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. 2021 Oct;4(5):e1384.
doi: 10.1002/cnr2.1384. Epub 2021 Apr 3.

A NIR fluorescent smart probe for imaging tumor hypoxia

Kenneth S Hettie  1   2 , Jessica L Klockow  1 , Eui Jung Moon  3 , Amato J Giaccia  3 , Frederick T Chin  1
Affiliations

A NIR fluorescent smart probe for imaging tumor hypoxia

Kenneth S Hettie et al. Cancer Rep (Hoboken). 2021 Oct.

Abstract

Background: Tumor hypoxia is a characteristic of paramount importance due to low oxygenation levels in tissue negatively correlating with resistance to traditional therapies. The ability to noninvasively identify such could provide for personalized treatment(s) and enhance survival rates. Accordingly, we recently developed an NIR fluorescent hypoxia-sensitive smart probe (NO2 -Rosol) 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 extreme stress by evaluating such under radiobiological hypoxic (pO2 ≤ ~0.5%) conditions, which is a far cry from representative levels for hypoxia for brain glioma (pO2 = ~1.7%) which fluctuate little from physiological hypoxic (pO2 = 1.0-3.0%) conditions.

Aim: We aimed to evaluate the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia in vitro and in vivo via assessing NTR activity in diverse GBM models under relevant oxygenation levels (pO2 = 2.0%) within physiological hypoxic conditions that mimic oxygenation levels in GBM tumor tissue in the brain.

Methods: 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 NO2 -Rosol in in vitro and tumor hypoxia imaging studies in vivo.

Results: The GBM39 cell line demonstrated the highest CAIX expression under hypoxic conditions representing that of GBM in the brain. NO2 -Rosol displayed an 8-fold fluorescence enhancement when evaluated in GBM39 cells (pO2 = 2.0%), thereby establishing its robustness and suitability for imaging hypoxia under relevant physiological conditions. We demonstrated the feasibility of NO2 -Rosol to afford tumor hypoxia imaging in vivo via it demonstrating a tumor-to-background of 5 upon (i) diffusion throughout, (ii) bioreductive activation by NTR activity in, and (iii) retention within, GBM39 tumor tissue.

Conclusion: We established the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia under relevant oxygenation levels in vitro and in vivo via assessing NTR activity in GBM39 models.

Keywords: NIR fluorescence; bioimaging; glioblastoma; hypoxia; smart probe.

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Conflict of interest statement

The authors have stated explicitly that there are no conflicts of interest in connection with this article.

Figures

FIGURE 1
FIGURE 1
Generalized representation of tumor tissue classification as a function of oxygen tension. Low oxygen tension (hypoxia) criteria are in accordance with measured glioma tumor tissue oxygenation levels (of low‐ and high‐grade including GBM)., The unit for a measured oxygenation level is represented by the partial pressure of oxygen (pO2) expressed as a percentage of total environmental pressure. Figure not drawn to scale
FIGURE 2
FIGURE 2
The spectroscopic and photophysical profile of NO 2 ‐Rosol in its inactive and active form (NH2‐Rosol). (A) Normalized absorption spectrum. (B) Normalized fluorescence emission spectrum (λ ex = 550 nm). (C) Normalized fluorescence excitation spectrum (λ ex = 500‐700 nm; λ em = 710 nm)
FIGURE 3
FIGURE 3
Western blot analysis and semi‐quantification of the hypoxia marker, CAIX, in panel of glioblastoma cell lines. GAPDH is used as a loading control. The GBM cell lines were incubated under normoxic conditions (pO2 = 20%) or mild but relevant physiological hypoxic conditions (pO2 = 2.0%) for 24 hours prior to cell lysis. CAIX signal was normalized to the loading control and the ratio of hypoxic‐to‐normoxic fluorescence intensity ratio was plotted (n = 2, **P < .01 using a one‐way analysis of variance followed by Tukey post hoc tests with the hypoxic‐to‐normoxic intensity of the U87 cells serving as the control)
FIGURE 4
FIGURE 4
Cell viability assay using varied concentration levels of NO 2 ‐Rosol with GBM39 cells. Cells receiving no treatment were used as a negative control and cells treated with 15% DMSO as a positive control. Cells were stained with Calcein‐AM, a live cell stain, and subsequently the fluorescence intensity was measured at 516 nm (λ ex = 494 nm, n = 5, *P < .05, ***P < .001, using a one‐way analysis of variance followed by Tukey post hoc tests with the intensity from the cells receiving no treatment serving as the control)
FIGURE 5
FIGURE 5
Confocal fluorescence microscopy images of GBM39 cells incubated with NO 2 ‐Rosol (10 μM) under normoxic (20% O2) or mild but relevant hypoxic (2% O2) conditions. (A) NIR fluorescence imaging. Emission was collected from 650 to 800 nm (λ ex = 550 nm). (B) Brightfield imaging. Scale bar = 25 μm. (C) Quantitative analysis of mean fluorescence intensity of the cells (n = 50, ***P < .001, by an unpaired t‐test with Welch's correction)
FIGURE 6
FIGURE 6
In vivo NIR fluorescence imaging of tumor hypoxia in murine GBM39 tumor model via NO 2 ‐Rosol. (A) NIR fluorescence imaging of hypoxia in murine GBM39 tumor model via the smart probe (25 μM, 50 μL) undergoing bioreductive activation by upregulated NTR activity and subsequently imaged at select time points (λ ex = 503‐555 nm). (B) Quantitative analysis from corresponding tumor hypoxia imaging experiment. Bars are expressed as a TBR (n = 3). There are no statistically significant differences in the TBR between the time points using a one‐way analysis of variance followed by Tukey post hoc tests with the pre‐injection TBR serving as the control
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