Two color imaging of different hypoxia levels in cancer cells

Hypoxia (low oxygen levels) exists in a wide range of biological contexts, including plants roots, bacterial biofilms, and solid tumors. In all cases, hypoxia elicits responses affecting the biological system that is experiencing low oxygen that impact on its survival. In the case of bacterial biofilms and tumors, hypoxia makes treating these conditions more difficult and has a negative impact on patient prognosis. Therefore, chemical probes that enable the study of biological hypoxia are valuable tools to help increase understanding of disease-related conditions that involve low oxygen levels, ultimately leading to improved diagnosis and treatment. While a number of small molecule-based hypoxia-sensing probes exist, the majority of these image only very severe hypoxia (≥1% O2), and therefore do not give a full picture of biological hypoxia, which is heterogenous. Commonly used antibody-based imaging tools for hypoxia are less convenient than small molecules, as secondary detection steps involving immunostaining are required. Here we report the synthesis, electrochemical properties, photophysical analysis, and biological validation of a range of indolequinone-based bioreductive fluorescent probes. We show that these compounds can image different levels of hypoxia in 2D and 3D cell culture. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Tokyo green-based probe 4 was only activated in severe hypoxia – 0.5% O2 and less. Simultaneous application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Compounds 2 and 4 are therefore useful tools to study hypoxia in a cellular setting and represent convenient alternatives to antibody-based imaging approaches.