Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Sounding Out Dysfunctional Oxygen Metabolism: A Small-Molecule Probe for Photoacoustic Imaging of Hypoxia Michael J. Stevenson and Marie C. Heffern* Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States
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tion. Second, HyP-1 detects hypoxic regions through photoacoustic (PA) imaging, a technology that is finding increasing favor for its excellent depth penetration and high spatial resolution. Third, HyP-1 functions as a multimodal imaging agent where both HyP-1 and the converted aniline, termed redHyP-1, are fluorescent in the near-infrared (NIR) region at distinct excitation wavelengths, permitting the application of HyP-1 as both a NIR probe and a PA probe. PA imaging agents convert absorbed light into heat that, in turn, induces thermoelastic expansions in surrounding tissues. These expansions generate ultrasound waves that can be detected by acoustic transducers. Activatable probes rely on signal enhancement of the product relative to the starting probe through either increasing the number of absorbed photons (molar absorptivity), decreasing the quantum yield and thus increasing the rate of conversion to heat, or shifting the maximum absorption wavelength for selective irradiation.5 Impressively, HyP-1 achieves activation by all three mechanisms, yielding highly sensitive PA signal enhancement. As compared to HyP-1, the reacted red-HyP-1 exhibits a 3.6-fold increase in molar absorptivity, a >2-fold decrease in quantum yield, and a resolved 90 nm shift in the absorbance maximum. In vitro assays in living cells demonstrated the intracellular compatibility of the probe and its hypoxia-responsive activation. To assess the in vivo applicability of HyP-1, the probe was applied to imaging chronic hypoxia in cancer using tumor allografts generated by subcutaneous flank injections of the highly metastatic 4T1 breast cancer line. Tumor-bearing flanks could be clearly distinguished by ratiometric NIR fluorescent imaging after intratumoral (or subcutaneous in control flank) injections of HyP-1 as well as by increased PA signal enhancement after tail-vein injection of HyP-1. Furthermore, three-dimensional reconstruction of the PA images revealed heterogeneous hot spots of the signal that are hypothesized to correspond to the hypoxic regions of the tumor. HyP-1 was then applied to a mouse model of hindlimb ischemia via artery constriction to evaluate its potential for imaging short-term acute hypoxia. These short time scales have posed challenges for nitroimidazole-based agents due to the complex biochemical pathways of probe activation.4 Intramuscular injection of HyP-1 1 h after surgical induction of ischemia showed a rapid identification of the hypoxic region in minutes via NIR imaging, rising to a near 15-fold turn-on 1 h after probe injection. Indeed, HyP-1 could also identify the hypoxic region with PA imaging, exhibiting a 3.1-fold increase in the intensity of the signal at the point of ischemia compared to the control limb.
xygen is essential to all mammals and requires tight control of its supply and consumption. Deprivation of sufficient oxygen from tissues disrupts normal cell metabolism in a pathological condition known as hypoxia. An improved understanding of oxygen homeostasis has unveiled important links between hypoxia and a range of disorders, including ischemic heart disease, cancer, kidney dysfunction, and brain damage.1 Key to this understanding is the emergence of methods for imaging hypoxia as research tools and for both diagnosis and prognosis of diseases. A recent report in Nature Communications led by Jefferson Chan at the University of Illinois at Urbana-Champaign has introduced a novel smallmolecule strategy for non-invasive multimodal in vivo imaging of hypoxic activation.2 Hypoxia research has widely relied on direct measurements of oxygen levels (pO2) in tissues [e.g., oxygen-sensitive electrodes (Figure 1A)].3 These methods, however, are invasive and challenging to apply for dynamic monitoring of hypoxic states in vivo.1 Visualizing time- and space-dependence of hypoxic states can impact clinical decision making, thus demanding strategies for real-time and dynamic imaging of oxygen status in tissues. To