Longitudinal optical imaging of tumor metabolism and hemodynamics

Abstract

An important feature of tumor hypoxia is its temporal instability, or "cycling hypoxia." The primary consequence of cycling hypoxia is increased tumor aggressiveness and treatment resistance beyond that of chronic hypoxia. Longitudinal imaging of tumor metabolic demand, hemoglobin oxygen saturation, and blood flow would provide valuable insight into the mechanisms and distribution of cycling hypoxia in tumors. Fluorescence imaging of metabolic demand via the optical redox ratio (fluorescence intensity of FAD/NADH), absorption microscopy of hemoglobin oxygen saturation, and Doppler optical coherence tomography of vessel morphology and blood flow are combined to noninvasively monitor changes in oxygen supply and demand in the mouse dorsal skin fold window chamber tumor model (human squamous cell carcinoma) every 6 h for 36 h. Biomarkers for metabolic demand, blood oxygenation, and blood flow are all found to significantly change with time (p<0.05). These variations in oxygen supply and demand are superimposed on a significant (p<0.05) decline in metabolic demand with distance from the nearest vessel in tumors (this gradient was not observed in normal tissues). Significant (p<0.05), but weak (r<or=0.5) correlations are found between the hemoglobin oxygen saturation, blood flow, and redox ratio. These results indicate that cycling hypoxia depends on both oxygen supply and demand, and that noninvasive optical imaging could be a valuable tool to study therapeutic strategies to mitigate cycling hypoxia, thus increasing the effectiveness of radiation and chemotherapy.

Figure 1

Hemoglobin saturation (HbSat) and redox images taken from a nontumor-bearing window chamber of a mouse breathing oxygen, air, and nitrogen (a) at 1 L∕min through a nose cone. To segment the vessels from background, the hemoglobin saturation images were thresholded by the total absorption image. The same mask was negatively applied to the redox images. Histograms of (b) the hemoglobin saturation and (c) redox images show that the hemoglobin saturation and redox ratio increase with oxygen breathing and decrease with nitrogen breathing, as expected. Image sizes are 2×1.5 mm.

Figure 2

(a) Hemoglobin saturation and (b) redox images taken from one mouse over the 36-h time course. The time of imaging in hours is indicated in the lower left corner of each hemoglobin saturation image. For the purposes of this figure only, the hemoglobin saturation images were thresholded by the total absorption image, and then by a 15-μm-diam circular kernel. The kernel excluded pixels with an absorption value smaller than the average of the 15-μm-diam kernel. The mask was applied to hemoglobin saturation images to isolate vessels from the nonvascular background, and the same vessel mask was negatively applied to the redox images. The tumor boundary is indicated by a white dashed line in (b). Image sizes are 2×1.5 mm.

Figure 3

An en-face view of the 3-D Doppler OCT volume (manually segmented in Amira software, Mercury Systems) from (a) the zero-hour time point of the same animal shown in Fig. 2 (2×1.5-mm imaged area, 0.6 mm depth). Plots of the cross sectional vessel velocity profile from one vessel [arrow in (a)] are shown along with the second-order polynomial fit and the R-squared value of the fit for each time point (b) through (h). The time of imaging in hours is indicated in the upper right corner of each plot.

Figure 4

Quantitative measures of (b) the redox ratio, (c) hemoglobin saturation, (d) vessel maximum velocity, (e) vessel inner diameter, (f) flow, and (g) shear rate over the 36-h time course in (a) six regions of interest from the same animal shown in Figs. 23. The mean and standard error are plotted for (b) the redox ratio and (c) hemoglobin saturation, and the mean (d) through (g) is plotted for the remaining variables.

Figure 5

Normalized redox ratio as a function of distance from the nearest vessel for tumors (compiled from n=94 line profiles corresponding to the 94 regions of interest from the three redox animals across the entire tumor time course), and for normal tissues (compiled from n=36 line profiles from six regions of interest from each of six normal animals). All profiles were normalized to the point closest to the vessel before averaging across all line profiles. For the tumors, there is a significant (p<0.05) decrease in the redox ratio between the point closest to the vessel (point zero) and all points greater than 30 μm from point zero. Normal tissues did not show a significant increase or decrease in the redox ratio as a function of the distance from the nearest vessel. Error bars are standard error.

Figure 6

Longitudinal variations in all biomarkers as a function of time. Values at each time point were averaged across all regions of interest and animals for the hemoglobin saturation (HbSat), flow, diameter, maximum velocity (V_max), and shear rate (n=21), as well as for the redox ratio (n=14). Note that the 36-h time point has four fewer samples (see Sec. 2). The linear mixed model determined that all biomarkers significantly change (p<0.05) with time, and that the vessel diameter is associated with the redox ratio of the adjacent tissue (p<0.05) after adjusting for the effect of time. Error bars are standard error.