Fig 5. Measuring therapy-induced tumor hypoxia in A549 non-small cell lung cancer xenografts.

(A) Fluorescence imaging of tumor hypoxia in representative mice from the IR-only and AuNP+IR groups with A549 xenografts. Mice were injected with HypoxiSense680 48 h before imaging. Tumor hypoxia is visualized pre- and post-10 Gy irradiation treatment. Representative images show the whole mouse and a magnified image of each tumor ROI. The color bar scale shows average radiant efficiency. (B) Plots show the mean quantification of each tumor ROI (Radiation Efficiency ([photon/sec]/μWatt/cm²)) on each day of imaging (Day -1, 2 and 13). In the IR-only group, the images show a 1.5-fold increase in signal 48 h post-IR compared to baseline while 11 days later the tumor hypoxia remained stable. The AuNP+IR group showed a 2.5-fold increase (**P<0.05) at 48 h post-IR and then a decrease to the level of IR-only signal 11 days later. Data presented as mean ± SD (n = 3). (C) The relative radiation efficiency intensity of HypoxiSense680 is similar in the control and GNP-only groups, indicating no interference from the attached fluorophore. (D) Qualitative histological assessment of tumor hypoxia by pimonidazole staining further confirmed the increase in the hypoxia following tumor vascular disruption at 48 h. Scale bar = 100 μm.