Tissue pO2 distributions in xenograft tumors dynamically imaged by Cherenkov-excited phosphorescence during fractionated radiation therapy

Abstract

Hypoxia in solid tumors is thought to be an important factor in resistance to therapy, but the extreme microscopic heterogeneity of the partial pressures of oxygen (pO₂) between the capillaries makes it difficult to characterize the scope of this phenomenon without invasive sampling of oxygen distributions throughout the tissue. Here we develop a non-invasive method to track spatial oxygen distributions in tumors during fractionated radiotherapy, using oxygen-dependent quenching of phosphorescence, oxygen probe Oxyphor PtG4 and the radiotherapy-induced Cherenkov light to excite and image the phosphorescence lifetimes within the tissue. Mice bearing MDA-MB-231 breast cancer and FaDu head neck cancer xenografts show different pO₂ responses during each of the 5 fractions (5 Gy per fraction), delivered from a clinical linear accelerator. This study demonstrates subsurface in vivo mapping of tumor pO₂ distributions with submillimeter spatial resolution, thus providing a methodology to track response of tumors to fractionated radiotherapy.

Fig. 1. Bio-distribution of Oxyphor PtG4 measured with IVIS instrument at different time points.

a Phosphorescence from different organs and tumors (n = 3) measured at 12 time points after IV injection of Oxyphor PtG4 (n = 3 mice per time point, n = 36 mice total). Ex vivo images of the phosphorescence from the excised organs (b) and their integrated intensities (c) at the 24 h time point after IV injection of Oxyphor PtG4 (n = 3). In vivo images of the phosphorescence (d) and the phosphorescence intensities in the tumors and regions of the normal tissue (marked by a black-dashed circle) (e) at 24 h after injection of Oxyphor PtG4. The mean signal from the tumors (1.97 ± 0.16 × 10⁴ counts, mean ± SE) was twice as high as that from the normal tissue (0.98 ± 0.09 × 10⁴ counts, mean ± SE) (n = 3). Error bars represent standard error of the mean. Significant difference was analyzed by two-sample t-test.

Fig. 2. Longitudinal in vivo imaging of phosphorescence during 5 days after a single IV injection of Oxyphor PtG4.

a Phosphorescence intensity images measured using wide-field optical excitation in a standard IVIS instrument. b Integrated intensity images of the Cherenkov light along with the phosphorescence of Oxyphor PtG4, acquired during the time of the radiation delivery. c Phosphorescence intensity images by CELI. Average intensity of the phosphorescence acquired by IVIS (d) and CELI (e) for the tumor area and for the normal tissue (n = 4). f The ratio of the signals tumor:normal tissue as imaged by IVIS and CELI (n = 4). Error bars represent standard error of the mean.

Fig. 3. CELI of oxygen in tumors and muscle in vivo before and 30 min after euthanasia.

Oxyphor PtG4 (50 μL of 200 μM solution) was locally injected into the MDA-MB-231 tumor (blue circle) and into muscle (yellow circle) before the imaging session. a, b Phosphorescence intensity images acquired at different delays relative to the radiation pulse before (a) and after euthanasia (b). The phosphorescence images are overlaid on a photograph of the mouse, which was taken before CELI. The tumor and muscle areas used for the analysis are encircled. Tissue oxygen maps (c, d) and pO₂ histograms (e, f) before and after euthanasia. g Average levels of oxygen in the tumor and muscle before and after euthanasia (n = 4). Boxplot shows median and interquartile range; whiskers indicate the range. Statistics was performed using two-sample t-test.

Fig. 4. In vivo imaging of pO₂ in mice 24 h after IV injection of Oxyphor PtG4.

a Phosphorescence intensity images acquired at different delays of 5, 10, 20, and 30 µs relative to the excitation pulse, overlaid with a photograph of the mouse. The regions corresponding to the tumor and the normal surrounding tissue are shown by the yellow and blue circles in the first image. b Tissue pO₂ map. c pO₂ histograms. d Median pO₂ values in the tumor and surrounding tissue (n = 3). Boxplot shows median and interquartile range; whiskers indicate the range. Statistics was performed using two-sample t-test. e Enlarged view of the tumor region in the pO₂ image. f pO₂ histograms of sub-regions (ROIs) outlined by black curves in e. g Areas shown in blue were characterized by pO₂ < 10 Torr. h Fraction of the tumor having pO₂ < 10 Torr.

Fig. 5. In vivo longitudinal pO₂ imaging of mice with two tumor lines during 5 days-long fractionated radiotherapy.

a, b Examples of pO₂ images (a) and of histograms (b) acquired during each day of the radiotherapy treatment. c, d Median pO₂ changes (c) and hypoxic fraction changes (d). Hypoxic fraction is defined here as the ratio of the area with pO₂ < 10 Torr to the total tumor area. The changes in the median pO₂ (c) and in the hypoxic fraction (d) are shown relative to the respective values on the first treatment day. e Relative tumor volume changes during and after the radiotherapy, normalized by the volume prior to the treatment. All data are shown as mean ± standard error of the mean (n = 3 in c, d, e).