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Posts Tagged ‘Tumour Microenvironment’

Clostridium difficile studies can be done in a Whitley Workstation

Hypoxic Snapshot Analysis in a 3D Engineered Tumour Scaffold

Researchers have long exposed 2 dimensional cells to varying degrees of hypoxia, often found in the tumour microenvironment. This can be great for showing overall hypoxic protein expression, but fails to account for cell to cell interactions, tissue gradients, and metabolic reprogramming. To more closely mimic in vivo conditions, a 3D culture environment is necessary. While 3D culture is nothing new, Rodenhizer et al in their paper “A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients” found a novel way to culture in a 3D environment while maintaining the ability to perform analyses that only function in 2D. These ingenious authors did all of this in their Hypoxystation by Don Whitley Scientific. They were able to analyse and map cellular metabolism and spatially identify known and novel metabolic response to hypoxia.

The team took carcinoma cells and seeded them onto a novel rolled scaffold system the authors termed “TRACER”. They exposed the scaffold to varying degrees of hypoxia, down to as low as 0.2% O2. After incubation they unrolled the 3D scaffold and started analysis to create a metabolic snapshot of the tumour. They found the 3D scaffold tumours displayed different concentrations of metabolites as compared to 2D tumour cells, implying cell to cell interactions plays a role in tumour metabolism, depending on the depth of the cell in the scaffold. The future of cancer research will be shaped by this innovative technique and oxygen control plays an important role with use of the Hypoxystation.

single layer TRACER

Image from Nature Materials Supplementary Information, “A Three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism phenotype in hypoxic gradients”

 

The Don Whitley Scientific Hypoxystation is ideal for many applications, as it can control oxygen down to 0.1% while providing a temperature and humidity controlled environment with ample working space. ISO class 3 clean room HEPA filtration is also available for long term cell culture applications.

Whitley Hypoxystation

Hypoxia and the Hallmarks of Cancer: Metabolic Reprogramming

Hanahan and Weinberg’s seminal papers on the Hallmarks of Cancer describe how cancer cells accommodate the frenzied growth characteristic of tumours. Low oxygen is eminently characteristic of tumours, and in this hypoxic environment, metabolism is reprogrammed to satisfy energetic and synthetic needs of the cells.

 

Our series on Hypoxia and the Hallmarks of Cancer has showcased research on how hypoxia in the tumour microenvironment affects 8 of the Hallmarks, and in the fifth and final chapter, we look more closely at how researchers are using the Hypoxystation to delineate the Hallmark Metabolic Reprogramming.

The Hypoxystation creates authentic cell culture conditions with regard to oxygen, CO2, temperature, and humidity.  Glove-less access to culture and manipulate cells under physiological atmosphere, in a HEPA-clean environment, allows cancer researchers to re-create the hypoxic tumour microenvironment. Hypoxystation user Dr Ali Tavassolli states that “We have only ever used the H35. I like the ease with which we can regulate and change the oxygen concentration”. And our user Dr. Brad Wouters at the Princess Margaret Cancer Centre in Toronto, who recently purchased his fourth Hypoxystation, says, “The continuous hypoxia we achieve in the workstation is a prerequisite for studies with hypoxia-activated drugs used in cancer therapy strategies.”

Hallmarks of Cancer

Metabolic Reprogramming

Changes in energy metabolism feature prominently in aggressive malignancy, and tumour hypoxia and the responding signalling pathways, featuring many HIF target genes, clearly interface with reprogrammed tumour metabolism. Reprogramming of conventional metabolic pathways serves to satisfy burgeoning energetic and anabolic needs of the tumour cells; many cancer cells may preferentially utilise glycolysis over oxidative phosphorylation, uncoupling mitochondrial metabolism from oxygen availability. Hypoxia-induced HIF’s attenuate mitochondrial function through diverse mechanisms, including down-regulation of enzymes in the electron transport chain and suppression of biogenesis of mitochondria. Signalling pathways involving HIF’s and many products of oncogenes and tumour suppressor genes interact to balance the energy needs of dividing cells with the requirement for bio-synthetic intermediates. Activation of lipid biosynthesis and other pathways with biosynthetic significance, such as the pentose phosphate pathway, is another metabolic consequence of hypoxia and HIF up-regulation. Reactive oxygen species ROS produced by the mitochondria stabilise HIF-1, influence redox homeostasis, and provide protective antioxidants to the cancer cells.

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Hypoxia and the Hallmarks of Cancer: Angiogenesis and Metastasis

The following was provided by HypOxygen, our distributor of Hypoxic Workstations in the US – Hanahan and Weinberg’s “Hallmarks of Cancer” are at the root of the multi-step progression of cancer, and they are all influenced by hypoxia in the tumor microenvironment. In this mini-review series, HypOxygen has been taking a closer look at the way Hypoxystation users worldwide are delineating the effects of hypoxia on the Hallmarks of Cancer: so far, we’ve showcased Avoiding Immune Destruction and Tumour Promoting Inflammation and Genome Instability and Mutation and Enabling Replicative Immortality.

