title: A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level.
abstract: Hypoxia is an intensively investigated condition with profound effects on cell metabolism, migration, and angiogenesis during development and disease. Physiologically, hypoxia is linked to tissue homeostasis and maintenance of pluripotency. Hypoxia also contributes to pathologies including cardiovascular diseases and cancer. Despite its importance, microscopic visualization of hypoxia is largely restricted to the detection of reductively activated probes by immunostaining. Here, we describe a novel family of genetically encoded fluorescent sensors that detect the activation of HIF transcription factors reported by the oxygen-independent fluorescent protein UnaG. It comprises sensors with different switching and memory behavior and combination sensors that allow the distinction of hypoxic and reoxygenated cells. We tested these sensors on orthotopically transplanted glioma cell lines. Using a cranial window, we could visualize hypoxia intravitally at cellular resolution. In tissue samples, sensor activity was detected in regions, which were largely devoid of blood vessels, correlated with HIF-1α stabilization, and were highly heterogeneous at a cellular level. Frequently, we detected recently reoxygenated cells outside hypoxic areas in the proximity of blood vessels, suggestive of hypoxia-promoted cell migration.
authors.affiliation: Mammalian Cell Signaling Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany Cluster of Excellence EXC 1003, Cells in Motion CiM, Münster, Germany.
authors.forename: Raghu
authors.initials: R
authors.lastname: Erapaneedi
authors.affiliation: Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Belousov
authors.affiliation: Cluster of Excellence EXC 1003, Cells in Motion CiM, Münster, Germany European Institute for Molecular Imaging - EIMI, Münster, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schäfers
authors.affiliation: Mammalian Cell Signaling Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany Cluster of Excellence EXC 1003, Cells in Motion CiM, Münster, Germany fkiefer@gwdg.de.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Kiefer
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D015374:Biosensing Techniques / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D004524:Eels; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D006801:Humans; D000860:Hypoxia; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D009369:Neoplasms / Q000473:pathology*; D011401:Promoter Regions, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A
keywords: UnaG; fluorescent protein; hypoxia; microscopy; sensor