Targeting the adaptive hypoxic response in multiple myeloma


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.

Read more: Targeting the adaptive hypoxic response in multiple myeloma

Brad Wouters: TRACER mimics oxygen and nutrient gradients in tumors

Interview with Brad Wouters, Princess Margaret Cancer Center, Toronto.  March 4, 2016

HypOxygen wanted to find out more about Brad Wouters' recent research, especially the interesting paper newly published describing his three-dimensional tumor TRACER project, published as “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' lab uses three H35 HypOxystations and one H45 HypOxystation.

The paper describes a new device that enables us to create naturally occurring oxygen gradients, such as the ones found in tumors. The TRACER (engineered tumour roll for analysis of cellular environment and response) developed by Alison McGuigan of the University of Toronto is basically an engineered tumour that is assembled by rolling a single-component biocomposite sheet. Respiration-induced oxygen and metabolite gradients are established inside the device, so it’s an alternative to other 3D models such as spheroids. The benefit is that the TRACER rolls can be rapidly disassembled, enabling you to interrogate the biological phenotypes or properties, characteristics, and metabolites of the cells in those defined locations within the gradient, in the six layers of the tracer. The cells can be removed in a variety of ways; you can remove the cells in a viable way and process them as you would any cells, to do flow cytometry, metabolomics, cell survival, proliferation.

Read more: Brad Wouters: TRACER mimics oxygen and nutrient gradients in tumors

Hypoxia signaling pathways in cancer at the Princess Margaret Cancer Centre


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.

Read more: Hypoxia signaling pathways in cancer at the Princess Margaret Cancer Centre

UT Southwestern explores the role of genetic and epigenetic mechanisms in the regulation of fetal lung development


Dr. Houda Benlhabib is in the Department of Biochemistry at UT Southwestern in Dallas, working on the role of genetic and epigenetic mechanisms in the regulation of fetal lung development as well as in reproductive and perinatal biology.

  1. “Estrogen-Related Receptor γ (ERRγ) Regulates Oxygen-Dependent Expression of Voltage-gated Potassium (K+) Channels and Tissue Kallikrein during Human Trophoblast Differentiation” (2013) Yanmin Luo, Premlata Kumar, and Carole R. Mendelson; Mol Endocrinol, Jun; 27(6): 940–952.

    Placental development during pregnancy is accompanied by increasing tissue oxygen levels, while persisting hypoxia contributes to the pathogenesis of pre-eclampsia and IUGR. HypOxystation user Carole Mendelson describes studies examining the expression patterns of genes for potassium channels and tissue kallikrein under hypoxic (2% O2) conditions. She found a novel role for estrogen-related receptor ϒ (ERRϒ) as an oxygen-responsive transcription factor during trophoblast differentiation.

  2. The c-Myc-Regulated MicroRNA-17∼92 (miR-17∼92) and miR-106a∼363 Clusters Target hCYP19A1 and hGCM1 To Inhibit Human Trophoblast Differentiation” (2013) Premlata Kumar, Yanmin Luo, Carmen Tudela, James M. Alexander, and Carole R. Mendelson; Mol Cell Biol. 2013 May; 33(9): 1782–1796.

    Mendelson’s group investigated the role of miRNA’s in regulating expression of genes involved in trophoblast differentiation. Oxygen tension and vascularization play a significant role in pre-eclampsia, one of the leading causes of fetal and maternal death. C-Myc expression was elevated at hypoxia, and translation of miR-17∼92 and miR-106a∼363 clusters was found to be increased, also. Together, these factors seem to promote stemness in the proliferating cytotrophoblasts and inhibit differentiation. Interesting commonalities with tumor cells with regard to proliferation and miRNA expression are discussed in this paper.

The lab has been using an HypOxystation for about 4 years now. HypOxygen asked how the lab uses the workstation for hypoxic cell culture.

Read more: UT Southwestern explores the role of genetic and epigenetic mechanisms in the regulation of fetal...

Cincinnati Children's Hospital using the HypOxystation for work on neuronal tumors


Prof. Biplab Dasgupta is at Cincinnati Children’s Hospital, UC Department of Pediatrics, working on neuronal tumors.

  1. “The AMPK inhibitor Compound C is a potent AMPK-independent anti-glioma agent” (2014) Xiaona Liu, Rishi Raj Chhipa, Ichiro Nakano, and Biplab Dasgupta;Mol Cancer Ther. 2014 Mar; 13(3): 596–605.
    The therapeutic value of Compound C (dorsomorphin) for glioblastoma was evaluated in this study by Liu et al. The lab uses an HypOxystation for cell culture based on the highly hypoxic nature of the tumor tissue. AMP Kinase, which functions as a cellular watchdog during metabolic crisis, is selectively inhibited by Compound C. Mechanistic studies revealed that the anti-proliferative effects of Compound C were AMPK-independent, and were significantly more pronounced in glioblastoma cells than in normal astrocytes.
  2. b.   “The tumour suppressor ​LKB1 regulates myelination through mitochondrial metabolism” (2014) Shabnam Pooya,Xiaona Liu,V.B. Sameer Kumar,Jane Anderson,Fumiyasu Imai,Wujuan Zhang,Georgianne Ciraolo,Nancy Ratner,Kenneth D.R. Setchell,Yutaka Yoshida,Michael P. Jankowski and Biplab Dasgupta; Nature Communications 5, Article number: 4993.
    Stem cell and other cellular differentiation processes are accompanied by a shift from glycolytic metabolism to oxidative, while de-differentiation in re-programming of pluripotent stem cells involves the transition back to a glycolytic metabolism. Many studies have shown that hypoxic culture increased cell survival, enhanced growth, increased cell yield, accelerated generation and self-renewal of stem cells, improved maintenance and increased longevity of stem cell pools. This study examines the role of the tumor suppressor Lkb-1 in differentiation of Schwann cells during myelination of peripheral axons. Deficient Lkb-1-mediated oxidative metabolism led to inadequate production of citrate and thus, impaired lipogenesis, and to peripheral neuropathy in mice.

The lab has been using an HypOxystation for approximately 5 years now. HypOxygen asked how the lab uses the workstation for hypoxic cell culture.

Read more: Cincinnati Children's Hospital using the HypOxystation for work on neuronal tumors