Fig 1.
Experimental timeline and design.
A schematic depiction of the experimental design to measure tumor hypoxia after radiation-induced tumor vascular damage is shown. In a human NSCLC xenograft model (A549) gold nanoparticles (AuNP, blue arrow) were i.v. administered to target tumor neovessels, and 10 Gy radiation was delivered (yellow arrow) 24 h post-AuNP injection. HypoxiSense680 fluorescence imaging (red arrow) was used to assess changes in tumor hypoxia pre (0 h) and post-treatment (48 h and 13 days) for all four groups (Control, AuNP, IR, and AuNP+IR). After imaging, mice were a) monitored for tumor progression or b) euthanized for immunohistochemistry studies to confirm tumor hypoxia.
Fig 2.
Tumor uptake and biodistribution studies.
(A) Tumor uptake of AuNP at 2 h post-incubation in A549 cells was measured using high-resolution TEM. Localized uptake in several sub-cellular compartments was observed following 2 h incubation. (B) High-resolution transmission electron microscopy was carried out on ex vivo A549 lung tumor sections 24 h after 1mg/g AuNP injection. White arrows indicate tumor cell/vasculature uptake of AuNP (at a magnification of 2500 - 10000x) and labels indicate the location of red blood cells and endothelial cells. (C) Longitudinal accumulation of AuNP in the tumor, kidney, liver, spleen, lungs, and heart was measured at 1, 4, 12, 24 h, and 30 days after i.v. administration of ~1mg/g of AuNP (n = 2–5). The amount of Au was quantified using ICP-MS and normalized with respective organ weights. Values represent mean ± SD, (**P<0.05).
Fig 3.
Evaluation of treatment toxicity.
H&E staining indicated no detectable toxicity in surrounding visceral organs such as spleen, kidney, lungs, heart, and liver due to either AuNP and/or radiation treatment. Scale bar: 50 μm.
Fig 4.
Therapeutic evaluation of tumor vascular disruption.
(A) Tumor growth relative to tumor size on day of irradiation is shown. A significant (**P<0.05) delay in tumor growth was observed for AuNP+IR treated mice compared to the controls up to 50 days (n = 9–10). Values represent mean growth ± SD. (B) Time-to-Tumor-Doubling (TTD) is shown with endpoint failure defined as tumor volume doubling relative to day of treatment. A significant (**P<0.05) delay in doubling, which is an indicator of a progressive disease, is seen for mice treated with AuNP+IR compared to all control groups (n = 9–10).
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.