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The ability to noninvasively identify such could provide for personalized treatment(s) and enhance survival rates. Accordingly, we recently developed an NIR fluorescent hypoxia\u2010sensitive smart probe (<jats:bold>NO<\/jats:bold><jats:sub><jats:bold>2<\/jats:bold><\/jats:sub><jats:bold>\u2010Rosol<\/jats:bold>) for identifying hypoxia via selectively imaging nitroreductase (NTR) activity, which could correlate to oxygen deprivation levels in cells, thereby serving as a proxy. We demonstrated proof of concept by subjecting a glioblastoma (GBM) cell line to <jats:italic>extreme<\/jats:italic> stress by evaluating such under <jats:italic>radiobiological<\/jats:italic> hypoxic (<jats:italic>p<\/jats:italic>O<jats:sub>2<\/jats:sub>\u2009\u2264\u2009~0.5%) conditions, which is a far cry from representative levels for hypoxia for brain glioma (<jats:italic>p<\/jats:italic>O<jats:sub>2<\/jats:sub>\u00a0=\u00a0~1.7%) which fluctuate little from <jats:italic>physiological<\/jats:italic> hypoxic (<jats:italic>p<\/jats:italic>O<jats:sub>2<\/jats:sub>\u00a0=\u00a01.0\u20103.0%) conditions.<\/jats:p><\/jats:sec><jats:sec><jats:title>Aim<\/jats:title><jats:p>We aimed to evaluate the robustness, suitability, and feasibility of <jats:bold>NO<\/jats:bold><jats:sub><jats:bold>2<\/jats:bold><\/jats:sub><jats:bold>\u2010Rosol<\/jats:bold> for imaging hypoxia in vitro and in vivo via assessing NTR activity in diverse GBM models under relevant oxygenation levels (<jats:italic>p<\/jats:italic>O<jats:sub>2<\/jats:sub>\u00a0=\u00a02.0%) within <jats:italic>physiological<\/jats:italic> hypoxic conditions that mimic oxygenation levels in GBM tumor tissue in the brain.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We evaluated multiple GBM cell lines to determine their relative sensitivity to oxygenation levels via measuring carbonic anhydrase IX (CAIX) levels, which is a surrogate marker for indirectly identifying hypoxia by reporting on oxygen deprivation levels and upregulated NTR activity. We evaluated for hypoxia via measuring NTR activity when employing <jats:bold>NO<\/jats:bold><jats:sub><jats:bold>2<\/jats:bold><\/jats:sub><jats:bold>\u2010Rosol<\/jats:bold> in in vitro and tumor hypoxia imaging studies in vivo.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The GBM39 cell line demonstrated the highest CAIX expression under hypoxic conditions representing that of GBM in the brain. <jats:bold>NO<\/jats:bold><jats:sub><jats:bold>2<\/jats:bold><\/jats:sub><jats:bold>\u2010Rosol<\/jats:bold> displayed an 8\u2010fold fluorescence enhancement when evaluated in GBM39 cells (<jats:italic>p<\/jats:italic>O<jats:sub>2<\/jats:sub>\u00a0=\u00a02.0%), thereby establishing its robustness and suitability for imaging hypoxia under relevant <jats:italic>physiological<\/jats:italic> conditions. We demonstrated the feasibility of <jats:bold>NO<\/jats:bold><jats:sub><jats:bold>2<\/jats:bold><\/jats:sub><jats:bold>\u2010Rosol<\/jats:bold> to afford tumor hypoxia imaging in vivo via it demonstrating a tumor\u2010to\u2010background of 5 upon (i) diffusion throughout, (ii) bioreductive activation by NTR activity in, and (iii) retention within, GBM39 tumor tissue.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>We established the robustness, suitability, and feasibility of <jats:bold>NO<\/jats:bold><jats:sub><jats:bold>2<\/jats:bold><\/jats:sub><jats:bold>\u2010Rosol<\/jats:bold> for imaging hypoxia under relevant oxygenation levels in vitro and in vivo via assessing NTR activity in GBM39 models.<\/jats:p><\/jats:sec>","DOI":"10.1002\/cnr2.1384","type":"journal-article","created":{"date-parts":[[2021,4,3]],"date-time":"2021-04-03T07:05:12Z","timestamp":1617433512000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["A <scp>NIR<\/scp> fluorescent smart probe for imaging tumor hypoxia"],"prefix":"10.1002","volume":"4","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0607-7944","authenticated-orcid":false,"given":"Kenneth S.","family":"Hettie","sequence":"first","affiliation":[{"name":"Department of Radiology Stanford University School of Medicine  Stanford California USA"},{"name":"Department of Otolaryngology \u2010 Head &amp; Neck Surgery Stanford University  Stanford California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica L.","family":"Klockow","sequence":"additional","affiliation":[{"name":"Department of Radiology Stanford University School of Medicine  Stanford 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