In the Hypoxystation, researchers working with cells in culture can mimic the physiological conditions that produce those characteristic Hallmarks. The Hypoxystation enables glove-less access to cultivate and manipulate cells under physiological conditions, in a HEPA-clean environment. Oxygen levels in the Hypoxystation can be reliably and accurately adjusted to below 1%, reflecting the high metabolism, low perfusion tumor microenvironment.

 

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Hallmarks Of Cancer
1. Inducing Angiogenesis

Angiogenesis and tumor-associated neo-vascularization are central to the progression of cancer, and hypoxia in the fast-growing, poorly perfused tumor setting is one of the main factors driving the formation of new vessels. Hypoxia in the tumor activates the hypoxia stress response, which is mediated at the cellular level by HIF, VEGF and many other cytokines, growth factors and guidance molecules. As a consequence, endothelial cells and pericytes proliferate and form new blood vessels, which are, however, disorderly and leaky, in turn exacerbating hypoxia in the tumor. Cancer treatment strategies striving to normalize tumor vessels for the purpose of improved drug delivery and alleviation of hypoxia in the tumor are showing great promise.

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2. Activating Invasion and Metastasis

As with the other Hallmarks of Cancer, metastasis and cancer progression are correlated with low oxygen levels in the tumor. HIF’s activate the expression of more than 1000 genes, numerous of which play a role in inducing genes involved in the EMT, through direct interactions with HRE’s at promotor sites and other mechanisms such as epigenetic alterations, like methylation/demethylation. Hypoxia promotes migration and invasion by facilitating the endothelial-mesenchymal transition, altering cell-cell contacts, and reducing adhesion to the extra-cellular matrix. Cancer cells and neighboring cells such as fibroblasts are all influenced by hypoxia, and all contribute to the restructuring of the tumor microenvironment. The effects of the Hallmarks of Cancer continually perturb and promote each other, as when hypoxia-driven metabolic reprogramming causes acidification of the extracellular microenvironment through increased production and secretion of lactate, in turn augmenting ECM remodeling and immune evasion. Similarly, formation of novel blood vessels enables extravasation and migration of cancer cells to form new tumors.

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The Hallmarks of Cancer: Genome Instability and Immortality

 

Dr Burga Kalz Fuller continues to look at the way the iconic “Hallmarks of Cancer“, as first described by Douglas Hanahan and Robert Weinberg, are influenced by hypoxia in the tumour microenvironment.

Oxygen around and within the tumour cells is central to metabolism, immunology, epigenetics and therapy resistance of all the cancers; in the lab, oxygen levels during tumour cell culture exert effects on metabolism, maintenance, cell yield, and cell survival. That’s why the authentic physiological cell culture conditions in the Hypoxystation help advance research into tumour progression and other events which determine malignancy and outcome of cancer diseases. The Hypoxystation enables glove-less access to cultivate and manipulate cells under physiological conditions, in a HEPA-clean environment.

In this mini-review series, we take a closer look at the way Hypoxystation users worldwide are delineating Hypoxia and the Hallmarks of Cancer. Previously, we had showcased research by Hypoxystation users involved with Avoiding Immune Destruction and Tumour Promoting Inflammation. Next, we want to show the many ways in which Hypoxystation users are researching the Hallmarks Genome Instability and Mutation and Enabling Replicative Immortality. One of those researchers, Dr. David Ho of the University of Miami, presented his results at the Cell Symposium on Cancer, Inflammation and Immunity in San Diego in June.


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David Ho

Dr. David Ho from the University of Miami with his poster presentation at the Cell Symposium on Cancer, Inflammation and Immunity

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1. Genome Instability and Mutation

Tumour hypoxia drives genomic instability both by increasing the volume of mutations (DNA strand breaks, base damage, and gene amplification) and by diminishing DNA repair efficiency. The low levels of oxygen typical of the tumor microenvironment decrease transcription of genes related to homologous repair and non-homologous end-joining, leading to the genetic instability observed in hypoxic tumour cells. Hypoxia induces production of reactive oxygen species ROS, which interact with nucleic acids, proteins and lipids, causing cellular damage and mutagenesis. Hypoxic activation of HIF-1 also upregulates expression of certain miRNA’s which suppress DNA repair pathways.