date, the most prevalent class of hypoxia-responsive imaging agents utilizes nitroimidazoles as the sensing moiety (Figure 1B).4 Cellular nitroreductases convert the -NO2 group of the nitroimidazole to a -NO2 radical, which is reoxidized back to -NO2 under normal pO2; in hypoxia, insufficient pO2 prevents reoxidation, leading to irreversible reduction of the -NO2 to a -NH2 group through various cellular processes (Figure 2A).4 A number of fluorescent sensors have been developed that exploit this biochemistry for hypoxia sensing, but as purely optical techniques, such probes are primarily limited to cellular imaging due to high scattering and poor depth penetration in tissues. Analogous agents for positron emission tomography (PET) imaging, including [18F]-MISO, that rely on accumulation of the -NH2 product for non-invasive whole-animal imaging have been proposed. Although such agents find limitations in high background and requisite exposure to ionizing radiation, the availability of such in vivo imaging modalities has accelerated our understanding, management, and predictions of hypoxia in pathophysiologies. To this end, the work by Chan and colleagues represents a new stride in hypoxia imaging technology with a threefold impact on the field.2 First, the probe, termed Hypoxia Probe 1 (HyP-1), utilizes an N-oxide sensing moiety that relies solely on direct chemical conversion (two-electron reduction) to the corresponding aniline (Figure 2B). The activation mechanism facilitates rapid detection of hypoxia on both long and short time scales and bypasses intermediate products otherwise observed with nitroimidazole that confound signal interpreta© XXXX American Chemical Society
Received: January 3, 2018
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DOI: 10.1021/acs.biochem.8b00011 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Current methods for monitoring hypoxia include (A) direct methods, such as oxygen-sensitive electrodes, and (B) imaging agents, many of which utilize nitroimidazole-based activation.
Figure 2. Activation mechanisms for hypoxia-sensitive molecular imaging agents. (A) Nitroimidazole probes,the most common agents, convert to the corresponding amines under hypoxic conditions. (B) Chan and co-workers have innovated a hypoxia-responsive trigger containing an N-oxide that undergoes a one-step two-electron reduction in hypoxia to generate the corresponding aniline. (2) Knox, H. J., Hedhli, J., Kim, T. W., Khalili, K., Dobrucki, L. W., and Chan, J. (2017) A bioreducible N-oxide-based probe for photoacoustic imaging of hypoxia. Nat. Commun. 8, 1794. (3) Wilson, W. R., and Hay, M. P. (2011) Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 11, 393−410. (4) Krohn, K. A., Link, J. M., and Mason, R. P. (2008) Molecular imaging of hypoxia. J. Nucl. Med. 49 (2), 129S−48S. (5) Reinhardt, C. J., and Chan, J. (2018) Development of Photoacoustic Probes for in Vivo Molecular Imaging. Biochemistry 57, 194−199.
Taken together, the work by Chan and co-workers importantly expands the approaches for studying hypoxia in disease. Though HyP-1 was initially intended for PA imaging, its multimodal capabilities showcase the impact of combining complementary modes to comprehensively monitor hypoxia localization and dynamics. With the ongoing efforts in imageguided surgery and targeted therapeutic strategies reliant on environmental stimuli like hypoxia, combinations of different modalities and sensing mechanisms are of ever-increasing relevance in basic and translational research. Applying unique platforms like HyP-1 in tandem with existing ones will unravel the kinetic and spatial complexities in the dynamics of dysfunctional oxygen metabolism.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: mcheff
[email protected]. ORCID
Marie C. Heffern: 0000-0001-7501-2741 Funding
Financial support was provided by the University of California, Davis. M.C.H. thanks the University of California Office of the President and the UC Davis Advance CAMPOS Faculty Scholar program for additional support. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Semenza, G. L. (2014) Oxygen Sensing, Hypoxia-Inducible Factors, and Disease Pathophysiology. Annu. Rev. Pathol.: Mech. Dis. 9, 47−71. B
DOI: 10.1021/acs.biochem.8b00011 Biochemistry XXXX, XXX, XXX−XXX
