The ability to accurately identify and characterize regions of hypoxia has been an active area of study due to the biological ramifications of low oxygen tension in different pathological conditions, including inflammation, infections, wound healing, cardiovascular conditions, kidney and pulmonary diseases, hepatic and neurological toxicities, and cancer. Although hypoxia contributes a significant role in these conditions, the ability to accurately and instantaneously monitor the presence of hypoxia in vivo and correlate comprehensive analysis of hypoxia-dependent molecular signatures in response to treatments regimens is lacking. With the advent of hypoxia-responsive contrast agents for magnetic resonance imaging (MRI), including the recent development and characterization by our team of a novel probe that involves both 19F and EuII, the capability to integrate direct hypoxia imaging in real-time with new technologies that enable spatial transcriptomic profiling has become a possibility. To assess the capability of this agent for studying hypoxia, we used osteosarcoma as a model. In this preliminary study we demonstrate two major results: First, we show that convection-enhanced delivery (CED) is a reliable and robust methodology to distribute MRI contrast agents throughout a tumor. Second, we show that integration of direct hypoxia-detecting imaging modalities and spatial biology enables real-time in vivo insights into biology and identification of biomarkers directly applicable to disease development and response to therapy. The ability to identify and define hypoxia-mediated biology using direct rather than indirect MRI methods has extremely significant implications in the care of a wide-range of pathological conditions. Consequently, the framework outlined in these preliminary studies is applicable to other pathological conditions and provides the basis for direct in vivo hypoxia detection, monitoring, and biological analyses with translational applications to patient care and management.

Radiographic mapping of hypoxia is needed to study a wide range of diseases. Complexes of Eu(II) are a promising class of molecules to fit this need, but they are generally limited by their rapid oxidation rates in vivo. Here, a perfluorocarbon-nanoemulsion perfused with N2 , forms an interface with aqueous layers to hinder oxidation of a new perfluorocarbon-soluble complex of Eu(II). Conversion of the perfluorocarbon solution of Eu(II) into nanoemulsions results in observable differences between reduced and oxidized forms by magnetic resonance imaging both in vitro and in vivo. Oxidation in vivo occurrs over a period of ≈30 min compared to <5 min for a comparable Eu(II)-containing complex without nanoparticle interfaces. These results represent a critical step toward delivery of Eu(II)-containing complexes in vivo for the study of hypoxia.

Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using Eu II -based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and Eu II . Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated Eu II/III -containing probes. We studied three different Eu II/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time ( T 1 ) and 19 F signal of solutions containing different ratios of Eu II - and Eu III -containing complexes was plotted against the percentage of Eu II -containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.

Anesthesia is often used in preclinical imaging studies that incorporate mouse or rat models. However, multiple reports indicate that anesthesia has significant physiological impacts. Thus, there has been great interest in performing imaging studies in awake, unanesthetized animals to obtain accurate results without the confounding physiological effects of anesthesia. Here, we describe a newly designed mouse holder that is interfaceable with existing MRI systems and enables awake in vivo mouse imaging. This holder significantly reduces head movement of the awake animal compared to previously designed holders and allows for the acquisition of improved anatomical images. In addition to applications in anatomical T2-weighted magnetic resonance imaging (MRI), we also describe applications in acquiring 31P spectra, manganese-enhanced magnetic resonance imaging (MEMRI) transport rates and resting-state functional magnetic resonance imaging (rs-fMRI) in awake animals and describe a successful conditioning paradigm for awake imaging. These data demonstrate significant differences in 31P spectra, MEMRI transport rates, and rs-fMRI connectivity between anesthetized and awake animals, emphasizing the importance of performing functional studies in unanesthetized animals. Furthermore, these studies demonstrate that the mouse holder presented here is easy to construct and use, compatible with standard Bruker systems for mouse imaging, and provides rigorous results in awake mice.

Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments.

The complexes described here serve as contrast agents for magnetic resonance imaging thermometry. The complexes differentially enhance contrast between 275 and 325 K. The basis of the temperature response of the fluorinated contrast complex is the modulation of water exchange caused by trifluoromethyl groups that can be chemically controlled.