Intracellular Detection of Low Oxygen Tension in Live Cells

Breathe new life into hypoxia research

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Oxygen homeostasis is an important physiological process that is required to maintain cellular health and function. Hypoxia is a condition of low oxygen tension in tissues and contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease, and chronic obstructive pulmonary disease. While generally associated with pathological conditions, hypoxia response pathways are also critical in the normal development of some cell types, such as hematopoietic stem cells. Although the significance of hypoxia in biological processes is well known, creating model systems to accurately control hypoxic conditions is extremely difficult for most researchers without access to elaborate instruments that allow precise control and maintenance of temperature, humidity, and gases (CO₂ and O₂) during an experiment. Fortunately, the EVOS FL Auto Imaging System with Onstage Incubator provides an easy-to-use platform that allows for the precise control of oxygen levels, thereby delivering an effective system for researchers to evaluate cellular responses to hypoxia by live-cell fluorescence imaging using the Image-iT Hypoxia Reagent.

Cellular responses to hypoxia

The growth patterns of solid tumors impose low oxygen concentrations on their core cells, and therefore adaptation to hypoxia is advantageous for tumor development and survival [1]. Conversely, disruption of these hypoxic responses can lead to leukemic transformation [2]. Hypoxic responses are also critical for the normal development of hematopoietic stem cells, which reside within a hypoxic bone marrow microenvironment. Hypoxia signaling pathways are used for cell fate decisions leading to normal hematopoiesis [3].

Adaptation to hypoxia is mediated largely by transcriptional activation of genes that facilitate short-term (e.g., glucose transport) and long-term (e.g., angiogenesis) adaptive mechanisms. A key regulator of cellular responses to hypoxic conditions is the transcription factor HIF-1 (hypoxia inducible factor-1), which functions as a master regulator of cellular and systemic homeostatic response to hypoxia by activating transcription of a wide array of genes, including those involved in energy metabolism, angiogenesis, and erythropoiesis, as well as genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia.

Methods for studying hypoxia

While the importance of studying the cellular signaling pathways involved in the hypoxic response is clearly understood in a wide range of biological applications, developing model systems to precisely study the effects of low oxygen levels on cells and tissues remains technically challenging for most laboratories. One common method for studying the downstream effects of HIF-1 depletion involves the addition of cobalt chloride (CoCl₂) to cells in culture. CoCl₂ can mimic the effects of hypoxia by stabilizing the HIF-1 complex and thereby activating HIF-1–inducible genes. However, CoCl₂ only impacts the HIF-1 pathway and may not affect other hypoxia-related pathways. In addition, other unknown cellular processes and functions may be disrupted by CoCl₂ treatment, inducing phenotypes that are unrelated to the hypoxic response. Techniques for imaging hypoxia include the use of invasive oxygen electrodes to measure tissue oxygen levels, HIF-1 or Glut1 tissue stains to look for indirect evidence of hypoxia in cells, and nitroimidazoles that bind to protein thiols in hypoxic tissue at acute reductions in oxygen levels.

Spheroid culture methods have enabled substantial contributions to both basic cell biology and cancer biology (see “Formation of uniform and reproducible 3D cancer spheroids in high-throughput plates"). The multicellular tumor spheroid (MCTS)—a 3D cell structure with a diameter of 200–500 μm—is a valuable model for cancer biology. Closely mimicking the physiology of small avascular tumors [4], spheroids in this model develop chemical gradients of oxygen, nutrients, and catabolites just like a tumor in vivo; they also possess histomorphological and functional features similar to those of tumors.

Both spheroids and tumors exhibit a heterogeneous distribution of cell types, expression patterns, and physiology. Cells located at the surface of a spheroid secrete specific compounds as a tumor would in vivo. Internally, spheroids possess the same hypoxic core seen in tumors; this hypoxic core is one of the most distinct characteristics of spheroid cultures that cannot be successfully reproduced with classic 2D culture methods. The MCTS model thus mimics in vivo solid tumors in which cells rapidly outgrow the blood supply, leaving the center of the tumor with an extremely low oxygen concentration (Figure 1).

The EVOS FL Auto Imaging System with Onstage Incubator: Live-cell imaging under precisely controlled oxygen levels

Incubation chambers have been used to lower oxygen concentrations to allow for long-term cell growth in hypoxic conditions. However, real-time visualization of cellular processes in response to hypoxic conditions becomes problematic when transferring cells from the incubator to a microscope for imaging. Not only is it difficult to achieve precise control of oxygen levels in an incubator, but reoxygenation may create misleading results during the time required to image hypoxic cells.