 

LITERATURE:

  • Jiang et al. (2016) “Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality” Int J Radiation Oncol Biol Phys, Vol. 95, No. 2, pp. 772 e781, 2016
    www.redjournal.org/article/S0360-3016(16)00056-0/abstract Hypoxystation user
  • Doherty et al. (2016) “Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand” Nature Scientific Reports 6:22668 (2016)
    www.ncbi.nlm.nih.gov/pmc/articles/PMC4778139/ Hypoxystation user
  • Hunter et al. (2016) “Hypoxia-activated prodrugs: paths forward in the era of personalised medicine” Br J Cancer. 2016 May 10; 114(10): 1071–1077
    www.ncbi.nlm.nih.gov/pmc/articles/PMC4865974/Hypoxystation user
  • Leszczynska et al. (2016) “Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1” Scientific Reports 6:21698 (2016
    www.ncbi.nlm.nih.gov/pmc/articles/PMC4753685/ Hypoxystation user
  • Timpano and Uniacke (2016) “Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation” Journal of Biological Chemistry 291(20):jbc.M116.717363
    www.jbc.org/content/291/20/10772.abstract Hypoxystation user
  • Haider et al. (2016) “Genomic alterations underlie a pan-cancer metabolic shift associated with tumour hypoxia“ Genome Biology (2016) 17:140
    www.ncbi.nlm.nih.gov/pubmed/27358048

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2. Enabling Replicative Immortality

Cancer is characterized by a nearly unlimited capacity of the tumour cells to proliferate. Hypoxia in the rapidly growing tumour supports immortalisation of a subset of cancer cells, the “cancer stem cells”. Factors such as hypoxia in the tumour microenvironment derail signals indicating senescence and initiating apoptosis, enabling an immortal lifespan. Telomerase, Notch, c-Myc, and OCT4 mediate the acquisition of a stem cell-like phenotype through down-regulation of differentiation genes and activation of stem genes, generating CSC’s with aggressive properties. These cancer stem cells residing in an hypoxic tumour niche are uniquely resistant to many therapies, where low oxygen promotes stemness, maintenance, and self-renewal of the CSC’s. Metastasis and invasion by these CSC’s induce the formation of secondary tumours, which in most cases dramatically worsen the prognosis for cancer patients.

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Scientist Working in Whitley Workstation

Hypoxia in the Tumour Microenvironment

Hypoxia in the tumour microenvironment affects all the characteristic Hallmarks of Cancer, significantly impacting progression of the cancer and the patients’ prognosis. Inflammation and immunity are both acutely influenced by the low oxygen typical of the tumour microenvironment: hypoxia creates an immune-suppressive network supporting tumour growth and metastasis, and it induces sustained inflammation in a “wound that never heals”.

Cancer research depends on recreating a physiologically accurate environment for cell cultures in the lab, and hypoxia in a closed workstation format such as a Whitley Hypoxystation is the best way to do that. Incubate, image, manipulate and assay – all inside the continuous, reliably stable hypoxic environment. HEPA filtered air scrubbed to ISO 14644 class 3 standards, sterile humidity, and containment options make the Hypoxystation the safest, cleanest workstation available for hypoxic cell culture down to 0.1% O2.

Our Hypoxystation users are investigating all aspects of the Hallmarks of Cancer and how they are shaped by hypoxia. We review their recent research on Avoiding Immune Destruction and Tumour Promoting Inflammation here.

 

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HypOxygen at Tumour Microenvironment Workshop in Miami

This article was written by Burga Kalz Fuller, join her and HypOxygen at the 15th International Tumour Microenvironment Workshop in Miami

In most cancers, the hypoxic microenvironment affects the development and progression of tumours, driving alterations in gene expression, metabolism and cell signalling, and significantly influencing the Hallmarks of Cancer. So what about in vitro cancer research, do culture parameters matter? Definitely! Numerous studies have shown that even very brief exposure to ambient oxygen levels and temperature significantly impacts cell culture, behaviour and function of cells in vitro.

HypOxygen will be exhibiting our Hypoxystation at the 15th International Tumour Microenvironment Workshop in Miami from 27th – 29th April. The special focus there is on “Hypoxia, Angiogenesis and Vasculature”, reflecting the critical importance of hypoxia in the context of cancer. With the Hypoxystation, cancer researchers have their finger on the pulse of physiological cell culture.

 

hypoxia

 

The Hypoxystation mimics the hypoxic conditions present in cancer, providing a closed workstation format with contiguous, stable low oxygen down to 0.1%. Precise oxygen, carbon dioxide, and humidity control within a temperature-controlled environment, as well as ample space for cellular manipulation, assays and microscopic observation, allow researchers to recreate physiological conditions. HEPA filtration, sterile steam humidification, and remote parameter monitoring are some of the features that make the Hypoxystation so unique.

Cancer research labs, who use a Hypoxystation to re-create hypoxic conditions in the tumour microenvironment, are publishing brilliant papers which demonstrate the influence of hypoxia on the Hallmarks of Cancer. Metabolic adaptation, sustained growth, resisting cell death, and angiogenesis are just some of the Hallmarks which are affected by hypoxia. Here are some recent highlights:

Hypoxystation users are showing that “culturing cells in ambient air, or ‘normoxia’ is far from physiological.

Visit HypOxygen at the 15th International Tumor Microenvironment Workshop in Miami