title: Electron spin resonance microscopic imaging of oxygen concentration in cancer spheroids.
abstract: Oxygen (O2) plays a central role in most living organisms. The concentration of O2 is important in physiology and pathology. Despite the importance of accurate knowledge of the O2 levels, there is very limited capability to measure with high spatial resolution its distribution in millimeter-scale live biological samples. Many of the current oximetric methods, such as oxygen microelectrodes and fluorescence lifetime imaging, are compromised by O2 consumption, sample destruction, invasiveness, and difficulty to calibrate. Here, we present a new method, based on the use of the pulsed electron spin resonance (ESR) microimaging technique to obtain a 3D mapping of oxygen concentration in millimeter-scale biological samples. ESR imaging requires the incorporation of a suitable stable and inert paramagnetic spin probe into the desirable object. In this work, we use microcrystals of a paramagnetic spin probe in a new crystallographic packing form (denoted tg-LiNc-BuO). These paramagnetic species interact with paramagnetic oxygen molecules, causing a spectral line broadening that is linearly proportional to the oxygen concentration. Typical ESR results include 4D spatial-spectral images that give an indication about the oxygen concentration in different regions of the sample. This new oximetry microimaging method addresses all the problems mentioned above. It is noninvasive, sensitive to physiological oxygen levels, and easy to calibrate. Furthermore, in principle, it can be used for repetitive measurements without causing cell damage. The tissue model used in this research is spheroids of Human Colorectal carcinoma cell line (HCT-116) with a typical diameter of ∼600μm. Most studies of the microenvironmental O2 conditions inside such viable spheroids carried out in the past used microelectrodes, which require an invasive puncturing of the spheroid and are also not applicable to 3D O2 imaging. High resolution 3D oxygen maps could make it possible to evaluate the relationship between morphological and physiological alterations in the spheroids, which would help understand the oxygen metabolism in solid tumors and its correlation with the susceptibility of tumors to various oncologic treatments.
authors.affiliation: Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Mada
authors.initials: M
authors.lastname: Hashem
authors.affiliation: Department of Biological Regulation - Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Weiler-Sagie
authors.affiliation: EPR Center for Viable Systems and the Geisel School of Medicine at Dartmouth, Departments of Radiology and Medicine, Lebanon, NH 03766, USA.
authors.forename: Periannan
authors.initials: P
authors.lastname: Kuppusamy
authors.affiliation: The Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel.
authors.forename: Gera
authors.initials: G
authors.lastname: Neufeld
authors.affiliation: Department of Biological Regulation - Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Neeman
authors.affiliation: Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa 32000, Israel. Electronic address: ab359@tx.technion.ac.il.
authors.forename: Aharon
authors.initials: A
authors.lastname: Blank
mesh_terms: D045744:Cell Line, Tumor; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology*; D008853:Microscopy / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D010092:Oximetry / Q000379:methods; D010100:Oxygen / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology; D014018:Tissue Distribution
chemical_list: D010100:Oxygen
keywords: Cancer spheroids; EPR; ESR; Imaging; Pulsed ESR