The EVOS FL Auto Imaging System with Onstage Incubator includes an environmental chamber allowing for the precise control of oxygen levels, temperature, and humidity, thereby delivering an effective system for researchers to evaluate cellular responses to hypoxia over long time periods by live-cell fluorescence imaging. The onstage incubator contains port connections for air, O₂, and N₂. Gas concentrations are controlled by the software on the EVOS FL Auto system, allowing cells to be cultured using precise O₂ concentrations over an extended period of time. The EVOS FL Auto Imaging System with Onstage Incubator is easy to use: Simply input the desired O₂ level and the onstage incubator will equilibrate to the selected conditions. The incubator is designed specifically for the EVOS automated imaging system, which combines live-cell imaging, area scanning, image stitching, and time-lapse imaging in a single user-friendly platform. EVOS imaging systems make multichannel fluorescence microscopy accessible to both novice users and high-throughput core imaging facilities (see “The EVOS FL Cell Imaging System: A key component of an imaging core facility").

Image-iT Hypoxia Reagent: A real-time oxygen detector

With the EVOS FL Auto Imaging System, live-cell imaging can be performed in real time under hypoxic conditions using the Image-iT Hypoxia Reagent. The Image-iT Hypoxia Reagent is a fluorogenic, cell-permeant compound for measuring hypoxia in live cells. This reagent is nonfluorescent in an environment with normal oxygen concentrations (approximately 20%) and becomes increasingly fluorescent as oxygen levels are decreased (Figure 2). Unlike nitroimidazoles (such as pimonidazole) that respond only to very low oxygen levels (<1%) [5,6], the Image-iT Hypoxia Reagent begins to fluoresce when oxygen levels drop below 5%.

Because it responds quickly to a changing environment, Image-iT Hypoxia Reagent can serve as a real-time oxygen detector, with a fluorescent signal that increases as atmospheric oxygen levels drop below 5% and decreases if oxygen concentrations increase. In addition, Image-iT Hypoxia Reagent is very easy to use; just add it to cell culture medium and image. These properties make this reagent an ideal tool for detecting hypoxic conditions in tumor cells, 3D cultures, spheroids, neurons, and other tissues used in hypoxia research. Reagents with similar applications have been reported to detect tumors in small animals, and their fluorescence properties have been shown to correspond with increased HIF-1α expression and translocation in hypoxic environments [7].

Immunodetection of HIF-1 in fixed cells

In fixed-cell imaging, HIF-1 expression is commonly used as a marker for monitoring cells following exposure to hypoxic conditions. The HIF-1α mouse monoclonal antibody provides a highly specific probe for evaluating HIF-1α expression in fixed-cell samples. We offer a number of Thermo Scientific Pierce antibodies that recognize HIF-1α and have been verified to perform in various applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), western blot analysis, and immunoprecipitation; Figure 3 shows an example of ICC using the anti–HIF-1α mouse monoclonal antibody, clone mgc3, which has citations describing its use in all of the above applications. Also available is the well-validated anti–HIF-1α mouse monoclonal antibody, clone H1alpha67, which has proven useful in at least 10 different types of immunoassays, including western blotting (Figure 4).

Accelerating the pace of hypoxia research

The EVOS FL Auto Imaging System with Onstage Incubator allows for the precise control of oxygen concentrations, overcoming technical hurdles that have plagued hypoxia research in the past. This easy-to- use system, in combination with the Image-iT Hypoxia Reagent, is ideal for studying the role of cellular responses to hypoxia in both basic biological processes and disease mechanisms.

References

1. Giaccia AJ, Simon MC, Johnson R (2004) The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease. Genes Dev 18:2183–2194.
2. Gezer D, Vukovic M, Soga T et al. (2014) Concise review: genetic dissection of hypoxia signaling pathways in normal and leukemic stem cells. Stem Cells 32:1390–1397.
3. Rankin EB, Giaccia AJ, Schipani E (2011) A central role for hypoxic signaling in cartilage, bone, and hematopoiesis. Curr Osteoporos Rep 9:46–52.
4. Kunz-Schughart LA (1999) Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor. Cell Biol Int 23:157–161.
5. Arteel GE, Thurman RG, Yates JM et al. (1995) Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver. Br J Cancer 72:889–895.
6. Gross MW, Karbach U, Groebe K et al. (1995) Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids. Int J Cancer 61:567–573.
7. Zhang S, Hosaka M, Yoshihara T et al. (2010) Phosphorescent light-emitting iridium complexes serve as a hypoxia-sensing probe for tumor imaging in living animals. Cancer Res 70:4490–4498.