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Review
. 2022 Feb 16;11(4):686.
doi: 10.3390/cells11040686.

Detection of Hypoxia in Cancer Models: Significance, Challenges, and Advances

Inês Godet  1   2   3 , Steven Doctorman  2 , Fan Wu  2 , Daniele M Gilkes  1   2   3   4
Affiliations
Review

Detection of Hypoxia in Cancer Models: Significance, Challenges, and Advances

Inês Godet et al. Cells. .

Abstract

The rapid proliferation of cancer cells combined with deficient vessels cause regions of nutrient and O2 deprivation in solid tumors. Some cancer cells can adapt to these extreme hypoxic conditions and persist to promote cancer progression. Intratumoral hypoxia has been consistently associated with a worse patient prognosis. In vitro, 3D models of spheroids or organoids can recapitulate spontaneous O2 gradients in solid tumors. Likewise, in vivo murine models of cancer reproduce the physiological levels of hypoxia that have been measured in human tumors. Given the potential clinical importance of hypoxia in cancer progression, there is an increasing need to design methods to measure O2 concentrations. O2 levels can be directly measured with needle-type probes, both optical and electrochemical. Alternatively, indirect, noninvasive approaches have been optimized, and include immunolabeling endogenous or exogenous markers. Fluorescent, phosphorescent, and luminescent reporters have also been employed experimentally to provide dynamic measurements of O2 in live cells or tumors. In medical imaging, modalities such as MRI and PET are often the method of choice. This review provides a comparative overview of the main methods utilized to detect hypoxia in cell culture and preclinical models of cancer.

Keywords: HIF; cancer; detection; hypoxia.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detecting hypoxia in vitro. (1) Immunolabeling of HIF-1α or HIF-2α protein. (2) Immunolabeling of downstream transcriptional targets of HIFs (e.g., CA-IX). (3) Immunolabeling of hypoxia probes delivered exogenously 3 h before fixation (e.g., pimonidazole). (4) Oxygen quenches phosphorescent probes (e.g., Rubidium) after excitation with 2-photon light. (5) DNA construct is transcriptionally regulated by HIFs that express any protein (e.g., GFP) in a hypoxia-dependent manner. (6) Fluorescent molecules activated by nitroreductases (NTR) exclusively under hypoxia.
Figure 2
Figure 2
The detection of hypoxia in vivo. (1) Immunolabeling of endogenous hypoxia markers in tumor sections by IF/IHC. (2) Immunolabeling of exogenous hypoxia probes delivered via I.P./I.V. injection 1–2 h before sacrificing the animal (e.g., pimonidazole) in tumor sections by IF/IHC or FC of tumor-dissociated cell suspension. (3) Hypoxia-dependent fluorescent reports are expressed in cancer cells used to generate tumors. Fluorescence can be acquired using a whole animal imaging approach, tumors sections can be imaged by fluorescent microscopy, or tumor cell suspension analyzed by FC. (4) Delivery of fluorescent molecules activated by NTRs exclusively under hypoxia. (5) Hypoxia-dependent bioluminescent reporter expression in cancer cells used to generate tumors. An animal is pre-injected with luciferin and imaged using IVIS. (6) Photoacoustic signals generated by the absorption of near-infrared photons in chromophores of O2-sensitive dyes delivered to the animal cause thermoelastic expansion recordable through a transducer. (7) Imaging of an O2-quenched phosphorescent molecule injected into the animal (e.g., PtG4) with Cherenkov-Excited Luminescence Imaging (CELI) that measures visible photons during radiotherapy. (8) Functional Magnetic Resonance Imaging (fMRI) technology that measures metabolic function via variations in oxyhemoglobin and deoxyhemoglobin ratios using blood-oxygen-level-dependent (BOLD) or tumor oxygenation level-dependent (TOLD) contrast methods. (9) Electron Paramagnetic Resonance Imaging (EPRI) is similar to MRI, but it uses an injected spin probe (e.g., C-labeled pyruvate). (10) Positron Emission Tomography (PET) is an imaging technology that uses 2-nitroimidazole radiolabeling tracers (e.g., 18F-FAZA) with computerized tomography. (11) Electrochemical oxygen sensor that can be implanted or inserted into a needle probe and detects ionization of O2 atoms via a reduction reaction at an electrode. (12) Optical invasive sensors contain an optical fiber with an O2-specific phosphorescent dye coated on the tip that, when excited, the emitted light is captured optically, and the ratio of captured over emitted light is converted to a specific O2 value.
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