning, just that we do go very low. Our goal in doing the in vitro assays is to look at processes in the animals. It’s much more difficult measuring oxygen or pH in mouse bone marrow, so this is why we are investing so much in the cell culture studies. When we get to the molecular biology and the biochemistry, and when we actually look at gene expression, where we see the fastest effect, that’s when we’ll be drilling down to minute differences.

We are looking at oxygen levels in the bone marrow because it’s known to be hypoxic, compared to other tissues, and the overall hypothesis is that this hypoxia either facilitates the tumors, or attracts the tumor cells, or makes them more resistant to treatment, as they like the hypoxic environment. Since it’s physiologically hypoxic, the stem cells responsible for hematopoiesis can thrive in this environment and produce the blood cells. This hypoxia may be why myeloma cells are in there. Cell lines are usually grown at ambient oxygen, because they’ve already adapted to these parameters, and cells that were originally adapted to hypoxia in vivo were lost or mutated over time.

There has never been any sort of contamination in our HypOxystation, in almost three years. We just wipe it down, just with a paper towel. We’ve never had any problems. It’s also pretty robust, it’s survived a move and a forklift and it never sprung a leak throughout.

I can see, and hear, the HypOxystation quietly working in the lab. If we shut the workstation down, the atmosphere stays stable, we don’t lose the hypoxia. It’s been a year since we replaced the CO2 bottle, and the compressed air. With pretty intense, daily work in the workstation, we use a 50 L bottle of nitrogen in about a month. That’s a good, reliably low consumption compared to the tank every day we were using with the box previously. Some workstations may be cheaper, but they are also more rinky-dink.